CN108939090B

CN108939090B - Liposome, preparation method and application

Info

Publication number: CN108939090B
Application number: CN201810770367.XA
Authority: CN
Inventors: 李良成; 郑滢; 钱丽霞
Original assignee: Xiamen University
Current assignee: Xiamen University
Priority date: 2018-07-13
Filing date: 2018-07-13
Publication date: 2021-01-01
Anticipated expiration: 2038-07-13
Also published as: CN108939090A

Abstract

The invention discloses a liposome, a preparation method and application thereof. The liposome contains two siRNAs, wherein the two siRNAs are S respectively_37/38、S_75/76Said S_37/38、S_75/76The sequences of (A) are SEQ ID NO 1-2 and SEQ ID NO 3-4, respectively. The invention utilizes DSPE-PEG₂₀₀₀Modification of liposomes with MTX as ligand, simultaneous encapsulation of S_37/38、S_75/76The two siRNAs can achieve the purposes of targeting ovarian cancer cells, thereby inhibiting the proliferation of tumor cells and activating the apoptosis of the tumor cells.

Description

Liposome, preparation method and application

Technical Field

The invention relates to the field of biology, in particular to a liposome, a preparation method and application.

Background

Ovarian cancer is a common malignant tumor of female reproductive organs, the incidence rate of the ovarian cancer is the third place and is only second to cervical cancer and uterine body cancer, but the mortality rate of the ovarian cancer is the first place of gynecological malignant tumor. Data have shown that the incidence of ovarian cancer has increased year by year in recent years. Ovarian cancer has hidden and inconspicuous early symptoms, but abdominal metastasis often occurs before symptoms appear, and 60-70% of patients are in late stage at the time of diagnosis. Treatment of ovarian cancer treatment options vary depending on the type of pathology, and surgical treatment is generally the preferred treatment followed by chemotherapy, radiation therapy, biological therapy, hormonal therapy, and the like. Although symptoms of ovarian cancer patients are relieved after primary surgical treatment and radiotherapy, most advanced patients relapse within 2-3 years and eventually develop chemotherapy resistance and die. Ovarian cancer chemotherapy resistance involves many mechanisms, of which abnormal expression of resistance-associated genes is an important factor.

Small interfering RNAs (siRNAs) are effector molecules in the phenomenon of RNA interference. RNA interference (RNAi) refers to gene silencing at the mRNA level induced by endogenous or exogenous RNA, with high specificity. siRNA is a novel gene drug, which can selectively target messenger RNA, combine and induce gene silencing after transcription of target genes, thereby effectively inhibiting expression of target genes. However, clinical use of siRNA has been limited due to non-specific uptake and low stability of cells. Naked siRNA encounters many obstacles when delivered in vivo, such as being easily degraded by rnases in serum and rapidly cleared by the kidneys; the problems of off-target effect, low transfection efficiency and the like exist; and the siRNA synthesized in vitro is easy to activate the natural immune system of human body to cause unnecessary toxic and side effects, etc. siRNA therefore needs to enter cells with the help of delivery vehicles to exert therapeutic effects.

At present, vectors used in clinical trials are generally classified into two types, namely viral vectors and non-viral vectors, wherein the expression level of the viral vectors is high and accounts for about 75% of the total amount of the vectors, but the viral vectors have potential safety problems, so the non-viral vectors are better choices. Various non-viral vectors have been used for gene therapy of tumors, such as naked DNA direct injection, liposomes, cationic polymers, etc. Wherein the liposome is a micro-vesicle formed by encapsulating a drug in a phospholipid bilayer. Has better development prospect in the fields of tumor drug resistance treatment, overcoming biological barriers, loading biological drugs and the like. The liposome mainly has the following functions as a drug carrier: the targeting of lymphatic system, passive targeting, active targeting after modifying antibody, hormone and receptor ligand, and physicochemical targeting after packaging some special substances. The cationic liposome and siRNA can form an amorphous compound through electrostatic adsorption, and the silencing effect of siRNA on target genes can be obviously improved. There have been several reports in clinical trials that liposome-encapsulated siRNA delivery is currently the most mature and close to clinically used siRNA treatment.

It has been reported that TRAIL is a member of Tumor Necrosis Factor (TNF) superfamily, and has the function of specifically inducing tumor cell apoptosis without toxic and side effects to normal tissue cells. In recent years, the application of molecular drugs targeting TNF-related apoptosis inducing ligand (TRAIL) signaling pathway in tumor therapy has been emphasized, and phase 2 clinical studies have been completed in FDA. Cell type death domain-containing Fas binding protein-like interleukin-1 beta converting enzyme inhibitor protein (c-FLIP) is a recently discovered inhibitor protein in the process of apoptosis, is a naturally occurring caspase inhibitor protein containing a Death Effector Domain (DED), and widely exists in various biological species, wherein the c-FLIP is a cell type death domain-containing cell type death domain-like interleukin-1 beta converting enzyme inhibitor protein (c-FLIP) inhibitor protein_LThe over-expression of the protein can inhibit cell apoptosis mediated by death receptors such as Fas and tumor necrosis factor-related apoptosis inducing ligand (TRAIL). The protein (MADD) containing mitogen-activated death domain kinase activation domain is a shearing isomer of insulinoma-glucagonoma clone20 (IG 20) gene, and is very important for tumor cell survival and TRAIL-mediated signal path inhibition of apoptosis. It has been reported that MADD is highly expressed in lung adenocarcinoma, thyroid cancer, ovarian cancer, breast cancer and other tissues, and the survival of tumor cells is promoted by inhibiting apoptosis. Laboratory design inhibits c-FLIP_LAnd siRNA sequence S expressed by MADD protein_37/38、S_75/76Thus, it is expected that apoptosis of tumor cells will be promoted.

Folate is a glycosylated phosphoinositide-linked membrane glycoprotein whose receptor is scarcely expressed in most normal tissues, but is overexpressed in many tumor cells. Methotrexate (MTX) is a folic acid antimetabolite and can also bind to a tumor surface folate receptor, and in order to further improve the delivery targeting property of the liposome carrier to siRNA, modification of a folate ligand can be added on the surface of the liposome, so that a targeted liposome carrier is constructed, and the targeting property of the liposome is further improved. The most common modification method of cationic liposome is polyethylene glycol (PEG), which can improve the stability of liposome, prolong the circulation time of liposome in blood, and also help the accumulation of liposome at tumor site.

Disclosure of Invention

The invention aims to provide a method for utilizing DSPE-PEG₂₀₀₀-MTX as ligand modified liposomes which are packaged S simultaneously in vivo_37/38、S_75/76The liposome can achieve the purposes of targeting ovarian cancer cells, inhibiting the proliferation of tumor cells and activating the apoptosis of the tumor cells.

In order to achieve the above object, the present invention provides a liposome, wherein the liposome comprises two siRNAs, each of the two siRNAs is S_37/38、S_75/76Said S_37/38、S_75/76The sequences of (A) are SEQ ID NO 1-2 and SEQ ID NO 3-4, respectively.

Further, the preparation method of the liposome comprises the following steps:

preparation of targeting ligand: adopts MTX, EDC, NHS and DSPE-PEG₂₀₀₀-NH₂Stirring with DMSO, separating by silica gel chromatography, filtering with membrane, rotary steaming, and lyophilizing to obtain final product;

preparation of targeted liposome samples: mixing DOTAP solution, DOPE solution, HSPC solution, cholesterol solution and DSPE-PEG₂₀₀₀Mixing the-MTX solution, adding CHCl₃Then placing on a rotary evaporator, vacuumizing, controlling pressure, performing rotary evaporation to enable the liposome to form a transparent and uniform film on the bottle wall, taking out after freezing, adding water for hydration, and performing ultrasonic treatment and film passing to obtain the target liposome sample.

Preparation of liposome/siRNA complexes: adding the obtained liposome into the siRNA of claim 1, mixing uniformly, and standing at room temperature to obtain the liposome/siRNA compound.

Furthermore, in the preparation of the targeting ligand, MTX, EDC, NHS and DSPE-PEG₂₀₀₀-NH₂And use of DMSOThe quantity proportion is as follows: 12 mg: 8 mg: 4.5 mg: 30 mg: 2.5 mL;

further, in the preparation of the targeting ligand, the preparation of the targeting ligand is that MTX, EDC and NHS are respectively weighed and dissolved in DMSO and stirred overnight in a dark place; mixing DSPE-PEG₂₀₀₀-NH₂Dissolving in DMSO, adding into the above solution, stirring overnight, separating with silica gel chromatography, collecting the desired sample, filtering, rotary evaporating, and freeze drying to obtain DSPE-PEG₂₀₀₀-a powder sample of MTX is the targeting ligand.

Further, in the preparation of the target liposome sample, a DOTAP solution, a DOPE solution, a HSPC solution, a cholesterol solution and DSPE-PEG₂₀₀₀MTX solution and CHCl₃The volume ratio of the dosage is as follows: 1.4: 1.488: 2: 1.6: 0.588: 6 mL.

Further, in the preparation of the targeted liposome sample, the DOTAP solution is CHCl of DOTAP₃Solution, DOPE solution is CHCl of DOPE₃Solution, HSPC solution is CHCl of HSPC₃Solution, Cholesterol solution is CHCl of Cholesterol₃Solutions, DSPE-PEG₂₀₀₀-DEPC solution with MTX DOTAP;

preferably, the DOTAP solution is 10mg DOTAP dissolved in 2mL HCL₃Performing the following steps; the DOPE solution is 10mgDOPE dissolved in 2mLCHCL₃Performing the following steps; the HSPC solution is 25.9mgHSPC dissolved in 2mLCHCL₃Performing the following steps; the cholesterol solution is 12.5mg cholesterol dissolved in 2mLCHCL₃Performing the following steps; DSPE-PEG₂₀₀₀The MTX solution is 10mgDSPE-PEG₂₀₀₀MTX was dissolved in 1mL DEPC.

Further, the target liposome sample is prepared by taking DOTAP solution, DOPE solution, HSPC solution, cholesterol solution and DSPE-PEG₂₀₀₀Mixing the-MTX solution, adding CHCl₃Adding the solution into an eggplant-shaped bottle, placing the bottle on a rotary evaporator, vacuumizing, controlling the pressure to be 0.01-0.04 MPa, and carrying out rotary evaporation for 30-40min at 42-48 ℃ to enable the liposome to form a transparent and uniform film on the wall of the bottle, freezing for 2-6 hours at-15-30 ℃, taking out the film, adding DEPC (diethyl phthalate) for hydration, carrying out ultrasonic treatment for 10-20 times by using a 200-mesh film, and then carrying out ultrasonic treatment for 10-20 times by using a 100-mesh film to obtain a targeted liposome sample.

Further, in the preparation of the liposome/siRNA complex, the nitrogen-phosphorus ratio of the liposome to the siRNA is 7: 1.

further, in the preparation of the liposome/siRNA complex, the siRNA is S37/38 and S75/76 according to a ratio of 1:1 molar ratio of the mixed composition.

The invention also provides the application of the liposome in tumor targeting.

The invention utilizes DSPE-PEG₂₀₀₀-MTX as ligand to modify liposomes with simultaneous encapsulation of S in the liposomes_37/38、S_75/76The purposes of targeting ovarian cancer cells, inhibiting the proliferation of tumor cells and activating the apoptosis of the tumor cells are achieved.

c-FLIP in the invention_LThe MADD specific shRNA sequence is as follows:

c-FLIP_L siRNA(S37/38):

Sense:5'-CUGCCUGAUAAUCGAUUGCtt-3' SEQ ID NO:1；

anti-sense:5'-GCAAUCGAUUAUCAGGCAGtt-3 SEQ ID NO:2；

MADD siRNA(S75/76)：

Sense:5'-GUACCAGCUUCAGUCUUUCtt-3' SEQ ID NO:3；

anti-sense:5'-GAAAGACUGAAGCUGGUACtt-3' SEQ ID NO:4。

ovarian cancer is a malignant tumor with high incidence and mortality, the incidence rate of ovarian cancer is also increasing year by year in recent years, and the drug resistance of ovarian cancer is a great difficulty for people to study and overcome cancer. Research has shown that an important regulatory mechanism for anti-apoptosis of tumor cells is to express anti-apoptotic proteins to make the cells resistant to apoptosis. TRAIL is a member of a newly discovered Tumor Necrosis Factor (TNF) superfamily, has no obvious toxic or side effect on normal cells, and can selectively induce tumor cells, transformed cells and virus-infected cells to generate apoptosis. Many studies in recent years have shown that one important reason why many tumor cells develop a TRAIL-resistant mechanism is that tumor cells synthesize apoptosis-inhibiting proteinsThereby producing resistance to apoptosis. c-FLIP_LIs a main inhibitory protein for resisting apoptosis, is over-expressed in a plurality of malignant tumor cells, ensures that the tumor cells have a certain escape mechanism, and is a very key action target point for treating tumors. MADD is a gene related to apoptosis inhibition activated by MAP kinase, and Mulherkarn, a scholarer, demonstrated that MADD gene is essential for cancer cell survival and that TRAIL-mediated apoptosis of cancer cells can be activated if the gene is knocked out.

In the research, a targeting ligand DSPE-PEG2000-MTX is synthesized and connected to a liposome, so that the active targeting property of the liposome is increased, and siRNA for inhibiting c-FLIPL and MADD expression is packaged in the liposome simultaneously, so that tumor cell apoptosis is promoted in multiple ways.

In the experimental process, a targeting ligand is synthesized, a targeting and non-targeting liposome is prepared by a film dispersion method, and various physical and chemical constants of the liposome and the drug loading ratio of siRNA are measured. In the targeting assay, two methods, flow cytometry and immunofluorescence, were chosen for validation. OVCAR-5 cells and targeted and non-targeted liposomes packaged with siRNA with fluorescence are incubated for 24h, and fluorescence intensity is detected by flow cytometry and immunofluorescence, so that MTX-LIP group can be endocytosed by OVCAR-5 cells faster than PEG-LIP group, and DSPE-PEG is verified₂₀₀₀Targeting of MTX. Meanwhile, the transfection efficiency of siRNA is detected by flow type, and the siRNA packaged in liposome has high transfection efficiency by flow type result analysis, and the ratio of siRNA to siRNA is 1:1 the siRNA encapsulated in the liposome can be well expressed. In vitro killing experiments, the same inhibitory c-FLIP was packaged_LCompared with PEG-LIP group, MTX-LIP group has more obvious killing effect on OVCAR-5 cells, which indicates that the siRNA expressed by MADD may have therapeutic effect on ovarian cancer.

In conclusion, MTX-LIP prepared by the invention can be taken up by OVCAR-5 cells more quickly, and inhibits c-FLIP_LAnd the liposome expressed by the MADD protein has the effect of obviously inhibiting the cell survival rate of the ovarian cancer cell strain OVCAR-5 cells, and the research lays an experimental basis for targeted therapy of ovarian cancerA foundation.

Drawings

FIG. 1 is targeting ligand DSPE-PEG₂₀₀₀-synthetic scheme for MTX.

FIG. 2 is DSPE-PEG₂₀₀₀Of MTX¹H-NMR spectrum;

FIG. 3 is a diagram of gel electrophoresis of non-targeted liposomes (PEG-LIP) diluted 5 times at different ratios;

FIG. 4 is a 10-fold dilution of non-targeted liposomes (PEG-LIP) by gel electrophoresis at different ratios;

FIG. 5 is a 10-fold dilution of non-targeted liposomes (PEG-LIP) by gel electrophoresis at different ratios;

FIG. 6 is a graph of gel electrophoresis at various ratios with five-fold dilution of targeted liposomes (MTX-LIP);

FIG. 7 is a representation of non-targeted liposomes (PEG-LIP);

FIG. 8 is a representation of targeted liposomes (MTX-LIP);

FIG. 9 is a graph of flow results of uptake of LIP + FAM siRNA by OVCAR-5 cells;

FIG. 10 is a graph of the flow results of uptake of LIP-CY5 siRNA by OVCAR-5 cells;

FIG. 11 is a graph of flow results of uptake of LIP-FAM siRNA + CY5 siRNA by OVCAR-5 cells;

FIG. 12 is a graph comparing the results of transfection of FAM siRNA alone;

FIG. 13 is a graph comparing the results of CY5 siRNA transfection alone;

fig. 14 is FAM siRNA: results of 1:1 double staining for CY5 siRNA ratio are compared;

FIG. 15 is a graph of transfection efficiency of siRNA by flow cytometry;

FIG. 16 is a graph showing the results of western blot protein expression level assay;

FIG. 17 is a graph showing the inhibition of MTT-detecting cells.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention. In the description of the present invention, "first", "second", "third", etc. are used for convenience of reference or description, and are not to be construed as being related in order or of relative importance, unless otherwise indicated, and "plurality", or "multiplicity" means two (or more) or more than two (or more). The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example (b):

1. targeting ligand DSPE-PEG₂₀₀₀Synthesis and characterization of MTX:

according to the synthetic route shown in figure 1, the targeting ligand DSPE-PEG is synthesized₂₀₀₀-MTX。

Respectively weighing 12mg of MTX, 8mg of EDC and 4.5mg of NHS, dissolving in 2mL of DMSO, and stirring overnight in the dark; 30mg of DSPE-PEG₂₀₀₀-NH₂Dissolving in 0.5mL DMSO, adding into the above solution, stirring overnight, separating with silica gel chromatography, collecting the desired sample, filtering with membrane, rotary evaporating, and freeze drying to obtain DSPE-PEG₂₀₀₀-powder samples of MTX. Subjecting the purified product to¹H-NMR analysis. The obtained spectrum is shown in FIG. 2, and it can be seen from FIG. 2 that 6.58(2H)7.59(2H),8.57(1H) are the corresponding chemical shift peaks of H on the benzene ring, indicating that MTX containing benzene ring has been successfully linked to DSPE-PEG₂₀₀₀In the above, the targeting ligand is successfully obtained.

2. Preparation of Targeted liposomes (MTX-LIP) and non-Targeted liposomes (PEG-LIP)

2.1 preparation of Targeted liposomes (MTX-LIP)

Respectively weighing DOTAP 10mg, DOPE 10mg, HSPC 25.9mg and cholesterol 12.5mg, and respectively using 2mL CHCl₃Dissolving, and DSPE-PEG₂₀₀₀MTX 10mg dissolved in 1mL DEPC water. Taking 1.4mL of DOTAP solution, 1.488mL of DOPE solution, 2mL of HSPC solution, 1.6mL of cholesterol solution and 0.588mL of DSPE-PEG₂₀₀₀-MTX solutionMixing the solution, adding 6mL CHCl₃. Adding the solution into an eggplant-shaped bottle, placing the bottle on a rotary evaporator, vacuumizing, controlling the pressure at 0.03MPa, carrying out rotary evaporation for 30-40min at 45 ℃ to enable the liposome to form a transparent and uniform thin film on the wall of the bottle, freezing for 4 hours at-20 ℃, taking out, adding 2.5mL of DEPC (diethylpyrocarbonate) for hydration, carrying out ultrasonic treatment (30% power, 28min, working for 5s and stopping for 10s) for 15 times, and then carrying out ultrasonic treatment on the film for 15 times and 100 times to obtain a targeted liposome sample.

2.2 preparation of non-Targeted liposomes (PEG-LIP)

Weighing DOTAP, DOPE, HSPC and cholesterol according to the prescription amount of preparation of the targeted liposome, dissolving in 2mL CHCl₃Medium, DSPE-PEG₂₀₀₀-NH₂210mg dissolved in 1mL DEPC water to prepare a solution, 1.4mL DOTAP solution, 1.488mL DOPE solution, 2mL HSPC solution, 1.6mL cholesterol solution and 0.588mL DSPE-PEG₂₀₀₀And (4) uniformly mixing the MTX solution, and preparing a non-target liposome sample by the same preparation method as the target liposome.

2.3 gel electrophoresis method for determining drug loading ratio of cationic liposome to siRNA

0.2mL of each of the prepared targeted and non-targeted liposomes was diluted 5-fold with PBS. And taking 6 EP tubes of the diluted targeted liposome group and the diluted non-targeted liposome group respectively according to the nitrogen-phosphorus ratio of the liposome to the siRNA of 0:1, 2:1, 4:1, 6:1, 8:1 and 10:1, wherein the siRNA is a composition of S37/38 and S75/76, S37/38: the molar ratio of S75/76 was 1: 1. adding the liposome diluted according to the corresponding proportion into 1 mu L of siRNA (0.264 mu g/mL), supplementing the volume to 11 mu L with double distilled water, blowing and beating for 3-5 times, uniformly mixing, and standing for 30min at room temperature to prepare liposome/siRNA complexes with different proportions. During this time, 2% RNA gel agarose gel and agarose loading buffer were prepared. After 30min, 2. mu.L of 6 XDNA loading buffer is added to each tube, mixed evenly, electrophoresed for 20min under the voltage of 80V for each tube/hole, and the agarose gel is taken out to be observed under ultraviolet.

The interaction between the positively charged cationic liposome and the negatively charged siRNA attracts the siRNA, causing the siRNA to be adsorbed and coagulated by the liposome to form a liposome/siRNA complex. When there are enough cationic liposomes with positive charge, siRNA can be completely loaded in the liposome. The loading capacity of the liposome to the siRNA is detected by a gel electrophoresis method, after EtBr is colored, the base pair of the siRNA shows high fluorescence under ultraviolet, and the fluorescence is reduced along with the combination with the liposome. FIG. 3 is a diagram of gel electrophoresis at different ratios after five-fold dilution of non-targeted liposomes (PEG-LIP). The liposome/siRNA nitrogen to phosphorus ratios in lanes 1-6 were 0/1, 2/1, 4/1, 6/1, 8/1, 10/1. Table 1 shows the liposome/siRNA volume ratios from left to right in fig. 3.

TABLE 1 Liposome/siRNA volume ratio table corresponding to FIG. 3

Lane lane	1	2	3	4	5	6
							Ratio of	0/1	2/1	4/1	6/1	8/1	10/1
Water (ul)	10	8	6	4	2	0
							PEG-LIP (5 Xdilution)	0	2	4	6	8	10
siRNA(0.264μg/μl)	1	1	1	1	1	1

FIG. 3 shows that, when the liposome/siRNA ratio was 2/1, siRNA had been completely encapsulated in liposome, because it could not be determined whether siRNA had been completely encapsulated in liposome before the ratio was 2/1, and PEG-LIP prepared was diluted 10-fold and then the 2.3 steps were repeated to perform the series of steps at different ratios. The results are shown in FIGS. 4 and 5. The liposome/siRNA ratios of lanes 1-7 in fig. 4 are 0/1, 0.5/1, 1/1, 2/1, 3/1, 4/1, 5/1, and the liposome/siRNA ratios of lanes 1-8 in fig. 5 are 0/1, 3/1, 4/1, 5/1, 6/1, 7/1, 8/1, 9/1. Table 2, table 3 shows the liposome/siRNA ratios from left to right in fig. 4 and fig. 5, respectively. From the results of fig. 4 and 5, it can be seen that under 10-fold dilution conditions, when the liposome/siRNA ratio is 7/1, siRNA is completely encapsulated in the liposome. FIG. 6 is a diagram of gel electrophoresis at various ratios after five-fold dilution of targeted liposomes (MTX-LIP). Wherein the liposome/siRNA ratios of lanes 1-6 are 0/1, 2/1, 4/1, 6/1, 8/1, 10/1. Table 4 shows the liposome/siRNA ratios from left to right in fig. 6. As can be seen in FIG. 6, when the liposome/siRNA ratio was 4/1, the siRNA was completely entrapped in the liposome.

TABLE 2 Liposome/siRNA ratio Table corresponding to FIG. 4

Lane lane	1	2	3	4	5	6	7
								Ratio of	0/1	0.5/1	1/1	2/1	3/1	4/1	5/1
Water (ul)	10	9.5	9	8	7	6	5
								PEG-LIP (10 Xdilution)	0	0.5	1	2	3	4	5
siRNA(0.264μg/μl)	1	1	1	1	1	1	1

TABLE 3 Liposome/siRNA ratio Table corresponding to FIG. 5

Lane lane	1	2	3	4	5	6	7	8
									Ratio of	0/1	3/1	4/1	5/1	6/1	7/1	8/1	9/1
Water (ul)	10	7	6	5	4	3	2	1
									PEG-LIP (10 Xdilution)	0	3	4	5	6	7	8	9
siRNA(0.264μg/μl)	1	1	1	1	1	1	1	1

TABLE 4 Liposome/siRNA ratio Table corresponding to FIG. 6

2.4 characterization of blank liposomes and Liposome/siRNA complexes

The particle size, potential and PDI index of the blank liposomes and the liposome/siRNA complexes at different ratios can be measured by a malvern particle sizer. The results are shown in FIGS. 7 and 8. FIG. 7 shows the data of non-targeted liposomes (PEG-LIP) and FIG. 8 shows the data of targeted liposomes (MTX-LIP). The results of fig. 7 and 8 show that the data for liposomes are within the normal range.

2.5 evaluation of Liposome Targeting

2.5.1 flow cytometry targeting of liposomes

OVCAR-5 cells were seeded in 6-well plates at 1.5-2X 10 per well⁵One well is provided with 1 hole of a blank control group, 3 holes of a PEG-LIP group and 3 holes of a MTX-LIP group, and 7 holes are provided. The plates were incubated at 37 ℃ with 5% CO₂The incubator was incubated overnight. Diluting the non-target liposome (PEG-LIP) prepared in 2.2 by 10 times with 1mL, and diluting the target liposome (MTX-LIP) by 5 times with 1 mL. When the cells grow to 30% -50%, the original culture medium is discarded, and 2mL of fresh serum-containing culture medium is added. The PEG-LIP group is prepared according to the liposome: siRNA nitrogen-phosphorus ratio of 7: 1 into three groups, adding FAM siRNA7.46 μ L, CY5 siRNA7.46 μ L, FAM siRNA3.73 μ L + CY5 siRNA3.73 μ L and diluted PEG-LIP liposome in corresponding proportion, and incubating for half an hour. MTX-LIP group was characterized by liposome siRNA of 4:1 nitrogen-phosphorus ratio is equal to that of the PEG-LIP group, corresponding siRNA and diluted MTX-LIP liposome are added, and then incubation is carried out for half an hour. During transfection, the prepared liposome/siRNA compound is added into a corresponding hole, the culture medium is discarded after 24 hours of transfection, PBS is used for washing for 2 times, trypsinization and centrifugation are carried out, PBS is used for washing for 3 times and centrifugation are carried out, and the fluorescence intensity of FAM and CY5 absorbed by cells is measured by a flow cytometer (the FAM excitation wavelength is 494nm, the emission wavelength is 522nm, the CY5 excitation wavelength is 646nm, and the emission wavelength is 664 nm). The results are shown in FIGS. 9, 10 and 11.

FIG. 9 is a graph showing the flow results of uptake of LIP + FAM siRNA into OVCAR-5 cells, FIG. 10 is a graph showing the flow results of uptake of LIP-CY5 siRNA into OVCAR-5 cells, and FIG. 11 is a graph showing the flow results of uptake of LIP-FAM siRNA + CY5 siRNA into OVCAR-5 cells simultaneously. As can be seen from the three results, the mean fluorescence intensity of the targeted liposome (MTX-LIP) group is about 1.5 times stronger than that of the PEG-LIP group, and the result indicates that the amount of siRNA mediated by the targeted liposome into OVCAR-5 cells is obviously higher than that of the non-targeted liposome, indicating that DSPE-PEG₂₀₀₀MTX modified liposomes may have some targeting properties.

2.5.2 immunofluorescence assay targeting of liposomes

OVCAR-5 cells were seeded in 12-well plates with 8.77X 10 cells per well⁴One well is provided with 1 hole of a blank control group, 3 holes of a non-targeting liposome (PEG-LIP) group and 3 holes of a targeting liposome (MTX-LIP) group, and the total number of the holes is 7. The plates were incubated at 37 ℃ and 5% CO₂Culturing in an incubator overnight until the cells grow to 30% -50%, discarding the original culture medium, and adding 1mL of fresh serum-containing culture medium. The PEG-LIP group was based on liposomes with siRNA of 7: 1 nitrogen phosphorus ratio of three groups, adding FAM siRNA3.79 μ L, CY5 siRNA3.79 μ L, FAM siRNA 1.89 μ L + CY5 siRNA 1.89 μ L and diluted PEG-LIP liposome in corresponding ratio, mixing and incubating for half an hour. MTX-LIP group was characterized by liposome siRNA of 4:1 and PEG-LIP group, adding corresponding siRNA and diluted MTX-LIP liposome, and then incubating for half an hour. During transfection, the prepared liposome/siRNA compound is added into a corresponding hole, the culture medium is discarded after 24 hours of transfection, PBS is used for washing three times, 4% paraformaldehyde is added to soak the small disc, the small disc is uniformly mixed in a 8-shaped manner, the small disc is fixed for 15-20min in a dark place, and the small disc is washed three times by PBS. Dropping a drop of glycerol containing DAPI on a glass slide, taking out a small round piece in a 12-hole plate, sucking excessive water, covering the side with cells opposite to the glass slide, removing air bubbles, sealing with nail polish, and observing the fluorescence intensity taken by the cells by a fluorescence confocal microscope.

DAPI, FAM and CY5 were excited with blue, green and red light, respectively, and immunofluorescence was performed in three groups. The results are shown in FIGS. 12, 13 and 14. FIG. 12 is a comparison of results of siRNA-FAM transfection alone, FIG. 13 is a comparison of results of siRNA-CY5 transfection alone, and FIG. 14 is a comparison of results of siRNA-FAM: the results of double staining with a molar ratio of siRNA-CY5 of 1:1 are compared in a graph. As can be seen from the results, after incubating siRNA with liposome for 24h, OVCAR-5 cells gradually took up siRNA, wherein the siRNA taken up by the non-targeted liposome (PEG-LIP) group was less, while the siRNA taken up by the targeted liposome (MTX-LIP) group was significantly more than that of the PEG-LIP group. This result also indicates that targeted liposomes were more likely to introduce siRNA into OVCAR-5 cells, further validating DSPE-PEG₂₀₀₀MTX modified liposomes may have some targeting properties.

2.6 flow cytometry measurement of transfection efficiency of siRNA

The transfection efficiency of targeted liposome-loaded siRNA was examined by flow cytometry. The results are shown in FIG. 15. Wherein a, a blank control group b, an MTX-LIP + FAM siRNA group c, an MTX-LIP + CY5 siRNA group d, an MTX-LIP + FAM siRNA + CY5 siRNA group e, a PEG-LIP + FAM siRNA group f, a PEG-LIP + CY5 siRNA group g, a PEG-LIP + FAM siRNA + CY5 siRNA group. As can be seen from fig. 15, the transfection efficiencies of the targeted ligand groups were all higher than those of the non-targeted ligand groups. FIGS. 15d and g are flow cytograms transfected with 2 siRNAs, and it can be seen from FIG. 15 that both transfected siRNAs were detectable in the cell.

2.7 Western blot vs. c-FLIP_LAnd downregulation of MADD expression

OVCAR-5 cells were inoculated into 6-well plates and divided into 8 groups according to the grouping of 2.6, and liposomes and siRNA were added to the corresponding groups after half an hour of incubation, and cultured for 48h at 37 ℃ with 5% CO 2. After 48h, adding 500 mu L of pancreatin into each well, digesting, centrifuging, adding 1mL of culture medium, blowing, transferring to a corresponding EP tube, centrifuging, discarding the supernatant, adding PBS, resuspending, centrifuging, discarding the supernatant, and depositing on ice to prepare a sample. Adding 50 μ L of lysine buffer into each tube, shaking once every 10min, centrifuging at 13000rpm for 30min at 4 deg.C for 15min, collecting supernatant, determining protein concentration, decocting for 5min, and freezing at-80 deg.C. After SDS-PAGE electrophoresis gel is prepared, electrophoresis is stopped after the sample is subjected to electrophoresis for 150min under the voltage of 80V, membrane transfer (90V, 90min) is carried out, and after milk is sealed for 1h, the antibody is incubated overnight. The membrane after the overnight is taken out the next day, washed three times with TBS for 10 min/time, washed three times after incubating the secondary antibody for 1h, and developed. And (5) stripping the antibody from the developed membrane and detecting the internal reference.

The results are shown in FIG. 16. Wherein a is c-FLIP_LThe expression level of the protein is a blank control group and PEG-LIP + S from left to right in sequence_37/38Group, MTX-LIP + S_37/38Group, PEG-LIP + S_37/38+S_75/76Group, MTX-LIP + S_37/38+S_75/76And (4) grouping. (ii) a And b is the expression level of the MADD protein. Wherein b is the expression level of the MADD protein, and a blank control group and PEG-LIP + S are sequentially arranged from left to right_75/76Group, MTX-LIP + S_75/76Group, PEG-LIP + S_37/38+S_75/76Group, MTX-LIP + S_37/38+S_75/76And (4) grouping. From A in FIG. 16, it can be seen that S is added_37/38The lighter band of group (2) indicates S_37/38Can obviously inhibit c-FLIP_LThe protein expression and the inhibition effect of the target ligand group are more obvious. And b in FIG. 16 shows addition of S_75/76Group of the strips is shallowAlso describes S_75/76Can inhibit the expression of MADD protein, and the inhibiting effect of the targeted liposome group is better than that of the non-targeted liposome group. In the group in which two siRNAs were packaged, it can be seen that both siRNAs down-regulate c-FLIP_LAnd expression of MADD, and the knockout effect of the targeted liposome group is more obvious.

2.8 MTT assay for cell inhibition

OVCAR-5 cells were seeded in 96-well plates and divided into blank control, negative control, non-targeted liposomes (PEG-LIP) + S_37/38Group, PEG-LIP + S_75/76Group, PEG-LIP + S_37/38+S_75/76Group, targeting liposome (MTX-LIP) + S_37/38Group, MTX-LIP + S_75/76Group, MTX-LIP + S_37/38+S_75/76Groups were 8 groups with 12 wells each. Four time points of the first, third, fifth and seventh days are set, and each time point is provided with 3 multiple holes. At the time point, 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazolium bromide [3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl-2-H-tetrazolium bromide, MTT, was added to each well]20 mu L of the solution is cultured for 4 hours, then the supernatant is discarded, 100 mu L of dimethyl sulfoxide (DMSO) is added into each well, and the mixture is uniformly mixed and then is transferred into another clean 96-well plate. The absorbance values were measured at 490nm wavelength with a microplate reader and the inhibition of cell growth was analyzed using statistical methods.

The inhibition of OVCAR-5 cells was examined by MTT assay. The absorbance values measured by the microplate reader at 490nm were analyzed by statistical analysis for inhibition of the cells and by t-test, the results are shown in FIG. 17, where P-value for t-test is < 0.05 and P-value < 0.01. The results show that liposomes encapsulating functional siRNA have an inhibitory effect on cell growth. Checking for LIP + S using t-test_37/38、LIP+S_75/76、LIP+S_37/38+S_75/76The difference between the targeted ligand group and the non-targeted ligand group in the three groups shows that the P is less than 0.01 at the fifth day and the seventh day, which reaches significant level, and the inhibition effect of the targeted liposome is more obvious than that of the non-targeted liposome.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made in the above embodiments by those of ordinary skill in the art without departing from the principle and spirit of the present invention.

SEQUENCE LISTING

<110> university of mansion

<120> liposome, preparation method and application

<130> XMDX-18011-CNI

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<170> PatentIn version 3.5

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<211> 21

<212> DNA

<213> Artificial Synthesis

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<213> Artificial Synthesis

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gcaaucgauu aucaggcagt t 21

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<212> DNA

<213> Artificial Synthesis

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guaccagcuu cagucuuuct t 21

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<212> DNA

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gaaagacuga agcugguact t 21

Claims

1. LipidThe liposome is characterized by comprising two siRNAs, wherein each of the two siRNAs is S_37/38、S_75/76Said S_37/38Has the sequence of SEQ ID NO 1 and SEQ ID NO 2, S_75/76The sequences of SEQ ID NO 3 and SEQ ID NO 4;

the preparation method of the liposome comprises the following steps:

preparation of targeted liposome samples: mixing DOTAP solution, DOPE solution, HSPC solution, cholesterol solution and DSPE-PEG₂₀₀₀Mixing the-MTX solution, adding CHCl₃Then placing on a rotary evaporator, vacuumizing, controlling pressure, performing rotary evaporation to enable the liposome to form a transparent and uniform film on the bottle wall, taking out after freezing, adding water for hydration, and performing ultrasonic treatment and film passing to obtain a target liposome sample;

preparation of liposome/siRNA complexes: and adding the obtained liposome into the siRNA, uniformly mixing, and standing at room temperature to obtain the liposome/siRNA compound.

2. The liposome of claim 1, wherein the targeting ligand is prepared from MTX, EDC, NHS, DSPE-PEG₂₀₀₀-NH₂And DMSO in the following ratio: 12 mg: 8 mg: 4.5 mg: 30 mg: 2.5 mL.

3. The liposome of claim 1 or 2, wherein the targeting ligand is prepared by dissolving MTX, EDC, NHS in DMSO separately under stirring overnight in the absence of light; mixing DSPE-PEG₂₀₀₀-NH₂Dissolving in DMSO, adding into the above solution, stirring overnight, separating with silica gel chromatography, collecting the desired sample, filtering, rotary evaporating, and freeze drying to obtain DSPE-PEG₂₀₀₀-a powder sample of MTX is the targeting ligand.

4. The liposome of claim 1, wherein in the preparation of the targeted liposome sample,DOTAP solution, DOPE solution, HSPC solution, cholesterol solution, DSPE-PEG₂₀₀₀MTX solution and CHCl₃The volume ratio of the dosage is as follows: 1.4: 1.488: 2: 1.6: 0.588: 6 mL.

5. The liposome of claim 1 or 4, wherein the targeted liposome sample is prepared by using CHCl in which DOTAP is present in a solution of DOTAP₃Solution, DOPE solution is CHCl of DOPE₃Solution, HSPC solution is CHCl of HSPC₃Solution, Cholesterol solution is CHCl of Cholesterol₃Solutions, DSPE-PEG₂₀₀₀-MTX is DSPE-PEG₂₀₀₀-DEPC solution of MTX.

6. The liposome of claim 5, wherein the solution of DOTAP is 10mg DOTAP dissolved in 2mL HCL₃Performing the following steps; the DOPE solution is 10mgDOPE dissolved in 2mLCHCL₃Performing the following steps; the HSPC solution is 25.9mgHSPC dissolved in 2mLCHCL₃Performing the following steps; the cholesterol solution is 12.5mg cholesterol dissolved in 2mLCHCL₃Performing the following steps; DSPE-PEG₂₀₀₀The MTX solution is 10mgDSPE-PEG₂₀₀₀MTX was dissolved in 1mL DEPC.

7. The liposome of claims 1 or 4, wherein the targeted liposome sample is prepared by taking DOTAP solution, DOPE solution, HSPC solution, cholesterol solution and DSPE-PEG₂₀₀₀Mixing the-MTX solution, adding CHCl₃Adding the solution into an eggplant-shaped bottle, placing the bottle on a rotary evaporator, vacuumizing, controlling the pressure to be 0.01-0.04 MPa, and carrying out rotary evaporation for 30-40min at 42-48 ℃ to enable the liposome to form a transparent and uniform thin film on the wall of the bottle, freezing for 2-6 hours at-15 ℃ to-30 ℃, taking out the thin film, adding DEPC (diethyl phthalate) for hydration, carrying out ultrasonic treatment for 10-20 times through a 200-mesh film, and then carrying out ultrasonic treatment for 10-20 times through a 100-mesh film to obtain a targeted liposome sample.

8. The liposome of claim 5, wherein the targeted liposome sample is prepared by taking DOTAP solution, DOPE solution, HSPC solution, cholesterol solution and DSPE-PEG₂₀₀₀Mixing the MTX solution and addingCHCL₃Adding the solution into an eggplant-shaped bottle, placing the bottle on a rotary evaporator, vacuumizing, controlling the pressure to be 0.01-0.04 MPa, and carrying out rotary evaporation for 30-40min at 42-48 ℃ to enable the liposome to form a transparent and uniform thin film on the wall of the bottle, freezing for 2-6 hours at-15 ℃ to-30 ℃, taking out the thin film, adding DEPC (diethyl phthalate) for hydration, carrying out ultrasonic treatment for 10-20 times through a 200-mesh film, and then carrying out ultrasonic treatment for 10-20 times through a 100-mesh film to obtain a targeted liposome sample.

9. The liposome of claim 1, wherein the liposome/siRNA complex is prepared such that the ratio of nitrogen to phosphorous of the liposome to siRNA is 7: 1.

10. the liposome of claim 1, wherein the liposome/siRNA complex is prepared wherein the siRNA is S_37/38And S_75/76According to the following steps of 1:1 molar ratio of the mixed composition.

11. Use of a liposome according to any of claims 1 to 10 for the preparation of a medicament for targeting a tumour.