CN110151702A - Polyethylene glycol modified influenza vaccine liposome and preparation method thereof - Google Patents

Abstract

The present invention provides polyethyleneglycol modified influenza vaccines liposome and preparation method thereof, which is made of influenza virus cracking inactivated vaccine, phosphatide, cholesterol, polyethylene glycol.The present invention prepares liposome with cholesterol and phosphatide, adds polyethylene glycol as water-wetted surface modifier, polyethyleneglycol modified influenza vaccines liposome is prepared using freeze thawing-desivac.The advantages that present invention has preparation method easy to operate, and stability is good, and cost is relatively low, Immune efficiency is high.

Description

Polyethylene glycol modified influenza vaccine liposome and preparation method thereof

Technical field:

The invention belongs to the field of biological technical preparations, in particular to the field of vaccine biological preparations, and mainly relates to a polyethylene glycol modified influenza vaccine liposome and a preparation method thereof.

Background technique:

Influenza (influenza, flu for short) is an acute respiratory infectious disease caused by infection with influenza virus that threatens human physical and mental health. Today, even with greatly improved preventive measures and medical technology, according to the World Health Organization, seasonal influenza causes 3-5 million severe cases and 300,000-500,000 deaths worldwide each year. China is a country with a high incidence of influenza viruses. Since 1957, the three world influenza pandemics have originated from my country. The scary thing about influenza is that it generally does not directly cause the death of the patient, but it can cause many complications that seriously endanger health, causing the patient's body to suffer varying degrees of damage, and even life-threatening in severe cases.

The current fight against influenza relies mainly on drugs and vaccines. Antiviral drugs can relieve symptoms and reduce mortality, but as amantadine drugs have been tolerated by H3N2 strains, oseltamivir (Tamiflu), an excellent neuraminidase inhibitor, is effective in some seasonal H1N1 Influenza is also almost ineffective, and zanamivir can cause bronchospasm in some people ^[1] . Drug treatment is the remedy for influenza infection, but influenza vaccine can help the vaccinated to build immunity before infection, and is currently recognized as the most effective way to reduce influenza morbidity and mortality.

However, the current influenza vaccine protection rate is only 10% to 60% ^[2] , and the protection period is only a few months ^[3] . During the 2014-2015 flu season, statistics from 2,321 people in 46 states in the United States showed that the vaccine protection rate was 22%, and the protection rate for people over 50 years old was only 14% ^[4] ; during the 2015-2016 flu season, 6,879 people in the United States had a vaccine protection rate of 22%. The survey showed that the vaccine protection rate was 48%, and the protection rate for people aged 50 to 64 was 26% ^[5] ; in the same year, a survey of 3842 people in the UK showed that the influenza vaccine protection rate was 52.4%, and the protection rate for people over 65 years old was 52.4%. was 29.1% ^[6] . The above data is far from the ideal protection rate of more than 75% and the protection period of 12 months, and the better protection rate of more than 90% and the protection period of 5 to 10 years, there is still a big gap ^[7] , especially for Protection rates are low in the elderly population most at risk of hospitalization and death from influenza ^[8] . Therefore, the immunization efficiency of existing influenza vaccines needs to be greatly enhanced.

At present, influenza vaccines are mainly split vaccines and subunit vaccines, which have weak immune activation ability, require a large number of vaccinations to induce the body to produce antibodies, and can hardly produce cellular immunity ^[9] . Furthermore, the widely used aluminum adjuvant is also difficult to induce the generation of cellular immunity ^[10] , so it is difficult to induce a long immune memory and obtain a certain degree of cross immunity, which is not conducive to fighting virus mutation. On the other hand, the intake of aluminum adjuvants also brings some safety risks. It is gratifying that liposome adjuvant can not only significantly promote the production of cellular immunity ^[9] , but also induce the body to produce an immune response by activating Toll-like receptor (TLR) ^[11] . TLR signaling is an important costimulatory signal for the immune response induced by influenza vaccine, but in aged or immunocompromised animal models, the effect of the vaccine is reduced due to the down-regulation of TLR ^[12] . In addition, liposomes are biodegradable, their size, charge, and surface modification materials are flexible and adjustable, and their release behavior is controllable. They can be prepared and loaded with vaccines with different water solubility under mild conditions without causing autoimmune reactions, so they are considered to be The most promising adjuvant system after aluminum adjuvant ^[13] .

Reasonable selection of vaccine adjuvants and design of technical routes are the keys to the research of split-inactivated vaccines. Polyethylene glycol (PEG) is a safe, non-toxic and invisible hydrophilic polymer. According to its steric stabilization effect, it can slow down the aggregation between particles, thereby enhancing the stability of the preparation during storage and application. , has been widely used in the field of medicine. Studies have also shown that embedding polyethylene glycol-distearate phosphatidylethanolamine on the liposome bilayer membrane can well prevent phagocytes from recognizing and taking up liposomes, thereby prolonging the circulation time of liposomes and facilitating drug entry. Lesion tissue, greatly improving the therapeutic index and efficacy.

PEG has also been used in the preparation of vaccines, as the closest patent: the patented micellar polypeptide vaccine with PEGylated phospholipids as a carrier (ZL201410570624.7) uses PEGylated phospholipids to prepare micelles, and the peptide vaccine is wrapped in In micelles, the design idea of this patent is mainly to use the solubilization effect of PEGylated phospholipids on protein polypeptides. The patented polyethylene glycol-inactivated vaccine mucosal immunizer (ZL200410017527.1) uses polyethylene glycol as a material in the production process of the vaccine stock solution, and is retained in the final vaccine product to enhance immunity.

Upon enquiry, before this application was filed, no technical solution identical to this application has been found.

references:

[1] Pop-Vicas A, Gravenstein S. Influenza in the elderly–A mini-review [J]. Gerontology, 2011, 57(5): 397-404.

[2] Seasonal Influenza Vaccine Effectiveness, 2005-2017 [M]. Centers for Disease Control and Prevention. 2018.

[3]Salvarani F,Turinici G.Optimal individual strategies for influenzavaccines with imperfect efficacy and durability of protection[J].Mathematical Biosciences&Engineering,2018,15(3):629-652.

[4] Flannery B, Clippard J, Zimmerman RK, et al. Early estimates of seasonal influenza vaccine effectiveness-United States, January 2015[J].MMWRM Morbidity and mortality weekly report, 2015,64(1):10-15.

[5] Jackson ML, Chung JR, Jackson LA, et al. Influenza vaccine effectiveness in the United States during the 2015–2016 season [J]. New England Journal of Medicine, 2017, 377(6):534-543.

[6] Pebody R, Warburton F, Ellis J, et al.Effectiveness of seasonalinfluenza vaccine for adults and children in preventing laboratory-confirmedinfluenza in primary care in the United Kingdom:2015/16 end-of-season results[J].Eurosurveillance , 2016, 21(38).

[7] Paules CI, Marston HD, Eisinger RW, et al. The Pathway to a Universal Influenza Vaccine [J]. Immunity, 2017, 47(4): 599-603.

[8]Torbic H,Roach EM.Current Influenza Vaccine Options for 2014[J].Current Emergency and Hospital Medicine Reports,2015,3(3):126-133.

[9] Petrovsky N, Aguilar JC. Vaccine adjuvants: current state and futuretrends [J]. Immunology & Cell Biology, 2004, 82(5): 488-496.

[10] Pasquale AD, Preiss S, Silva FTD, et al. Vaccine adjuvants: from 1920 to 2015 and beyond[J]. Vaccines, 2015, 3(2): 320-343.

[11] She Zhennan, Zhai Wenjun, Deng Yihui. Analysis of the immune mechanism in the phenomenon of "accelerating blood flow clearance" [J]. Journal of Shenyang Pharmaceutical University, 2011, (9): 760-768.

[12]Renshaw M,Rockwell J,Engleman C,et al.Cutting edge:impaired Toll-like receptor expression and function in aging[J].The Journal of Immunology,2002,169(9):4697-4701.

[13] Schwendener RA. Liposomes as vaccine delivery systems: a review of the recent advances[J]. Therapeutic advances in vaccines, 2014, 2(6): 159-182.

Invention content:

The purpose of the present invention is to provide a polyethylene glycol-modified influenza vaccine liposome, and at the same time disclose a preparation method that is simple, easy to implement, controllable in quality, and capable of industrial production.

The object of the present invention is achieved through the following technical solutions: polyethylene glycol modified influenza vaccine liposome, which is composed of influenza virus split inactivated vaccine, phospholipids, cholesterol and polyethylene glycol, etc.; wherein the quality of the influenza virus split inactivated vaccine The mass ratio of HA to phospholipid is 1:60～1:200, the mass ratio of cholesterol to phospholipid is 1:2～1:5, and the mass ratio of polyethylene glycol to cholesterol is 1:1～1: 3.

Preferably, in the polyethylene glycol modified influenza vaccine liposome of the present invention, the mass ratio of the influenza virus split inactivated vaccine to phospholipid is 1:60-1:100 when the mass of the influenza virus split and inactivated vaccine is calculated as HA, and the cholesterol and phospholipid are in a mass ratio of 1:60 to 1:100. The mass ratio of polyethylene glycol to cholesterol is 1:3 to 1:4, and the mass ratio of polyethylene glycol to cholesterol is 1:1 to 1:3; more preferably, the mass ratio of polyethylene glycol to cholesterol is 1:2.

As most preferably, in the polyethylene glycol modified influenza vaccine liposome of the present invention, the mass ratio of the influenza virus split inactivated vaccine to phospholipid is 1:70～1:80 when the mass of the influenza virus split and inactivated vaccine is calculated as HA, and the cholesterol and The mass ratio of phospholipids was 1:3.75, and the mass ratio of polyethylene glycol to cholesterol was 1:2.

Preferably, the molecular weight of the polyethylene glycol of the present invention is 1000-10000.

Preferably, the polyethylene glycol is polyethylene glycol-1000, polyethylene glycol-2000, polyethylene glycol-3000, polyethylene glycol-4000, polyethylene glycol-6000, polyethylene glycol- A mixture of one or more of 8000 and PEG-10000. As more preferably, the polyethylene glycol is one of polyethylene glycol-2000, polyethylene glycol-3000, polyethylene glycol-4000, polyethylene glycol-6000, polyethylene glycol-8000 or Various mixes.

Preferably, the phospholipid of the present invention is selected from a mixture of one or more of soybean lecithin, hydrogenated soybean lecithin, egg phospholipid, phosphatidylcholine, and phosphatidylethanolamine. More preferably, the phospholipid is selected from soybean lecithin.

The present invention also provides a kind of preparation method of polyethylene glycol modified influenza vaccine liposome at the same time, and its technology comprises the following steps:

(a) Dissolve phospholipid and cholesterol with ethanol at 50-60°C, and the amount of ethanol is subject to complete dissolution of phospholipid and cholesterol, then rotate under reduced pressure at 30-40°C to remove the ethanol solvent to obtain a phospholipid membrane.

(b) adding phosphate buffer to the phospholipid membrane to make the phospholipid concentration 10 mg/mL, and adding polyethylene glycol according to the prescribed amount, and then performing rotary hydration at 50-60 ° C to obtain the first liposome product . Preferably, the pH value of the phosphate buffer is 7.0±0.2, and the hydration time is 40-50 min.

(c) reducing the particle size of the first liposome product through ultrasonic treatment with a probe to obtain a liposome suspension.

(d) Mix the liposome suspension with the split and inactivated influenza B virus vaccine in an appropriate proportion, and freeze and thaw 2 to 4 times between -40°C and 25°C; then pre-freeze it at about -40°C for 8 ~12h; finally freeze-dried on a freeze dryer at -52°C and 0.06mbar for more than 28h to obtain a white loose lyophilized powder, which is polyethylene glycol-modified influenza vaccine liposomes, and stored at 4°C.

Compared with the prior art, the present invention has the following advantages:

(1) Compared with the vaccine stock solution without adjuvant, or the vaccine with conventional aluminum adjuvant, and conventional vaccine liposomes, polyethylene glycol-modified influenza vaccine liposomes have stronger cellular immunogenicity and can induce The body produces stronger cellular immunity.

(2) Compared with the vaccine stock solution water injection without adjuvant, or the conventional vaccine liposome freeze-dried powder, the polyethylene glycol modified influenza vaccine liposome freeze-dried powder has better storage stability.

(3) The polyethylene glycol modified influenza vaccine liposome provided by the present invention has a simple preparation process, does not contact toxic organic solvents during the preparation process, and does not require high temperature and severe operation in the whole process, which can effectively protect the self structure of the influenza vaccine from being destroyed.

(4) The present application uses cholesterol and phospholipids to prepare liposomes, then adds polyethylene glycol as a hydrophilic surface modifier, and finally adopts a freeze-thaw-lyophilization method to prepare polyethylene glycol-modified influenza vaccine liposomes. Compared with the technical solution described in CN201410570624.7, the present invention does not need to use expensive PEGylated phospholipids, and has no micelle encapsulation step, the preparation process is simpler, and the process is more controllable. Compared with CN200410017527.1, the present invention combines antigen (vaccine), liposome and polyethylene glycol, which can better increase the immune activation effect of antigen, and meanwhile, the process is simple and easy to industrialize.

Detailed ways:

In order to understand the present invention more clearly, the present invention will be further described in detail below with reference to the embodiments completed according to the technical solutions of the present invention given by the inventor. The present invention is not limited to these embodiments, and any form modifications and/or changes made to the present invention will fall within the protection scope of the present invention.

In the present invention, all equipment or raw materials, etc. can be obtained from the market or commonly used in the industry. Among them, influenza virus lysate was provided by Jiangsu Watson Biotechnology Co., Ltd.; standard calf serum albumin BSA was purchased from Sigma in the United States; soybean lecithin, hydrogenated soybean lecithin, egg lecithin, hydrogenated egg yolk lecithin, phosphatidylcholine Alkali and phosphatidylethanolamine were purchased from Beijing Meiyas Phospholipid Technology Co., Ltd.; cholesterol was purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.; polyethylene glycol was purchased from Shanghai McLean Biochemical Technology Co., Ltd.

The abbreviations for each component used in the following examples are as follows:

SPC: Soy Lecithin

EPC: egg yolk lecithin

HSPC: Hydrogenated Soy Lecithin

DSPC: Distearoyl Phosphatidyl Choline

DPPC: dipalmitoyl choline

DSPE: Distearoylphosphatidylethanolamine

DPPE: dipalmitoyl phosphatidylethanolamine

CHOL: cholesterol

PEG-1000: Polyethylene Glycol-1000

PEG-2000: polyethylene glycol-2000

PEG-3000: polyethylene glycol-3000

PEG-4000: polyethylene glycol-4000

PEG-6000: Polyethylene Glycol-6000

PEG-8000: Polyethylene Glycol-8000

PEG-10000: Polyethylene Glycol-10000

PBS buffer: Phosphate buffer

HA: Hemagglutinin

Embodiment 1 PEG-modified influenza vaccine liposome preparation prescription 1:

Recipe 2:

Recipe 3:

Recipe 4:

Recipe 5:

Recipe 6:

Recipe 7:

Recipe 8:

Recipe 9:

Prescription 10:

Recipe 11:

Recipe 12:

Recipe 13:

Recipe 14:

Recipe 15:

Recipe 16:

Recipe 17:

Recipe 18:

In the present embodiment, the preparation method of the polyethylene glycol modified influenza liposome vaccine of prescription 1 to prescription 18 is as follows:

(1) Disperse cholesterol and phospholipid in 18-22 mL of absolute ethanol, and fully dissolve them in a water bath at 50-60°C.

(2) The above dissolved mixed solution is evaporated under reduced pressure at 30-40° C. to remove ethanol, and the rotation speed is 40-50 r/min until the film is formed.

(3) Add about 25-35 mL of pH 7.0 ± 0.2 PBS buffer solution to the lipid film formed by cholesterol and phospholipid, add the prescribed amount of PEG, and then perform rotary hydration at 50-60 ° C for 40-50 min, that is, The first liposomes were obtained.

(4) Use the probe ultrasonic technology to process the first liposome: put the ultrasonic probe into the first liposome for intermittent ultrasonic treatment (ultrasonic 30s, pause for 30s, ultrasonic frequency 40-50%), the total ultrasonic treatment The time was 2 min, and after the ultrasonic wave was over, it was placed in a water bath at 50 to 60° C. for 1 to 2 hours to obtain a milky white liposome suspension.

(5) Fully mix the suspension obtained above and the influenza vaccine stock solution of corresponding quality (calculated by the mass of HA) in an appropriate proportion, and distribute them in ampoules, 2ml/bottle; quickly freeze in a -40°C low-temperature refrigerator After 1 to 2 hours, the ampoule containing the drug-loaded liposome is slowly thawed at room temperature (one freeze-thaw number), and then thawed and then frozen in a -40°C low temperature refrigerator for 8 to 12 hours.

(6) Put the pre-frozen drug-loaded liposomes on a freeze dryer (0.06mbar, -52°C) and freeze-dried for more than 28 hours to obtain a freeze-dried powder. The freeze-dried powder is sealed and stored at 4°C. , that is.

The resulting lyophilized product is loose, white or nearly white in appearance. After redissolving, the particle size is 1-2 μm.

Example 2 Preparation of unmodified influenza vaccine liposomes

Recipe 19:

The preparation method of the unmodified influenza liposome vaccine of prescription 19 in this example is the same as that in example 1, except that the step of adding PEG is not included in the whole preparation process. The prepared unmodified influenza liposome vaccine will be used for animal immunogenicity experiments and stability evaluation.

Example 3 Experimental study on animal immunogenicity

1. Animal grouping and immunization

Mice were randomly divided into polyethylene glycol-modified influenza vaccine liposome group, PBS blank control group, vaccine stock solution group, and unmodified influenza vaccine liposome group, with 3 mice in each group. The PBS was the negative blank control group, and the vaccine stock solution was the positive control group. The dose of the vaccine administration group was calculated as 6μg/vaccine based on HA. After anesthesia, one of pulmonary administration immunization and intraperitoneal administration immunization was used for immunization on 0d, and then immune-related indexes were detected on 7d, 14d, and 28d after that. The vaccine used in the polyethylene glycol-modified influenza vaccine liposome group was prepared according to prescription 2 and prescription 8 in Example 1; the vaccine used in the unmodified influenza vaccine liposome group was prepared according to prescription 19 in Example 2, and the vaccine stock solution The group and the PBS group were directly formulated into solutions and administered.

2. Spleen lymphocyte proliferation assay

Under sterile conditions, the prepared mouse spleen lymphocyte suspension was suspended with 3 mL of 10-15% fetal bovine serum, and the number of cells was adjusted to about 3.0×10 ⁶ /mL. Add 100 μL/well of prepared mouse spleen lymphocyte suspension to each well and blank control well, and the final cell concentration of each well is 3.0×10 ⁶ /mL; then add 20 μg/mL concanavalin A solution to the experimental well. (ConA solution), RPMI-1640 medium was added to the blank control wells, both at 100 μL/well, and 3 duplicate wells were set in both the experimental group and the blank group. After dosing, incubate for 48 hours in a 37°C, 5% CO ₂ cell incubator, take it out, add 20 μL/well MTT (5 mg/mL) on a sterile ultra-clean workbench in the dark, mix gently, and put in cells After culturing in the incubator for 4 hours, the reaction was terminated, and 100 μL/well triple solution was added to dissolve the purple crystals. After 8 to 12 hours, the OD value of each well was measured at the experimental wavelength of 570 nm and the reference wavelength of 630 nm by a microplate reader, and the average OD of each mouse ConA well was used. The average OD value of the value/blank control well was the stimulation index (SI) to judge the proliferation level of mouse spleen lymphocytes.

3. Flow cytometry to detect surface markers of T lymphocytes

Under sterile conditions, the prepared mouse spleen lymphocyte suspension was suspended with 3 mL of PBS to adjust the number of cells to about 3×10 ⁶ /mL, and 1 mL of the cell suspension was taken into an EP tube, and divided into 4 groups, the first group The cell suspension was a blank control without dye added. In the second group, 1 μL of Anti-mouse CD4 PE (L3T4, 0.2 mg/mL, eBioscience ^TM , thermofisher) was added to the cell suspension to mix well, and 1 μL was added to the third group of cell suspensions. Anti-mouse CD8a FITC (Ly-2, 0.5mg/mL, eBioscience ^TM , thermofisher) was mixed, and 1 μL of Anti-mouse CD4 PE and 1 μL of Anti-mouse CD8a FITC were added to the cell suspension of group 4 and mixed well , labeled with tin foil for 30 minutes in the dark, and then detected by flow cytometry, grouped by SS and FS, and analyzed by Beckman Coulter CXP. The CD4+/CD8+ ratio was detected by flow cytometry to investigate its cellular immunogenicity.

4. Experimental results

Validated by spleen lymphocyte proliferation assay and T lymphocyte expression labeling assay. Using the spleen lymphocyte proliferation test (Table 1) and the T lymphocyte surface labeling test (Table 2), the results show that the polyethylene glycol-modified influenza vaccine liposome has the effect of enhancing cellular immunity, and the effect is better than that of the unmodified influenza vaccine Liposome group. The cellular immunogenicity of PEG-modified influenza vaccine liposomes is stronger, which can induce stronger cellular immunity in the body. Compared with the vaccine stock solution, the unmodified influenza vaccine liposome also enhanced the cellular immunogenicity of the body.

Table 1 Results of spleen lymphocyte proliferation assay

Table 2 T lymphocytes indicate labeling test results 7d, 14d, 28d (n=3)

Embodiment 4 Influenza vaccine liposome stability experiment

Polyethylene glycol modified influenza vaccine liposome suspension and lyophilized powder were prepared according to recipe 2 in Example 1, and unmodified influenza vaccine liposome lyophilized powder was prepared according to recipe 19.

The prepared polyethylene glycol modified influenza vaccine liposome suspension, polyethylene glycol modified influenza vaccine liposome lyophilized powder and unmodified influenza vaccine liposome lyophilized powder were placed at different temperatures (37± 2°C, 25±2°C, and 4°C) to conduct temperature acceleration experiments, take samples at set time points (0, 5, 10, 15, 30d), redissolve liposome lyophilized powder in PBS, and then reconstitute the lyophilized powder at 4 days. ℃, 45000r/min high-speed centrifugation for 1h, the protein content was determined by Lowry method, and the encapsulation efficiency was calculated. To investigate the changes in the encapsulation efficiency of polyethylene glycol modified influenza vaccine liposome suspension, polyethylene glycol modified influenza vaccine liposome lyophilized powder and unmodified influenza vaccine liposome lyophilized powder at different time points , the encapsulation efficiency results are expressed as (X±SD)%.

The unmodified influenza vaccine liposome freeze-dried powder placed at 40±2°C was sampled on 10d, and the unmodified influenza vaccine liposome freeze-dried powder placed at 25±2°C was sampled on 30d, and the micro-hemagglutination inhibition ( HI) method was used to measure mouse serum for a certain period of time after immunization, and H ₁ N _I influenza vaccine standard antigen was used to measure corresponding antibodies, and the immunogenicity of unmodified influenza vaccine liposome freeze-dried powder after being placed at high temperature was investigated.

The measurement results are shown in Table 3, Table 4, Table 5 and Table 6, respectively. According to the results, as time goes by, compared with the polyethylene glycol-modified influenza vaccine liposome suspension without freeze-dried, the freeze-dried polyethylene glycol-modified influenza vaccine liposome lyophilized powder and the non-lyophilized Modified influenza vaccine liposome lyophilized powder has higher encapsulation efficiency. It shows that the physical stability of the influenza vaccine liposomes with or without polyethylene glycol modification after lyophilization is significantly increased compared with that before lyophilization. From Table 4, it can be seen that the reduction value of the encapsulation rate at 0-15 days: the polyethylene glycol-modified influenza vaccine liposomes are expected to achieve the purpose of reducing the pressure of cold chain transportation regardless of whether before or after lyophilization. It can be seen from the biological stability data of unmodified influenza vaccine liposomes in Table 6: the influenza vaccine liposome freeze-dried powder is placed at room temperature for 30 days, and the ratio of antibody titers after immunization and pre-immunization at 40±2°C for 10 days is greater than 4. The immune system is still effective.

Table 3 Test results of liposome stability of vaccines in each group (37±2℃)

Table 4 Test results of liposome stability of vaccines in each group (25±2℃)

Table 5 Test results of liposome stability of vaccines in each group (4±2℃)

Table 6 Unmodified influenza vaccine liposome biological stability test results

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of the present invention. within the scope of protection.

Claims (7)

1. polyethyleneglycol modified influenza vaccines liposome, which is characterized in that solid by influenza virus cracking inactivated vaccine, phosphatide, gallbladder Pure and mild polyethylene glycol is made；Wherein the quality of influenza virus cracking inactivated vaccine calculates the mass ratio of Shi Qiyu phosphatide with HA as 1: The mass ratio of 60 ~ 1:200, cholesterol and phosphatide is 1:2 ~ 1:5, and the mass ratio of polyethylene glycol and cholesterol is 1:1 ~ 1:3.

2. polyethyleneglycol modified influenza vaccines liposome as described in claim 1, which is characterized in that the influenza virus cracking The quality of inactivated vaccine is 1:60 ~ 1:100 with the mass ratio that HA calculates Shi Qiyu phosphatide, and the mass ratio of cholesterol and phosphatide is 1: The mass ratio of 3 ~ 1:4, polyethylene glycol and cholesterol is 1:1 ~ 1:3.

3. polyethyleneglycol modified influenza vaccines liposome as described in claim 1, which is characterized in that point of the polyethylene glycol Son amount is 1000-10000.

4. polyethyleneglycol modified influenza vaccines liposome as claimed in claim 3, which is characterized in that the polyethylene glycol is poly- Ethylene glycol -1000, Polyethylene glycol-2000, polyethylene glycol -3000, polyethylene glycol-4000, polyethylene glycol-6000, polyethylene glycol - 8000, one of polyethylene glycol-1000 0 or a variety of mixing.

5. polyethyleneglycol modified influenza vaccines liposome as described in claim 1, which is characterized in that the phosphatide is selected from soybean One of lecithin, hydrogenated soy phosphatidyl choline, egg phosphatide, phosphatidyl choline, phosphatidyl-ethanolamine or a variety of mixing.

6. polyethyleneglycol modified influenza vaccines liposome as claimed in claim 5, which is characterized in that the phosphatide is soybean ovum Phosphatide.

7. the preparation method of the polyethyleneglycol modified influenza vaccines liposome as described in claim 1-6 is any, which is characterized in that The following steps are included:

(a) phosphatide and cholesterol are dissolved in 50 ~ 60 DEG C with ethyl alcohol, phosphatide and cholesterol can be completely dissolved by ethanol consumption Subject to, in 30 ~ 40 DEG C of decompression rotary evaporations, alcohol solvent is removed, immobilized artificial membrane is obtained；

(b) phosphate buffer is added in immobilized artificial membrane, so that phospholipid concentration is 10 mg/mL, and poly- second two is added by recipe quantity Alcohol carries out rotation aquation at 50-60 DEG C later to get liposome first product.

(c) liposome first product is reduced into partial size by Probe Ultrasonic Searching processing, obtains liposome turbid liquor.

(d) liposome turbid liquor is mixed with influenza B virus cracking inactivated vaccine by suitable proportion, between -40 DEG C to 25 DEG C Multigelation 2 ~ 4 times；Later in -40 DEG C or so 8 ~ 12h of low temperature pre-freeze；Finally on freeze drier with -50 ~ -60 DEG C, 28 h or more are lyophilized under the conditions of 0.06 mbar, obtain white loose shape freeze-drying powder, as polyethyleneglycol modified influenza vaccines Liposome sets 4 DEG C of storages.