A nano-liposome with synergistic effect of double adjuvants, and a preparation method and application thereof

Abstract

The application belongs to the technical field of pharmaceutical preparations, and discloses a nano-liposome with synergistic effect of double adjuvants and a preparation method and application thereof.The preparation method of the nano-liposome with synergistic effect of double adjuvants comprises the following steps: (1) mixing manganese chloride solution, cationic liposome, distearoyl phosphatidylcholine, distearoyl phosphatidyl ethanolamine-polyethylene glycol, cholesterol and a solvent to react, and evaporating to obtain a lipid film; (2) mixing the lipid film, human unmethylated oligodeoxynucleotide and water to heat, and filtering to obtain the nano-liposome with synergistic effect of double adjuvants.The obtained nano-liposome with synergistic effect of double adjuvants has excellent sustained release property.The obtained nano-liposome with synergistic effect of double adjuvants has stable physicochemical properties after being prepared into a nano-liposome vaccine adjuvant, and is convenient to store and transport.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical preparations, in particular to a nano-liposome with synergistic effect of double adjuvants and a preparation method and application thereof. BACKGROUND

[0002] Cancer is a major public safety issue worldwide, is the main cause of death in countries, and is one of the important obstacles to prolonging human life. The current traditional treatment methods for cancer mainly include radiotherapy, surgical resection and systemic therapy. Each type of treatment has advantages and disadvantages. In most cancers, multiple treatment regimens must be used together to achieve the best results. The means of treating cancer by activating the immune system in the body has been carried out for a long time. More and more evidence shows that the innate immune and adaptive immune systems make an important contribution to the anti-tumor effect of traditional chemotherapy and radiotherapy, and the introduction of immunotherapy can make up for the defects of traditional therapy such as insufficient targeting and serious side effects. However, the premise of immunotherapy depends on the activation of the immune system, and the activated immune system can better play an anti-tumor role. In order to better play the tumor killing effect, the use of adjuvants is inevitable. An immune adjuvant is an immune response enhancer, and a vaccine is usually added with an adjuvant to enhance the immune response of the body to the antigen. Adjuvants are diverse, and common adjuvants include aluminum salt adjuvants, oil emulsion adjuvants and Toll-like receptor agonists. The activation of the immune system by a single adjuvant is sometimes weak, so the use of a double adjuvant synergistic preparation can provide a new dimension to solve the current problems.

[0003] Nanobiotechnology is considered to be a unique fusion of nanotechnology and biotechnology. With decades of continuous development, nanotechnology has been widely used in tumor treatment. Among them, nano-liposomes stand out in tumor treatment due to their good biocompatibility and biodegradability, and their low biological toxicity. Liposomes are closed spherical structures with a double (single) and / or concentric multiple double (multiple) closed central water cavity formed by self-assembly of lipids. The basic components of liposomes are usually amphiphilic phospholipids and cholesterol, and the amphiphilic phospholipids form a bilayer structure, and the cholesterol supports and maintains the bilayer structure.

[0004] Therefore, there is an urgent need in the art to develop a nano-liposome with synergistic effect of double adjuvants that can significantly improve the utilization rate of adjuvants. SUMMARY

[0005] The purpose of the present application is to provide a nano-liposome with synergistic effect of double adjuvants and a preparation method and application thereof, in order to solve the problems of weak activation of the immune system by the existing single adjuvant, low utilization rate of adjuvants, and weak effect of adjuvants in enhancing the immune response of the body to antigens.

[0006] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0007] The application provides a preparation method of a nano-liposome with a double adjuvant synergistic effect, which comprises the following steps:

[0008] (1) mixing a manganese chloride solution, cationic liposome, distearoyl phosphatidyl choline, distearoyl phosphatidyl ethanolamine-polyethylene glycol, cholesterol and a solvent, and then reacting to obtain a lipid film through evaporation;

[0009] (2) mixing the lipid film, human unmethylated oligodeoxynucleotide and water, and then performing heat treatment to obtain the nano-liposome with the double adjuvant synergistic effect through filtration.

[0010] Preferably, the manganese chloride solution is an ethanol solution of manganese chloride; the concentration of the manganese chloride solution is 8-12 muM; and the solvent comprises chloroform, methanol, ethanol or isopropyl alcohol.

[0011] Preferably, the mass-volume ratio of the manganese chloride solution, cationic liposome, distearoyl phosphatidyl choline, distearoyl phosphatidyl ethanolamine-polyethylene glycol, cholesterol and solvent is 0.05-0.2 mL: 30-40 mg: 7-8.5 mg: 2-4 mg: 13-15 mg: 2-5 mL.

[0012] Preferably, the reaction is carried out in the dark, the stirring speed is 100-400 r / min, and the reaction time is 20-40 min.

[0013] Preferably, the evaporation temperature is 40-60 DEG C, and the evaporation time is 3-5 h.

[0014] Preferably, the mass-volume ratio of the lipid film, human unmethylated oligodeoxynucleotide and water is 10-30 mg: 300-350 mu g: 2-10 mL.

[0015] Preferably, the heat treatment temperature is 40-60 DEG C, the heat treatment time is 0.5-2 h, the heat treatment is carried out under ultrasonic conditions, and the ultrasonic frequency is 25-50 kHz.

[0016] Preferably, the filter membrane used in the filtration is a polycarbonate filter membrane, the pore size of the filter membrane is 0.22-0.45 mu m, and the filtration temperature is 40-60 DEG C.

[0017] The application further provides the nano-liposome with the double adjuvant synergistic effect prepared by the preparation method.

[0018] The application further provides application of the nano-liposome with the double adjuvant synergistic effect in preparation of a drug for treating cancer through immunotherapy.

[0019] Compared with the prior art, the present application has the following beneficial effects:

[0020] The present application dissolves the cationic liposome, distearoylphosphatidylcholine, distearoylphosphatidyl ethanolamine-polyethylene glycol, cholesterol and manganese chloride in a solvent by Bangham method, removes the solvent by evaporation to form a lipid film, adds an aqueous medium containing human unmethylated oligodeoxynucleotide to the lipid film at the phase transition temperature of phospholipid to hydrate and drop the lipid film, and self-assembles to form a closed spherical structure, i.e. liposome. Then the size of the liposome is controlled by polycarbonate membrane extrusion technology to obtain the double adjuvant nano-liposome. The double adjuvant nano-liposome obtained by the present application can significantly improve the utilization rate of adjuvant, provides a controllable scheme for tumor immunotherapy, and enhances the immune response of the body to antigen. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the provided drawings.

[0022] Figure 1 The scanning electron microscope images of the double adjuvant synergistic effect nano-liposome obtained in Example 1 and the nano-liposomes obtained in Comparative Examples 1-2, wherein a is the double adjuvant synergistic effect nano-liposome obtained in Example 1, b is the nano-liposome obtained in Comparative Example 1, and c is the nano-liposome obtained in Comparative Example 2;

[0023] Figure 2 The particle size distribution graphs of the double adjuvant synergistic effect nano-liposome obtained in Example 1 and the nano-liposomes obtained in Comparative Examples 1-2;

[0024] Figure 3 The average particle size statistical result graphs of the double adjuvant synergistic effect nano-liposome obtained in Example 1 and the nano-liposomes obtained in Comparative Examples 1-2;

[0025] Figure 4 The average Zeta potential statistical result graphs of the double adjuvant synergistic effect nano-liposome obtained in Example 1 and the nano-liposomes obtained in Comparative Examples 1-2;

[0026] Figure 5 The Zeta potential distribution graphs of the double adjuvant synergistic effect nano-liposome obtained in Example 1 and the nano-liposomes obtained in Comparative Examples 1-2;

[0027] Figure 6 The stability test result graphs of the double adjuvant synergistic effect nano-liposome obtained in Example 1;

[0028] Figure 7 Gel image of stability of the aqueous solution of the nanoliposome with the synergistic effect of the double adjuvants and the human unmethylated oligodeoxynucleotide (CpG-ODN) obtained in Example 1 in the culture medium;

[0029] Figure 8 Graph of stability results of the aqueous solution of the nanoliposome with the synergistic effect of the double adjuvants obtained in Example 1 and the human unmethylated oligodeoxynucleotide (CpG-ODN) in the culture medium;

[0030] Figure 9 Graph of phagocytosis of the nanoliposome with the synergistic effect of the double adjuvants obtained in Example 1 on the mouse macrophage cell line RAW264.7;

[0031] Figure 10 Graph of phagocytosis of the nanoliposome with the synergistic effect of the double adjuvants obtained in Example 1 on the mouse bone marrow-derived dendritic cells;

[0032] Figure 11 Graph of phagocytosis efficiency of the nanoliposome with the synergistic effect of the double adjuvants obtained in Example 1, wherein (a) is the phagocytosis efficiency on the mouse macrophage cell line RAW264.7, and (b) is the phagocytosis efficiency on the mouse bone marrow-derived dendritic cells;

[0033] Figure 12 Graph of activation effect of PBS on dendritic cells;

[0034] Figure 13 Graph of activation effect of NLP on dendritic cells;

[0035] Figure 14 Graph of activation effect of Mn@NLP on dendritic cells;

[0036] Figure 15 Graph of activation effect of Mn@CpG@NLP on dendritic cells;

[0037] Figure 16 Graph of cytokine transcription level detection results of PBS, NLP, Mn@NLP and Mn@CpG@NLP;

[0038] Figure 17 Graph of biosafety evaluation results of the nanoliposome with the synergistic effect of the double adjuvants obtained in Example 1. DETAILED DESCRIPTION

[0039] The present application provides a preparation method of a nanoliposome with a synergistic effect of double adjuvants, comprising the following steps:

[0040] (1) mixing manganese chloride solution, cationic liposome, distearoylphosphatidylcholine, distearoylphosphatidyl ethanolamine-polyethylene glycol, cholesterol and solvent, and then reacting, evaporating to obtain a lipid film;

[0041] (2) mixing the lipid film, human unmethylated oligodeoxynucleotide and water, and then heat treating, filtering to obtain the nano-liposome with synergistic effect of double adjuvants.

[0042] In the present application, the manganese chloride solution is an ethanol solution of manganese chloride; the concentration of the manganese chloride solution is preferably 8-12 μM, further preferably 9-11 μM, and more preferably 9.5-10 μM; and the solvent includes chloroform, methanol, ethanol or isopropyl alcohol.

[0043] In the present application, the preparation of the manganese chloride solution comprises the following steps: ultrasonic dispersion of manganese chloride in ethanol to obtain the manganese chloride solution; and the frequency of ultrasonic is preferably 25-50 kHz, further preferably 30-45 kHz, and more preferably 35-40 kHz.

[0044] In the present application, the mass-volume ratio of the manganese chloride solution, cationic liposome, distearoylphosphatidylcholine, distearoylphosphatidyl ethanolamine-polyethylene glycol, cholesterol and solvent is preferably 0.05-0.2 mL: 30-40 mg: 7-8.5 mg: 2-4 mg: 13-15 mg: 2-5 mL, further preferably 0.1-0.18 mL: 32-35 mg: 7.5-8 mg: 2.5-3 mg: 13.5-14.5 mg: 3-4 mL, and more preferably 0.12-0.15 mL: 32.1-33.5 mg: 7.6-7.9 mg: 2.81-2.9 mg: 14 mg: 3.5 mL.

[0045] In the step (1) of the present application, the mixing is carried out under ultrasonic condition, and the frequency of ultrasonic is preferably 25-50 kHz, further preferably 30-45 kHz, and more preferably 35-40 kHz.

[0046] In the present application, the reaction is carried out in the dark, and the stirring speed of the reaction is preferably 100-400 r / min, further preferably 200-300 r / min, and more preferably 230-280 r / min; and the reaction time is preferably 20-40 min, further preferably 25-35 min, and more preferably 30-32 min.

[0047] In the present application, the evaporation temperature is preferably 40-60 °C, further preferably 45-55 °C, and more preferably 50-53 °C; and the evaporation time is preferably 3-5 h, further preferably 3.5-4.5 h, and more preferably 4 h.

[0048] In the present application, the mass-volume ratio of the lipid film, the human unmethylated oligodeoxynucleotide and water is preferably 10-30 mg: 300-350 μg: 2-10 mL, further preferably 15-25 mg: 310-340 μg: 4-8 mL, more preferably 20 mg: 320-330 μg: 5-7 mL.

[0049] In the present application, the temperature of the heat treatment is preferably 40-60℃, further preferably 45-55℃, more preferably 50℃; the time of the heat treatment is preferably 0.5-2 h, further preferably 1-1.5 h; the heat treatment is carried out under ultrasonic condition, and the frequency of the ultrasonic is preferably 25-50 kHz, further preferably 30-45 kHz, more preferably 35-40 kHz.

[0050] In the present application, the filter membrane used for the filtration is a polycarbonate filter membrane, and the pore size of the filter membrane is preferably 0.22-0.45 μm, further preferably the temperature of the filtration is 40-60℃.

[0051] The present application also provides a preparation method of the nano-liposome with the synergistic effect of double adjuvants.

[0052] The present application also provides an application of the nano-liposome with the synergistic effect of double adjuvants in the preparation of a drug for treating cancer by immunotherapy.

[0053] The technical solutions provided by the present application are described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.

[0054] Example 1

[0055] (1) Manganese chloride was dispersed in anhydrous ethanol under ultrasonic condition at a frequency of 45 kHz to obtain a manganese chloride solution with a concentration of 10 μM. 5 mL of chloroform was added into a round-bottom flask, and 32.10 mg of cationic liposome (Dlin-MC3-DMA), 7.9 mg of distearoyl phosphatidylcholine (DSPC), 2.81 mg of distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 (DSPE-PEG-2000), 14.89 mg of cholesterol and 0.1 mL of the manganese chloride solution were dispersed in chloroform, and the raw materials were completely mixed under ultrasonic condition at a frequency of 25 kHz, and then the mixture was stirred at a speed of 230 r / min in the dark for 30 min to obtain a mixed solution; the mixed solution was vacuum rotary evaporated at 50℃ for 4 h to completely volatilize the organic solvent, and a lipid film was obtained;

[0056] (2) The human unmethylated oligodeoxynucleotide (CpG-ODN) was dissolved in deionized water at 50°C, and vortexed to disperse, to obtain a human unmethylated oligodeoxynucleotide solution; the human unmethylated oligodeoxynucleotide solution and the lipid film were mixed (the mass-volume ratio of the lipid film, human unmethylated oligodeoxynucleotide and water was 20 mg:330 μg:5 mL), and then the lipid film was uniformly distributed in the human unmethylated oligodeoxynucleotide solution by ultrasonic treatment (ultrasonic frequency was 50 kHz) at 50°C for 1 h, to obtain a liposome solution; the liposome solution was sequentially passed through 0.45 μm polycarbonate filter membrane and 0.22 μm polycarbonate filter membrane at 50°C, and the obtained solution was the nano-liposome with synergistic effect of double adjuvants, denoted as Mn@CpG@NLP.

[0057] The scanning electron microscope image of the nano-liposome with synergistic effect of double adjuvants obtained in the example is shown in FIG. 1. Figure 1

[0058] Example 2

[0059] (1) The manganese chloride was dispersed in anhydrous ethanol by ultrasonic treatment at a frequency of 30 kHz, to obtain a manganese chloride solution with a concentration of 12 μM. 5 mL of chloroform was added to a round-bottom flask, and 32.10 mg of cationic liposome (Dlin-MC3-DMA), 7.9 mg of distearoyl phosphatidylcholine (DSPC), 2.81 mg of distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 (DSPE-PEG-2000), 14.89 mg of cholesterol and 0.1 mL of the manganese chloride solution were dispersed in chloroform, and the raw materials were completely mixed by ultrasonic treatment at a frequency of 30 kHz, and then stirred at a speed of 200 r / min for 30 min in the dark, to obtain a mixed solution; the mixed solution was vacuum rotary evaporated at 55°C for 3.5 h, to completely volatilize the organic solvent, and to obtain a lipid film;

[0060] (2) The human unmethylated oligodeoxynucleotide (CpG-ODN) was dissolved in deionized water at 55°C, and vortexed to disperse, to obtain a human unmethylated oligodeoxynucleotide solution; the human unmethylated oligodeoxynucleotide solution and the lipid film were mixed (the mass-volume ratio of the lipid film, human unmethylated oligodeoxynucleotide and water was 20 mg:330 μg:5 mL), and then the lipid film was uniformly distributed in the human unmethylated oligodeoxynucleotide solution by ultrasonic treatment (ultrasonic frequency was 45 kHz) at 50°C for 1 h, to obtain a liposome solution; the liposome solution was sequentially passed through 0.45 μm polycarbonate filter membrane and 0.22 μm polycarbonate filter membrane at 50°C, and the obtained solution was the nano-liposome with synergistic effect of double adjuvants.

[0061] Example 3

[0062] ​(1) Manganese chloride was dispersed in anhydrous ethanol under ultrasonic condition at a frequency of 45 kHz to obtain a manganese chloride solution with a concentration of 10 μM. 5 mL of chloroform was added into a round bottom flask, and 35 mg of cationic liposome (Dlin-MC3-DMA), 7.6 mg of distearoyl phosphatidylcholine (DSPC), 2.81 mg of distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 (DSPE-PEG-2000), 14.89 mg of cholesterol and 0.2 mL of the manganese chloride solution were dispersed in chloroform. The raw materials were mixed completely under ultrasonic condition at a frequency of 25 kHz, and then the mixture was stirred at a speed of 230 r / min for 30 min in the dark to obtain a mixed solution. The mixed solution was evaporated under vacuum at 50 °C for 4 h to completely volatilize the organic solvent, and a lipid film was obtained.

[0063] (2) Human unmethylated oligodeoxynucleotide (CpG-ODN) was dissolved in deionized water at 50 °C and vortexed to disperse, to obtain a human unmethylated oligodeoxynucleotide solution. The human unmethylated oligodeoxynucleotide solution and the lipid film were mixed (the mass-volume ratio of the lipid film, human unmethylated oligodeoxynucleotide and water was 20 mg:330 μg:5 mL) and then ultrasonicated (ultrasonic frequency was 50 kHz) at 50 °C for 1 h to uniformly distribute the lipid film in the human unmethylated oligodeoxynucleotide solution, to obtain a liposome solution. The liposome solution was sequentially passed through 0.45 μm and 0.22 μm poly carbonate filter membranes at 50 °C, and the solution obtained was the nano-liposome with synergistic effect of double adjuvants.

[0064] Comparative Example 1

[0065] The difference from Example 1 is that the addition of the manganese chloride solution and the human unmethylated oligodeoxynucleotide solution was omitted, and the other steps were the same as those in Example 1. A nano-liposome was obtained, which was denoted as NLP.

[0066] Comparative Example 2

[0067] The difference from Example 1 is that the addition of the human unmethylated oligodeoxynucleotide solution was omitted, and the other steps were the same as those in Example 1. A nano-liposome was obtained, which was denoted as Mn@NLP.

[0068] Application Example 1

[0069] The nano-liposome with synergistic effect of double adjuvants obtained in Example 1 and the nano-liposomes obtained in Comparative Examples 1-2 were observed by scanning electron microscopy. The observation method and results are as follows.

[0070] Observation method: NLP, Mn@NLP and Mn@CpG@NLP were dropped on the sample stage, air-dried for 24 h, and then sprayed with gold for 3 times, and observed by SEM. The results are shown in Figure 1 .

[0071] From the SEM images, it can be seen that the nano-liposomes obtained in Example 1 and Comparative Example 2 are spherical, and the size is uniform. The size of the nano-liposomes obtained in Example 1 is smaller than that of the nano-liposomes obtained in Comparative Example 2.Figure 1 It can be known that the particle size of the nanoliposome with the synergistic effect of double adjuvants obtained by the application is about 150 nm, and has good ability to enter the draining lymph nodes, so that the nanoliposome with the synergistic effect of double adjuvants can better activate the immune response in the body.

[0072] Application Example 2

[0073] The nanoliposomes with the synergistic effect of double adjuvants obtained in Example 1 and the nanoliposomes obtained in Comparative Examples 1-2 were subjected to DLS particle size and Zeta potential analysis, and the analysis method and results are as follows.

[0074] Analysis method: NLP, Mn@NLP and Mn@CpG@NLP were each diluted to 1 mL with ultrapure water to maintain a concentration of 1 mg / mL, added to a quartz glass dish and a Zeta potential detection cell, and subjected to DLS determination, and the test results are shown in Table 1. Figures 2-5

[0075] As can be known from FIG. Figures 2-5 , the particle sizes of NLP, Mn@NLP and Mn@CpG@NLP are 167±2.0 nm, 191±3.08 nm and 146.8±1.49 nm, respectively. With the addition of Mn 2+ , the particle size of the nanoparticles increases, because the addition of positive charges increases the hydrophobic interaction and the internal cavity of the liposome increases. As a negatively charged molecule, CpG is complexed by the attraction of positive and negative charges, and most of the nanoparticles are slightly smaller in size due to the interaction between CpG and the liposome. The PDI of NLP, Mn@NLP and Mn@CpG@NLP is 0.15±0.02, 0.22±0.01 and 0.14±0.03, respectively, and the PDI of the three is less than 0.25, which has good water dispersibility. After interaction with the CpG molecule, the PDI of the nanomolecule decreases, because the liposome molecule and the CpG molecule complex produce better heterogeneity.

[0076] Application Example 3

[0077] The nanoliposomes with the synergistic effect of double adjuvants obtained in Example 1 and the nanoliposomes obtained in Comparative Examples 1-2 were subjected to encapsulation rate determination, and the determination method and results are as follows.

[0078] Determination method: 3000 Da ultrafiltration tubes were used to centrifuge NLP, Mn@NLP and Mn@CpG@NLP at 4°C at high speed (5000 r / min) for 30 min, and the filtered liquid was subjected to nucleic acid concentration determination, and the free nucleic acid content in the remaining liquid was detected, and the encapsulation rate was calculated according to "encapsulation rate (%) = total nucleic acid content (C0) - (free nucleic acid content (C1)) / total nucleic acid content (C0) x 100%", and the results are shown in Table 1.​

[0079] Table 1. Encapsulation efficiency of nanoliposomes with synergistic effect of dual adjuvants obtained in Example 1 and nanoliposomes obtained in Comparative Examples 1-2.

[0080] [C0 (pg / pL)] [C1 (pg / μL)] Encapsulation efficiency (%) Mn@CpG@NLP 1.024 0.025 97.56 Mn@NLP 1.052 0.033 96.86 NLP 0.922 0.045 95.12

[0081] As shown in Table 1, the nanoliposomes with synergistic dual adjuvant effect obtained in this invention have good encapsulation capacity.

[0082] Application Example 4

[0083] The stability of the nanoliposomes with synergistic effect of dual adjuvants obtained in Example 1 was tested. The test methods and results are as follows.

[0084] Test method: 50 μL Mn@CpG@LNP was added to 1 mL of NaCl solutions with concentrations of 0 mM, 50 mM, 100 mM, 150 mM, 200 mM, and 250 mM, respectively. The light scattering intensity (SLI) of Mn@CpG@LNP in the aqueous solution was measured using a dynamic light scattering instrument. The stability constant (K) of Mn@CpG@LNP was calculated according to the formula: "Stability constant (%) = (SLI value (I) in different concentrations of sodium chloride solution / SLI value (I0) measured in the initial aqueous solution)) × 100%". The results are shown below. Figure 6 As shown.

[0085] Depend on Figure 6 It is evident that the nanoliposomes with synergistic dual adjuvant effect obtained in this invention maintain good stability under different NaCl solution concentrations, especially when the ionic strength is within the physiological environment (150 mM), remaining in the range of 0.9–1. These results indicate that nanovaccines incorporating the nanoliposomes with synergistic dual adjuvant effect obtained in this invention possess excellent stability after self-assembly and can be used under normal physiological conditions.

[0086] Application Example 5

[0087] The protective effect of the nanoliposomes with synergistic effect of dual adjuvants obtained in Example 1 was tested. The test methods and results are as follows.

[0088] Test method: The Mn@CpG@NLP (containing 500 μg of human unmethylated oligodeoxynucleotide (CpG-ODN)) and the aqueous solution of human unmethylated oligodeoxynucleotide (CpG-ODN) (containing 500 μg of human unmethylated oligodeoxynucleotide (CpG-ODN)) obtained in Example 1 were respectively dissolved in 1 mL of 1640 culture medium containing 50% serum, and incubated at 37°C for 24 h. Sampling was performed at different time intervals (0 h, 2 h, 4 h, 8 h, 12 h and 24 h), and 20 μL was taken each time. 5 μL of 20% SDS and 5 μL of Loading Buffer were added to the obtained samples to obtain a mixture; 15 μL of each mixture was taken and loaded, and electrophoresis was performed in a 2% agarose gel at 110 V, and after 15 min, the content of human unmethylated oligodeoxynucleotide (CpG-ODN) was observed by a gel imaging instrument, and the obtained results are shown in Figure 7 and Figure 8 .

[0089] As can be seen from Figure 7 and Figure 8 , part of the free human unmethylated oligodeoxynucleotide (CpG-ODN) is degraded in the 1640 culture medium containing 50% serum in 4 h, and basically no band of human unmethylated oligodeoxynucleotide (CpG-ODN) can be observed in 12 h, indicating that the adjuvant is completely degraded. In contrast, the human unmethylated oligodeoxynucleotide (CpG-ODN) in the Mn@CpG@NLP can remain stable for 24 h, and the band does not disappear with the extension of time. The results show that the Mn@CpG@NLP has good protection for human unmethylated oligodeoxynucleotide (CpG-ODN).

[0090] Application Example 6

[0091] The dual-adjuvant synergistic nanoliposomes obtained in Example 1 were subjected to in vitro phagocytosis test, and the test method and results are as follows:

[0092] Test method: Mouse bone marrow-derived dendritic cells and macrophage cell lines were cultured in vitro and inoculated into a confocal dish, and after 12 h of culture, CpG-ODN, Mn@NLP and Mn@CpG@NLP were added, respectively, and after 24 h of continuous culture, cell fixation and cell nucleus staining were performed, and observation was performed using a confocal microscope. The test results are shown in Figure 9 and Figure 10 .

[0093] As can be seen from Figure 9 and Figure 10 , the CpG-modified cyanine dye Cy5 (red) and the cholesterol-modified fluorescein isothiocyanate FITC (green) were labeled, and the cell nucleus was labeled with DAPI. Figure 9Phagocytosis effect of mouse macrophage cell line RAW264.7, Figure 10 Phagocytosis effect of mouse bone marrow-derived dendritic cells. When CpG-ODN, Mn@NLP and Mn@CpG@NLP were co-incubated with mouse bone marrow-derived dendritic cells for 24 h, the phagocytosis efficiency of mouse bone marrow-derived dendritic cells to CpG-ODN was not very high, and only weak fluorescence was observed after 24 h, which was caused by the degradation of CpG-ODN by nucleases existing in the serum and the low phagocytosis efficiency of itself. In contrast, in Mn@NLP and Mn@CpG@NLP, there was significantly enhanced red fluorescence and green fluorescence. The phagocytosis efficiency of mouse macrophage cell line RAW264.7 was observed by the same experimental means, and the experimental results were similar to those of mouse bone marrow-derived dendritic cells, and the fluorescence in the Mn@NLP and Mn@CpG@NLP groups was significantly stronger. The results showed that Mn@CpG@NLP could be well phagocytosed by antigen-presenting cells.

[0094] Application Example 7

[0095] The phagocytosis efficiency of the double adjuvant synergistic nanoliposomes obtained in Example 1 was tested, and the test method and results are as follows:

[0096] Test method: mouse bone marrow macrophage cell line was cultured in vitro, and CpG-ODN, NLP, Mn@NLP and Mn@CpG@NLP were added respectively, and flow cytometry analysis was performed after 24 h of continuous culture. The test results are shown in Figure 11 .

[0097] As can be seen from Figure 11 , the CpG-modified cyanine dye Cy5.5 (red) and the cholesterol-modified fluorescein isothiocyanate FITC (green) were added. Figure 11 (a) is the green fluorescence FITC intensity, Figure 11 (b) is the red fluorescence Cy5.5 intensity. After RAW264.7 cells phagocytosed Mn@NLP and Mn@CpG@NLP nanoliposomes, the intensity of green fluorescence increased significantly, indicating that the nanoliposomes had good phagocytosis uptake ability. Compared with CpG-ODN, the red fluorescence intensity of the Mn@CpG@NLP group also increased significantly, indicating that the nanoliposomes had good protection effect on CpG-ODN, and the results obtained by laser confocal were similar. The results showed that Mn@CpG@NLP could be well phagocytosed by antigen-presenting cells and had good protection effect on CpG-ODN.

[0098] Application Example 8

[0099] In vitro dendritic cell activation experiment was performed on the nanoliposomes with double adjuvant synergistic effect obtained in Example 1, and the experimental method and results are as follows:

[0100] Experimental method: mouse bone marrow-derived dendritic cells were inoculated in 24-well plates, and the experiment was divided into 4 groups, namely PBS group, NLP group, Mn@NLP group and Mn@CpG@NLP group. Staining used 100 μL system, and the amount of mouse flow antibody was CD16 / CD32 (0.25 μg), CD11c-FITC (0.25 μg), CD80-APC (0.25 μg) and CD86-PerCP (0.25 μg). The proportion of CD80 + / CD86 + cells in CD11c population was detected by flow cytometry. The test results are shown in Figures 12-15 .

[0101] As can be seen from Figures 12-15 , compared with PBS group, NLP group and Mn@NLP group, the proportion of CD80 + / CD86 + cells in Mn@CpG@NLP group was significantly increased, and Mn@CpG@NLP could well promote the maturation of BMDC.

[0102] Application Example 9

[0103] The cytokine transcription level of the double adjuvant synergistic effect of the nanoliposomes obtained in Example 1 was detected, and the detection method and results are as follows:

[0104] Detection method: mouse bone marrow-derived dendritic cells were inoculated in 6-well plates, and the test was divided into 4 groups, namely PBS group, NLP group, Mn@NLP group and Mn@CpG@NLP group. After 24 h of culture, RNA was extracted and reverse transcription and real-time fluorescence quantitative PCR experiment was carried out. The test results are shown in Figure 16 .

[0105] As can be seen from Figure 16 , compared with PBS group, NLP group and Mn@NLP group, the expression amount of IL-6, IL-12, IFN-α, IFN-β and CXCL9 in Mn@CpG@NLP group was significantly up-regulated, Mn@CpG@NLP could well promote the transcription level of cytokines to rise, and cause better immune response.

[0106] Application Example 10

[0107] The biological safety of the double adjuvant synergistic effect of the nanoliposomes obtained in Example 1 was evaluated, and the evaluation method and results are as follows:

[0108] Evaluation method: HUVEC cells were co-incubated with different concentrations of Mn@CpG@NLP (0 mM, 0.025 mM, 0.05 mM, 0.075 mM, 0.1 mM, and 0.2 mM) in 96-well plates for 24 h. CCK-8 reagent was then added, and the absorbance at 450 nm was measured. Cell viability was calculated. Evaluation results are as follows: Figure 17 As shown.

[0109] Depend on Figure 17 It can be seen that as the concentration of Mn@CpG@NLP increases, the survival rate of HUVEC cells does not show a significant downward trend, indicating that Mn@CpG@NLP has no obvious cytotoxicity and can be used in in vivo experiments.

[0110] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a nano-liposome with a synergistic effect of double adjuvants, characterized in that, The method comprises the following steps: (1) mixing manganese chloride solution, cationic liposome, distearoylphosphatidylcholine, distearoylphosphatidylethanolamine-polyethylene glycol, cholesterol and solvent, and then reacting to obtain a lipid film through evaporation; (2) mixing the lipid film, human unmethylated oligodeoxynucleotide and water, and then performing heat treatment to obtain the double adjuvant synergistic nano-liposome through filtration; the particle size of the double adjuvant synergistic nano-liposome is 146.8±1.49nm; The mass-volume ratio of the manganese chloride solution, the cationic liposome, the distearoylphosphatidylcholine, the distearoylphosphatidylethanolamine-polyethylene glycol, the cholesterol and the solvent is 0.1mL:32.1mg:7.9mg:2.81mg:14.89mg:5mL; The reaction is carried out in the dark, the stirring speed is 230r / min, and the reaction time is 30min; The evaporation temperature is 50℃, and the evaporation time is 4h; The mass-volume ratio of the lipid film, the human unmethylated oligodeoxynucleotide and the water is 20mg:330μg:5mL; The heat treatment temperature is 50℃, the heat treatment time is 1h, the heat treatment is carried out under ultrasonic condition, and the ultrasonic frequency is 50kHz; The filtration is sequentially carried out through 0.45μm polycarbonate filter membrane and 0.22μm polycarbonate filter membrane, and the filtration temperature is 50℃; The manganese chloride solution is an ethanol solution of manganese chloride; The concentration of the manganese chloride solution is 10μM; and the solvent is chloroform.

2. The double adjuvant synergistic nano-liposome prepared by the preparation method of the double adjuvant synergistic nano-liposome of claim 1.

3. The double adjuvant synergistic nano-liposome of claim 2 is applied to the preparation of a drug for treating cancer through immunotherapy.

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