Therapeutic liposome with targeting effect as well as preparation method and application thereof
By designing a targeted therapeutic liposome that uses the drug molecule's own ionizable amine group to load siRNA, the problems of low bioavailability and adverse reactions to cationic lipid carriers have been solved, and efficient and safe siRNA delivery and controlled release have been achieved.
Patent Information
- Application Number
- CN202411932120.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-25
- Publication Date
- 2025-05-09
AI Technical Summary
Existing siRNA therapies have low bioavailability due to unmodified siRNA being filtered by nuclease activity and renal filtration, and the use of cationic lipids as carriers may lead to adverse reactions.
A targeted therapeutic liposome was designed to utilize the ionizable amine group carried by the drug molecule itself to provide a driving force for the load of siRNA, avoiding the use of cationic lipids, thereby reducing the risk of adverse reactions. The liposome is a phospholipid bilayer structure, embedded with cholesterol, amphiphilic molecules, cRGD and amine-containing drugs, and siRNA is embedded in the hydrophilic cavity.
Effective targeted delivery and controlled release of siRNA are achieved, which improves the bioavailability and efficacy of drugs, while reducing the risk of adverse reactions and improving the clinical treatment administration method.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of medical technology, in particular to therapeutic liposomes with targeting effect, and a preparation method and application thereof. Background Art
[0002] siRNA therapy is a new type of therapy with the potential to treat a variety of diseases. In theory, siRNA has the potential to target any gene and can mediate the silencing of specific genes. However, unmodified siRNA is rapidly cleared from the circulation by nuclease activity and renal filtration, resulting in low bioavailability after systemic administration. Therefore, choosing a suitable delivery vehicle to safely and effectively deliver siRNA to target cells has become a key issue. We chose the most advanced non-viral carrier lipid nanostructure carrier in clinical practice, which is an efficient carrier of siRNA.
[0003] Liposome drug delivery system is a new drug delivery system with relatively mature research. Liposome is an artificial membrane with a phospholipid bilayer structure, which has great development potential as a drug carrier. It has the following characteristics: (1) Liposome can carry water-soluble, fat-soluble and amphoteric drugs. Through the characteristics of its lipid bilayer, the drug can be better released and absorbed in the body, enhancing the efficacy and reducing toxicity; (2) Liposome has good biocompatibility with biological tissues and cells; (3) Liposome can achieve targeted delivery and controlled release of drugs through various pathways, improving the stability and efficacy of drugs in the body.
[0004] The composition of lipid drug delivery system includes ionizable cationic lipids, PEG-lipids, phospholipids and cholesterol. Ionizable cationic lipids play an important role in the delivery of siRNA by lipid drug delivery system, but ionizable cationic lipids have potential side effects and may cause some adverse reactions in clinic.
[0005] Therefore, it is necessary to design a new therapeutic liposome with targeted effect. Summary of the invention
[0006] The present invention provides therapeutic liposomes with targeting effects, and preparation methods and applications thereof. Under the premise of ensuring the targeting effect, the ionizable amino groups carried by the drug molecules themselves are used to provide driving force for the loading of siRNA, thereby avoiding the defects of the prior art that cationic lipids are required and have potential toxic side effects.
[0007] In view of this, the scheme of the present invention is:
[0008] The first aspect of the present invention is to propose a therapeutic liposome with a targeted effect, wherein the liposome is a phospholipid bilayer structure; cholesterol, amphiphilic molecules, cRGD and amine-containing drugs are embedded in the phospholipid bilayer structure; siRNA is embedded in the hydrophilic cavity of the phospholipid bilayer structure, and the siRNA is used to reduce the expression of disease-related genes.
[0009] Furthermore, the liposome includes, by weight, 1-2 parts of the amine-containing drug, 0.01-0.1 parts of siRNA, 8-16 parts of phospholipids, 2-4 parts of cholesterol, 2-4 parts of amphiphilic molecules, and 0.6-1.2 parts of cRGD.
[0010] Furthermore, the hydrophobic end of the amphiphilic molecule is distearoylphosphatidylethanolamine, and the hydrophilic end is polyethylene glycol; and / or, the amine-containing drug is selected from tamoxifen, berberine or corynepine.
[0011] Furthermore, the diameter of the liposome is 50-200 nm.
[0012] The second aspect of the present invention is to provide a method for preparing the therapeutic liposomes described in the first aspect, the steps comprising:
[0013] S1. dissolving phospholipids, cholesterol, amphiphilic molecules, cRGD and amine-containing drugs in an organic solvent to obtain an oil phase, and dissolving siRNA in an acidic buffer to obtain an aqueous phase;
[0014] S2. Based on microscale flow, the aqueous phase and the oil phase are mixed at different flow rates to obtain a liposome solution;
[0015] S3. The organic solvent in the liposome solution is removed to obtain therapeutic liposomes with targeting effect.
[0016] Furthermore, in step S2, the water phase flow rate is 300-8100 μL / min, and the oil phase flow rate is 100-900 μL / min.
[0017] Furthermore, in step S3, the process of removing the organic solvent is performed using dialysis.
[0018] Preferably, the molecular cutoff of the dialysis process is 200-14000 MW, the dialysis time is 2-4 hours, and the external phase is replaced every 1 hour; and / or, the external phase used in the dialysis is a PBS buffer solution with a pH of 7.4 or physiological saline.
[0019] The third aspect of the present invention is to propose the use of the therapeutic liposomes described in the first aspect in the preparation of drugs.
[0020] Furthermore, when the amine-containing drug is selected from tamoxifen and conoxine, it is used to inhibit cancer cells, such as treating breast cancer and ovarian cancer, and breast cancer includes recurrent and metastatic breast cancer and advanced breast cancer; the siRNA targets the PIK3CA gene, and the siRNA sequence from 3' to 5' end is preferably as shown in SEQ ID NO: 1.
[0021] Furthermore, the amine-containing drug is selected from berberine, and is used to treat autoinflammatory diseases or inflammatory diseases caused by pathogenic microorganisms, such as systemic vasculitis, arteritis, Hashimoto's thyroiditis, intervertebral disc degeneration, arthritis and other diseases; inflammatory diseases caused by pathogenic microorganisms include upper respiratory tract infection, pneumonia, urethritis, soft tissue infection and other diseases. The siRNA targets the TLR4 gene, and preferably the siRNA sequence from 5' to 3' end is as shown in SEQ ID NO: 2.
[0022] Furthermore, the drug also includes a pharmaceutically acceptable adjuvant.
[0023] The therapeutic liposome with targeted effect described in the present invention uses a liposome carrier system to encapsulate siRNA and amine-containing drugs, which can exert the synergistic therapeutic effect of siRNA and drugs. It can not only deliver drugs in a targeted manner, maintain the release time of drugs, and improve the bioavailability of drugs, but also improve the clinical treatment drug administration method and reduce the pain of patients undergoing drug administration, and has significant application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 This is the hydrated particle size distribution diagram of the tamoxifen-siRNA liposomes in Test Example 1.
[0025] Figure 2 This is the electron micrograph of the tamoxifen-siRNA liposome in Test Example 1.
[0026] Figure 3 and Figure 4 They are standard curves of tamoxifen and model nucleic acid drug in Test Example 1 respectively.
[0027] Figure 5 This is the standard curve of berberine in Test Example 1.
[0028] Figure 6 This is the NMR image of each component of tamoxifen-siRNA liposome in Test Example 2.
[0029] Figure 7 These are the RT-qPCR results of the three amine-containing drug-siRNA liposomes in Test Example 3.
[0030] Figure 8These are the experimental results of the particle size and PDI stability of tamoxifen-siRNA liposomes in Experimental Example 1.
[0031] Fig. 9 This is a graph showing the in vitro release results of tamoxifen-siRNA liposomes in Experimental Example 1.
[0032] Fig.10 and Fig.11 They are the fluorescence results and average fluorescence intensity results of MCF-7 uptake of tamoxifen-siRNA liposomes in Experimental Example 2, respectively.
[0033] Fig.12 This is a fluorescence efficiency diagram of BV-2 taking up berberine-siRNA liposomes in Experimental Example 3.
[0034] Fig.13 This is the result of TNF-α enzyme-linked immunosorbent assay in Experimental Example 4 of the present invention.
[0035] Fig.14 These are the results of testing the serum ALT, AST, ALP and creatinine levels of mice in different drug treatment experimental groups in Experimental Example 5.
[0036] Fig.15 and Fig.16 They are the mouse tumor picture and mouse tumor growth curve in Experimental Example 5 respectively.
[0037] Fig.17 Figure 5 shows the changes in body weight of mice in different drug treatment experimental groups in Experimental Example 5 of the present invention.
[0038] Fig.18 These are H&E stained sections of the heart, liver, spleen, lung, and kidney of mice in different drug treatment experimental groups in Experimental Example 5 of the present invention.
[0039] Fig.19 These are the results of cell survival rate of mice in different drug treatment experimental groups in Experimental Example 6.
[0040] Fig. 20 These are the flow cytometry results for monitoring the macrophage polarization type in Experimental Example 7. DETAILED DESCRIPTION
[0041] The technical solution of the present invention will be clearly and completely described below in conjunction with the preferred embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0042] In one embodiment, a therapeutic liposome with a targeted effect is proposed, wherein the liposome is a phospholipid bilayer structure; cholesterol, amphiphilic molecules, cRGD and amine-containing drugs are embedded in the phospholipid bilayer structure; siRNA is embedded in the hydrophilic cavity of the phospholipid bilayer structure, and the siRNA is used to reduce the expression of disease-related genes. The therapeutic liposome uses a liposome carrier system to encapsulate siRNA and amine-containing drugs, which can exert a synergistic therapeutic effect of siRNA and drugs, not only can the targeted delivery of drugs, but also the release time of drugs can be maintained, the bioavailability of drugs can be improved, and the clinical treatment administration method can be improved.
[0043] In a preferred embodiment, the liposome includes, by weight, 1-2 parts of the amine-containing drug, 0.01-0.1 parts of siRNA, 8-16 parts of phospholipids, 2-4 parts of cholesterol, 2-4 parts of amphiphilic molecules, and 0.6-1.2 parts of cRGD.
[0044] In a preferred embodiment, the hydrophobic end of the amphiphilic molecule is distearoylphosphatidylethanolamine, and the hydrophilic end is polyethylene glycol; and / or, the amine-containing drug is selected from tamoxifen, berberine or coryneline. The siRNA is an siRNA of a therapeutic target gene related to the corresponding indications of tamoxifen, berberine, and coryneline. Those skilled in the art can select and design siRNA according to known specific therapeutic targets.
[0045] In a preferred embodiment, for tamoxifen-siRNA liposomes and conoxine-siRNA liposomes, the siRNA targets PIK3CA, and the siRNA sequence from 3' to 5' end is:
[0046] GACAAUGAAUUAAGGGAAATT (SEQ ID NO: 1)
[0047] For berberine--siRNA liposome siRNA targeting TLR4, the siRNA sequence from 5' to 3' end is:
[0048] GAAAUGAGCUGGUAAAGAATT (SEQ ID NO: 2).
[0049] In a preferred embodiment, the method for preparing the therapeutic liposomes described above comprises the following steps:
[0050] S1. dissolving phospholipids, cholesterol, amphiphilic molecules, cRGD and amine-containing drugs in an organic solvent to obtain an oil phase, and dissolving siRNA in an acidic buffer to obtain an aqueous phase;
[0051] S2. Based on microscale flow, the water phase flow rate was controlled to be 300-8100 μL / min, and the oil phase flow rate was controlled to be 100-900 μL / min to mix and obtain a liposome solution;
[0052] S3. The organic solvent in the liposome solution is removed by dialysis to obtain therapeutic liposomes with targeted effects.
[0053] In a preferred embodiment, the preparation process compares different liposome preparation methods to determine the optimal drug-lipid ratio of 1:6 (the ratio of the sum of the mass of the amine-containing drug to phospholipids, cholesterol, amphiphilic molecules and cRGD), and the optimal formula ratio is the mass ratio of egg yolk phosphatidylcholine, cholesterol, DSPE-PEG and cRGD of 10:2:3:0.9.
[0054] The following are preferred implementation examples and experimental examples. If the manufacturer of reagents or instruments is not specified in the examples, conventional products can be purchased from the market. If specific conditions are not specified, conventional conditions or conditions recommended by the manufacturer can be used.
[0055] Preparation Example Preparation of amine-containing drug-siRNA liposomes
[0056] 1) Tamoxifen-siRNA liposome 1
[0057] 15 mg of egg yolk lecithin, 3 mg of cholesterol, 4.5 mg of DSPE-PEG2000, 4.75 mg of tamoxifen, and 1.35 mg of cRGD were weighed respectively, and the above materials were dissolved in 8.5 mL of anhydrous ethanol for later use; 20 μg of siRNA was dissolved in 7 mL of acetamine buffer solution for later use, and the acetamine buffer solution was obtained by diluting the acetamine buffer solution with a pH of 5 ten times; the anhydrous ethanol and the acetamine buffer solution were mixed in a microfluidic chip at a flow rate ratio of 1:6 and a total flow rate of 300 μL / min, and the 60% liposome solution in the middle was collected into a dialysis bag with a molecular cutoff of 14000; the dialysis bag was placed in a PBS buffer solution with a pH of 7.4 for 3 hours, and the external phase was replaced every 1 hour; after the dialysis was completed, the liposome solution in the dialysis bag was collected to obtain tamoxifen-siRNA liposomes.
[0058] 2) Tamoxifen-siRNA liposome 2
[0059] 15 mg of egg yolk lecithin, 4.5 mg of cholesterol, 3 mg of DSPE-PEG2000, 4.75 mg of tamoxifen, and 0.9 mg of cRGD were weighed respectively, and the above materials were dissolved in 8.5 mL of anhydrous ethanol for later use; 20 μg of siRNA was dissolved in 7 mL of acetamine buffer solution for later use, and the acetamine buffer solution was obtained by diluting the acetamine buffer solution with a pH of 5 ten times; the anhydrous ethanol and the acetamine buffer solution were mixed in a microfluidic chip at a flow rate ratio of 1:3 and a total flow rate of 500 μL / min, and the middle 60% liposome solution was collected into a dialysis bag with a molecular cutoff of 2000; the dialysis bag was placed in physiological saline for dialysis for 3 hours, and the external phase was replaced every 1 hour; after the dialysis was completed, the liposome solution in the dialysis bag was collected to obtain tamoxifen-siRNA liposomes.
[0060] 3) Tamoxifen-siRNA liposome 3
[0061] 15 mg of egg yolk lecithin, 4.5 mg of cholesterol, 3 mg of DSPE-PEG2000, 4.75 mg of tamoxifen, and 0.9 mg of cRGD were weighed respectively, and the above materials were dissolved in 8.5 mL of anhydrous ethanol for later use; 20 μg of siRNA was dissolved in 7 mL of acetamine buffer solution for later use, and the acetamine buffer solution was obtained by diluting the acetamine buffer solution with a pH of 5 by ten times; the anhydrous ethanol and the acetamine buffer solution were mixed in a microfluidic chip at a flow rate ratio of 1:9 and a total flow rate of 300 μL / min, and the 60% liposome solution in the middle was collected into a dialysis bag with a molecular cutoff of 5000; the dialysis bag was placed in a PBS buffer solution with a pH of 7.4 for 3 hours, and the external phase was replaced every 1 hour; after the dialysis was completed, the liposome solution in the dialysis bag was collected to obtain tamoxifen-siRNA liposomes.
[0062] 4) Berberine--siRNA liposome
[0063] The lipid material was dissolved in 8.5 mL of anhydrous ethanol, and 20 μg of siRNA was dissolved in 7 mL of acetamine buffer solution for later use, wherein the acetamine buffer solution was obtained by diluting the acetamine buffer solution with a pH of 5 ten times; the anhydrous ethanol and the acetamine buffer solution were mixed in a microfluidic chip at a flow rate ratio of 1:6 and a total flow rate of 400 μL / min, and the 60% liposome solution in the middle was collected into a dialysis bag with a molecular cutoff of 8000; the dialysis bag was placed in a PBS buffer solution with a pH of 7.4 and dialyzed for 3 hours, and the external phase was replaced every 1 hour; after the dialysis was completed, the liposome solution in the dialysis bag was collected to obtain berberine-siRNA liposomes.
[0064] 5) Conosine--siRNA liposome
[0065] Take the lipid material and dissolve it in 8.5mL of anhydrous ethanol, and take another 20μg of siRNA and dissolve it in 7mL of acetamine buffer solution for standby use, the acetamine buffer solution is obtained by diluting the acetamine buffer solution of pH=5 ten times; the above-mentioned anhydrous ethanol and acetamine buffer solution are mixed in a microfluidic chip at a flow rate ratio of 1:6 and a total flow rate of 400μL / min, and the 60% liposome solution in the middle is collected into a dialysis bag with a molecular cutoff of 14000; the above-mentioned dialysis bag is placed in a PBS buffer solution of pH=7.4 for 3h, and the external phase is replaced every 1h; after the dialysis is completed, the liposome solution in the dialysis bag is collected to obtain LNP-corynoxine-siRNA liposomes. LNP-corynoxine and c-LNP-corynoxine-siRNA are both obtained by this preparation method.
[0066] Test Example 1 Characterization of amine-containing drug-siRNA liposomes
[0067] 1. Determination of particle size, PDI and Zeta potential
[0068] 1) Determination of particle size:
[0069] For amine-containing drug-siRNA liposomes, the particle size of the liposomes was measured using a laser scattering particle size analyzer. The hydrated particle size distribution diagram is shown in Figure 1 , polydispersity indicates the uniformity of particle size. The smaller the polydispersity, the more uniform the particles.
[0070] 2) Determination of Zeta potential
[0071] After the particle size was measured, the amine-containing drug-siRNA liposomes were transferred to a potential cup, and the measurement temperature was set to 25° C. The final measurement result of the sample was the average of 20 measurement results.
[0072] Table 1 shows the particle size and potential of different amine-containing drug-siRNA liposomes. The average particle size is within the range of 100 nm (generally 30 nm to micron level). For intravenous administration, the particle size should be less than 200 nm. The polydispersity index (PDI) is between 0.176-0.243, and the Zeta potential is between -6.78 and -1.35 mV (generally between -50 mV and 50 mV). For intravenous administration, the negative Zeta potential can reduce the conditioning and phagocytosis of the RES system on the lipid drug delivery system.
[0073] Table 1:
[0074]
[0075] 2. Morphological Observation
[0076] Transmission electron microscopy (TEM) observation. Using PBS buffered saline solution, pH = 7.4 as the dispersion medium, tamoxifen-siRNA liposomes (lipid concentration of 0.25 mg / mL) were dropped onto a copper grid covered with a carbon film, left to dry overnight at room temperature, and then observed and recorded using a transmission electron microscope at 120 kV. The results are shown in Figure 1. Figure 2 It was shown that under transmission electron microscopy, tamoxifen-siRNA liposomes appeared to be spherical in shape with a particle diameter of about 70 nm, which was consistent with the particle size results.
[0077] 3. Encapsulation Efficiency Determination
[0078] 1) Determination of encapsulation efficiency of amine-containing drugs
[0079] The tamoxifen encapsulation efficiency was determined by HPLC (standard curve see Figure 3 , Y = 18834x + 965.7, R 2 =1, Y is the peak area, x is the concentration of tamoxifen), the encapsulation efficiency in liposomes is close to 100%; the berberine encapsulation efficiency was determined by UV spectrophotometer (standard curve see Figure 5 , Y = 0.0499x - 0.0014, R 2 =0.9999, y is the absorbance at 347nm, x is the berberine concentration), and the encapsulation efficiency in liposomes is about 75%.
[0080] 2) siRNA encapsulation efficiency determination
[0081] The siRNA encapsulation efficiency was determined by nanodrop (standard curve see Figure 4 , Y = 20.607x-0.0033, y is the absorbance at A260, x is the siRNA concentration), the highest encapsulation efficiency of nucleic acid in tamoxifen-siRNA liposomes is 81%
[0082] Test Example 2 Characterization of Tamoxifen-siRNA Liposomes (LNP-TA-siRNA) by H-NMR Spectroscopy
[0083] Phosphatidylcholine, cholesterol, DSPE-PEG-Mal, tamoxifen and tamoxifen-siRNA liposomes were dissolved in deuterated chloroform and sent to NMR, while cRGD and model nucleic acid drugs were dissolved in heavy water and sent to NMR. The results of H NMR showed that Figure 6The CH2=CH2 chemical shifts in lecithin are 5.14ppm and 5.28ppm, the CHR=CHR chemical shift in cholesterol is 5.27ppm, the OCH2CH2 chemical shift in DSPE-PEG-Mal is 3.61ppm, the benzene ring chemical shift in cRGD is 7.27ppm, the H on the benzene ring in tamoxifen is 6.9-7.5ppm, and the H on the deoxyribose ring in the model nucleic acid drug is 3.85-4.4ppm. All of these special H can be found in the H NMR spectrum of tamoxifen-siRNA liposomes.
[0084] Test Example 3 RT-qPCR Data
[0085] 1. RT-qPCR data of LNP-SM102-siRNA and LNP-TA-siRNA
[0086] 1) SM102 is a commercially available ionizable cationic lipid. Its standard formulation was used to prepare LNP-SM102-siRNA for comparison with LNP-TA-siRNA. LNP-SM102-siRNA was prepared by mixing SM102, phosphatidylcholine, cholesterol, and DSPE-PEG at a molar ratio of 50:10:38.5:1.5 using microfluidics;
[0087] 2) RT-qPCR experiments on MCF-7 cell line
[0088] ① Seed plate: MCF-7 was plated at 4×10 5 The cells were seeded in 24-well plates and cultured until the cell density was appropriate.
[0089] ②Administration: Control group, LNP-SM102-siRNA group and LNP-TA-siRNA group were treated for 4 hours, and then cells were collected for RT-qPCR detection. The results are as follows Figure 7 As shown in A.
[0090] 2. qPCR experiment of berberine-siRNA liposome (LNP-BBR-siRNA) on RAW 264.7 cell line
[0091] ① Type 1: RAW 264.7 at 4×10 5 The cells were inoculated at a density of 1.5 μg / ml in 24-well plates and cultured to an appropriate density.
[0092] ②Administration: Control group, LPS group and LNP-BBR-siRNA group were treated for 4 h, and then cells were collected for RT-qPCR detection. The results are as follows Figure 7 B.
[0093] 3. Corynoxine-siRNA liposomes (LNP-corynoxine-siRNA) 1) qPCR experiments on MCF-7 cell lines
[0094] ① Seed plate: MCF-7 was plated at 4×10 5 The cells were inoculated at a density of 1.5 μg / ml in 24-well plates and cultured to an appropriate density.
[0095] ②Administration: The control group, LNP-SM102-siRNA and LNP-corynoxine-siRNA groups were treated for 4 hours, and then the cells were collected for RT-qPCR detection. The results are as follows Figure 7 C.
[0096] The following uses the tamoxifen-siRNA liposome 1 and berberine-siRNA liposome obtained in the preparation example as examples to verify the relevant pharmacological properties.
[0097] Experimental Example 1 Stability study and in vitro release of tamoxifen-siRNA liposomes
[0098] 1) Average hydrated particle size and stability of tamoxifen-siRNA liposomes
[0099] Dynamic light scattering was used to continuously measure the changes in the hydrated particle size of tamoxifen-siRNA liposomes stored in FBS at 4°C over 7 days. Figure 8 As shown, the particle size and PDI of tamoxifen-siRNA liposomes did not change much during 7 days when stored in FBS at 4°C.
[0100] 2) In vitro release of tamoxifen-siRNA liposomes
[0101] The in vitro release of tamoxifen in tamoxifen-siRNA liposomes was determined by dialysis: the in vitro release of the drug at 37°C was determined by high performance liquid chromatography. Take 3 mL of the newly prepared tamoxifen-siRNA liposomes, first determine the total amount of tamoxifen encapsulated in the liposomes by HPLC, then place it in a 14000MW dialysis bag and tie it tightly, use 200 mL of PBS with pH = 7.4 and PBS with pH = 5.8 as the release phase, take 2 mL of the sample to be tested at the specified time 1, 2, 4, 8, 10, 24h, and fill with 2 mL of PBS, immediately detect the tamoxifen concentration in the external phase by HPLC, and draw the release curve of tamoxifen with the time point as the horizontal axis and the tamoxifen release amount corresponding to the time point as the vertical axis. The results are shown in Figure 2. Fig. 9As shown in Figure 3, the release of TA depends on the pH value of the buffer solution and the release time. At pH 7.4, the maximum release of TA is close to 70%, while at pH 5.8, the maximum release of TA reaches 100%, which is due to the protonation of TA occurring at a lower pH value. In addition, the surface charge of the lipid drug delivery system becomes positive at a lower pH value, weakening the electrostatic interaction with TA and promoting the drug release process.
[0102] Experimental Example 2 Study on cellular uptake of tamoxifen-siRNA liposomes
[0103] 1) Preparation of coumarin liposomes
[0104] The preparation method of coumarin-labeled liposomes is as follows: accurately weighing coumarin and lipid materials according to a mass ratio of coumarin to lipid material of 2:75, dissolving them in 8.5 mL of anhydrous ethanol, and taking 20 μg of siRNA and dissolving them in 7 mL of acetamine buffer solution for standby use, wherein the acetamine buffer solution is obtained by diluting an acetamine buffer solution with a pH of 5 by ten times; mixing the anhydrous ethanol and the acetamine buffer solution in a microfluidic chip at a flow rate ratio of 1:6 and a total flow rate of 300 μL / min, collecting 60% of the liposome solution in the middle into a dialysis bag with a molecular cutoff of 14000; placing the dialysis bag in a PBS buffer solution with a pH of 7.4 for dialysis for 3 hours, and replacing the external phase every 1 hour; after the dialysis is completed, collecting the liposome solution in the dialysis bag to obtain coumarin-labeled tamoxifen-siRNA liposomes.
[0105] 2) Cellular uptake experiments on MCF-7 cell line
[0106] ① Seed plate: MCF-7 was plated at a ratio of 1×10 5 The cells were inoculated at a density of 1.5 μg / ml in 24-well plates and cultured to an appropriate density.
[0107] ② Administration: The non-administered group was used as a blank control. The coumarin-labeled tamoxifen-siRNA liposomes were incubated with cells for 0.5h, 1h, 2h, 4h and 6h, and the administration was done in reverse order.
[0108] ③ Fluorescence microscopy: Fluorescence microscopy was used to directly observe the efficiency of cell uptake of coumarin-labeled tamoxifen-siRNA liposomes. The experimental results are as follows: Fig.10 As shown, the fluorescence brightness gradually increases with time.
[0109] ④ Flow cytometry detection: Digest the cells in the 24-well plate, collect the precipitate by centrifugation and resuspend it in PBS, and then detect it on the flow cytometer. The experimental results are as follows: Fig.11 As shown, the average fluorescence intensity gradually increased with time.
[0110] Experimental Example 3 Study on Cellular Uptake of Berberine-siRNA Liposomes
[0111] 1) Preparation of ICG liposomes
[0112] The preparation method of ICG-labeled liposomes is as follows: ICG and lipid material are accurately weighed according to the ratio of ICG: lipid material mass ratio of 10:75, and dissolved in 8.5 mL of anhydrous ethanol; 20 μg of siRNA is dissolved in 7 mL of acetamine buffer solution for standby use, and the acetamine buffer solution is obtained by diluting the acetamine buffer solution of pH=5 ten times; the anhydrous ethanol and the acetamine buffer solution are mixed in a microfluidic chip at a flow rate ratio of 1:6 and a total flow rate of 400 μL / min, and the middle 60% liposome solution is collected into a dialysis bag with a molecular cutoff of 8000; the dialysis bag is placed in a PBS buffer solution of pH=7.4 for 3 hours, and the external phase is replaced every 1 hour; after the dialysis is completed, the liposome solution in the dialysis bag is collected to obtain ICG-labeled berberine-siRNA liposomes.
[0113] 2) Cellular uptake experiments on BV-2 cell line
[0114] ① Plate: Spread the slides in a 24-well plate and add BV-2 at a concentration of 1×10 5 The cells were inoculated at a density of 1.5 μg / ml in 24-well plates and cultured to an appropriate density.
[0115] ② Administration: The group without administration was used as blank control. The ICG-labeled berberine-siRNA liposomes were incubated with cells for 6 hours.
[0116] ③ Staining preparation and detection: After incubation, fix with 4% paraformaldehyde for 30 minutes and stain with DAPI staining solution for 5 minutes. Then observe the efficiency of cell uptake of berberine-siRNA liposomes under laser confocal and total internal reflection imaging system. The experimental results are as follows Fig.12 As shown, berberine-siRNA liposomes were taken up into BV-2 cells.
[0117] Experimental Example 4 TNF-α ELISA of berberine-siRNA liposomes
[0118] 1) Preparation of berberine-siRNA liposomes
[0119] The lipid material was dissolved in 8.5 mL of anhydrous ethanol, and 20 μg of siRNA was dissolved in 7 mL of acetamine buffer solution for later use, wherein the acetamine buffer solution was obtained by diluting the acetamine buffer solution with a pH of 5 ten times; the anhydrous ethanol and the acetamine buffer solution were mixed in a microfluidic chip at a flow rate ratio of 1:6 and a total flow rate of 400 μL / min, and the 60% liposome solution in the middle was collected into a dialysis bag with a molecular cutoff of 8000; the dialysis bag was placed in a PBS buffer solution with a pH of 7.4 and dialyzed for 3 hours, and the external phase was replaced every 1 hour; after the dialysis was completed, the liposome solution in the dialysis bag was collected to obtain berberine-siRNA liposomes.
[0120] 2) Collection of cell culture supernatant
[0121] ① Type plate: RAW264.7 with 5×10 3 The cells were inoculated at a density of 1.5 μg / mL and cultured in 96-well plates until they reached an appropriate density.
[0122] ②Administration: Three groups were set up, namely blank control group, LPS group and berberine-siRNA liposome group. The LPS concentration in the LPS group was 100 ng / mL, the LPS concentration in the berberine-siRNA liposome group was 100 ng / mL, and the berberine concentration was 1.25 μg / mL. The supernatant was collected after 24 hours of culture.
[0123] 3) TNF-α ELISA
[0124] The blank control group was added to the diluent, and the supernatant of the LPS group and the berberine-siRNA liposome group was added to the standard wells. The reaction wells were sealed with sealing tape and incubated at 37°C in a constant temperature box in the dark for 90 minutes. Prepare the biotinylated antibody working solution 20 minutes in advance and wash the plate 5 times. Add the biotinylated antibody diluent to the blank control wells, and add the biotinylated antibody working solution (100μL / well) to the remaining wells. Seal the reaction wells with new sealing tape and incubate at 37°C in a constant temperature box in the dark for 60 minutes. Prepare the enzyme conjugate working solution 20 minutes in advance. Place at room temperature (22-25°C) in the dark. Wash the plate 5 times. Add the enzyme conjugate diluent to the blank control wells, and add the enzyme conjugate working solution (100μL / well) to the remaining wells. Seal the reaction wells with new sealing tape and incubate at 37°C in a constant temperature box in the dark for 30 minutes. Turn on the power of the microplate reader, preheat the instrument, and set the detection program. Wash the plate 5 times. Add 100 μL / well of chromogenic substrate (TMB), incubate in a 37°C incubator, away from light for 15 minutes. Add 100 μL / well of reaction stop solution, mix well, and measure the OD450 value immediately (within 3 minutes). Fig.13 As shown, the berberine-siRNA liposome group significantly reduced the TNF-α content in RAW264.7 cells.
[0125] Experimental Example 5 Pharmacodynamic Study of Tamoxifen-siRNA Liposomes
[0126] 1) Construction of MCF-7 subcutaneous tumor model
[0127] Collect MCF-7 cells and resuspend them at 3 × 10 7 150 μL of cell resuspension was injected subcutaneously into the right skin of healthy balb / c nude mice under the armpit. When the tumor grew to 50-100 mm 3 You can then conduct relevant experiments and calculate the volume using V = a × b. 2 / 2 (a: length of the tumor, b: width of the tumor).
[0128] 2) Growth inhibition of MCF-7 subcutaneous tumors by tamoxifen-siRNA liposomes and in vivo safety evaluation
[0129] The day of tumor implantation was recorded as day 0. When the tumor grew to 50-100 mm 3 The mice were randomly divided into four groups, namely, saline group, TA Tablets group, c-LNP-TA group, and c-LNP-TA-siRNA group, with six mice in each group. The mice were given the drug once every two days for five times, and the gavage group was given the drug every day for ten times. The tumor volume and body weight were measured every two days. The drug was administered from the 8th day by tail vein injection. The tamoxifen content in the TA Tablets group was 10 mg / kg / d, and the tamoxifen content in the c-LNP-TA group and the c-LNP-TA-siRNA group was 0.1125 mg / kg / d.
[0130] After the administration, blood was collected from the eyeball on the 18th day, and the serum was separated by centrifugation and stored at -80°C. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and creatinine were measured using a blood biochemical test kit. The experimental results are as follows Fig.14 As shown in the figure, the levels of ALT, AST, ALP and creatinine were all within the normal reference range (ALT (10.06-96.47U / L), AST (36.31-235.48U / L), ALP (22.52-474.35U / L), creatinine (10.91-85.09)), indicating that tamoxifen-siRNA liposomes did not cause liver and kidney damage. The mice were then killed, and the tumors, heart, liver, spleen, lungs, and kidneys were extracted by dissection. They were washed with PBS and dried on weighing paper, weighed, and the tumors were photographed. The experimental results are shown in the figure. Fig.15 , Fig.16 , Fig.17As shown in the figure, it can be seen that the weight of mice in each group fluctuated steadily during the administration period, and there was no obvious difference in the weight of mice in each group. Then, hematoxylin-eosin (H&E) stained sections of each organ were prepared, observed and photographed under an inverted fluorescence microscope, and the experimental results are shown in the figure. Fig.18 As shown, it can be seen that the myocardial cell structure of each group is intact, and the structures of hepatic sinusoids, hepatic lobules, splenic sinusoids, alveolar capillaries and glomeruli are similar to those of the Saline group. There is no cell enlargement and lymphocyte infiltration, and the cell structure is intact, indicating that tamoxifen-siRNA has no obvious tissue toxicity.
[0131] Experimental Example 6 Study on the cytotoxicity of LNP-corynoxine-siRNA
[0132] The LNP-corynoxine-siRNA liposomes, NP-corynoxine, and c-LNP-corynoxine-siRNA obtained in the preparation example were subjected to CCK8 experiments on MCF-7 cells to study cytotoxicity. The specific process is as follows:
[0133] ① Seed plate: MCF-7 was plated at 5×10 3 The cells were inoculated into 96-well plates and cultured to an appropriate density.
[0134] ② Drug administration: After 24 hours of drug administration according to the set concentration gradient, the drug was discarded. After that, pure culture medium containing 10% CCK8 was added to each well and incubated at 37°C for 2 hours.
[0135] ③ Detection: Place the incubated 96-well plate in an ELISA reader and detect at a wavelength of 450nm. The experimental results are shown in Fig.19 Compared with the group without siRNA addition, the cell survival rate of the group with siRNA added was significantly reduced, indicating that siRNA can target the target gene to exert a therapeutic effect.
[0136] Experimental Example 7 Monitoring of LNP-BBR-siRNA Macrophage Polarization Type
[0137] The berberine-siRNA liposomes obtained in the preparation example were used to perform a macrophage polarization type monitoring experiment on RAW264.7 cells. The steps are as follows:
[0138] ① Type 1: RAW264.7 at 4×10 5 The cells were inoculated at a density of 1.5 μg / mL in 6-well plates and cultured to an appropriate density.
[0139] ②Administration: set up three groups: control, LPS, and LNP. The concentration of LPS was 100 ng / mL, and the concentration of LNP was 1.25 μg / mL. After 24 hours of culture, the cells were digested and incubated with antibodies, and the flow cytometer was used for detection. The experimental results are shown in Fig. 20,After the addition of LNP-BBR-siRNA to the LPS group, M1 polarization was significantly reduced, proving that LNP-BBR-siRNA has a certain anti-inflammatory effect.
[0140] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.
Claims
1. A therapeutic liposome with targeting effect, characterized in that: The liposome is a phospholipid bilayer structure; cholesterol, amphiphilic molecules, cRGD and amine-containing drugs are embedded in the phospholipid bilayer structure; siRNA is embedded in the hydrophilic cavity of the phospholipid bilayer structure, and the siRNA is used to reduce the expression of disease-related genes.
2. The therapeutic liposome according to claim 1, characterized in that The liposome composition is as follows by weight: 1-2 parts of an amine-containing drug, 0.01-0.1 parts of siRNA, 8-16 parts of phospholipids, 2-4 parts of cholesterol, 2-4 parts of amphiphilic molecules, and 0.6-1.2 parts of cRGD.
3. The therapeutic liposome according to claim 1, characterized in that The hydrophobic end of the amphiphilic molecule is distearoylphosphatidylethanolamine, and the hydrophilic end is polyethylene glycol; And / or, the amine-containing drug is selected from tamoxifen, berberine or corynepine.
4. The therapeutic liposome according to claim 1, characterized in that The diameter of the liposome is 50-200 nm.
5. The method for preparing the therapeutic liposome according to any one of claims 1 to 4, characterized in that the steps include: S1. dissolving phospholipids, cholesterol, amphiphilic molecules, cRGD and amine-containing drugs in an organic solvent to obtain an oil phase, and dissolving siRNA in an acidic buffer to obtain an aqueous phase; S2. Based on microscale flow, the aqueous phase and the oil phase are mixed at different flow rates to obtain a liposome solution; S3. The organic solvent in the liposome solution is removed to obtain therapeutic liposomes with targeting effect.
6. The preparation method according to claim 5, characterized in that: In step S2, the water phase flow rate is 300-8100 μL / min, and the oil phase flow rate is 100-900 μL / min.
7. The preparation method according to claim 5, characterized in that: In step S3, the process of removing the organic solvent is performed using dialysis.
8. The preparation method according to claim 7, characterized in that: The molecular retention capacity of the dialysis process is 200-14000MW, the dialysis time is 2-4h, and the external phase is replaced every 1h; and / or, the external phase used in the dialysis is a PBS buffer solution with a pH of 7.4 or physiological saline.
9. Use of the therapeutic liposome according to any one of claims 1 to 4 in the preparation of a drug, characterized in that: The medicament is used to treat cancer or inflammatory diseases.
10. The use according to claim 9, characterized in that: The drug also includes pharmaceutically acceptable adjuvants.