CN110151702A - Polyethyleneglycol modified influenza vaccines liposome and preparation method thereof - Google Patents
Polyethyleneglycol modified influenza vaccines liposome and preparation method thereof Download PDFInfo
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- CN110151702A CN110151702A CN201910551492.6A CN201910551492A CN110151702A CN 110151702 A CN110151702 A CN 110151702A CN 201910551492 A CN201910551492 A CN 201910551492A CN 110151702 A CN110151702 A CN 110151702A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5252—Virus inactivated (killed)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16211—Influenzavirus B, i.e. influenza B virus
- C12N2760/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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
Technical field:
The invention belongs to biotechnology formulation arts, specially vaccine field of biological, relate generally to a kind of poly- second two
Alcohol modifies influenza vaccines liposome and preparation method thereof.
Background technique:
Influenza (influenza, abbreviation influenza) is that a kind of threat mankind body and mind that influenza virus infection causes is strong
The Acute respiratory infectious disease of health, infectiousness is extremely strong, and the outburst of " spanish influenza " causes the life of global about 40,000,000 people to be walked
To termination.Nowadays, even if precautionary measures and medical technology are all greatly improved, but according to World Health Organization, every year
Because seasonal influenza causes global 3,000,000-500 ten thousand several cases and 300,000-50 ten thousand people dead.And China is exactly influenza virus
Multiple state, 3 pandemics influenzas since nineteen fifty-seven are all derived from China.The fearful place of influenza is exactly, from
Body will not generally directly result in death, but it can cause many complication for seriously endangering health, cause patient's body by
Different degrees of damage, even threat to life when severe.
Drug and vaccine are relied primarily on to anti influenza at present.Antiviral drugs can be relieved symptom and reduce the death rate, still
If adamantane amine drug is resistant to by H3N2 strain, the Oseltamivir (Tamiflu) of excellent effect in neuraminidase inhibitor
It also almost fails to certain seasonality H1N1 influenzas, and zanamivir can lead to part population bronchial spasm[1].Drug therapy
It is the remedial measure infected after influenza, but influenza vaccines can help inoculator to establish immunity before infection, be current public affairs
That recognizes reduces the most effectual way of flu episode rate and the death rate.
However, current influenza vaccines protective rate only has 10%~60%[2], and the protection period only has the several months[3].2014~
Influenza season in 2015, the data statistics of 46 2321 people of state in the U.S. showed that vaccine protective rate is 22%, for 50 years old or more crowd
Protective rate only has 14%[4]；2015~2016 years influenza seasons, the investigation of 6879 people of the U.S. show that vaccine protective rate is 48%, for
The protective rate of 50~64 years old crowds is 26%[5]；The application form free flow influenza vaccine protective rate in same year, 3842 people of Britain is
52.4%, the protective rate for over-65s crowd is 29.1%[6].Above-mentioned data apart from ideal 75% or more protective rate
With 12 months protection periods, and more preferably 90% or more protective rate and 5~10 years protection periods, there are also very big gaps[7],
Especially for most easily because influenza cause it is also very low with the protective rate of dead elderly population in hospital[8].Therefore, existing influenza epidemic disease
The Immune efficiency of seedling also needs to greatly reinforce.
Influenza vaccines are mainly split vaccine and subunit vaccine at present, their immune activation ability is weaker, are needed big
Amount inoculation just can induce body and generate antibody, and can hardly generate cellular immunity[9].Furthermore very widely used today aluminium assistant
Agent is also difficult to the generation of inducing cellular immune[10], therefore, it is difficult to induce longer immunological memory and obtain certain cross immunity,
It is unfavorable for fighting virus variation.On the other hand, and the intake of aluminium adjuvant also brings along the risk in terms of some safeties.It is worth glad
Happiness, Liposome Adjuvant can not only remarkably promote the generation of cellular immunity[9], can also be by activating Toll-like receptor (Toll
Like receptor, TLR) mode induce body generate immune response[11].TLR signal is that influenza vaccines induce immune response
Important costimulatory signal, but under causing vaccine to act on due to the downward of TLR in old or the mice model
Drop[12].In addition, liposome is biodegradable, size, charge, face finish material are flexibly adjustable, and release behavior is controllable, energy
Water-soluble different vaccine is prepared and loaded in a mild condition, does not cause autoimmune response, therefore be considered as aluminium adjuvant
Most promising adjuvant system later[13]。
It reasonably selects vaccine adjuvant and designing technique route is the key that cracking inactivated vaccine research.And polyethylene glycol
(PEG) it is hydrophilic polymer a kind of safe and non-toxic and with recessive role, particle can be slowed down according to the effect of its stereoscopic stable
Between aggregation, thus enhance preparation storage and application process in stability, had been widely used in field of medicaments.
Research also indicates that, polyethylene glycol-distearyl acid phosphatidyl-ethanolamine is inlayed on liposome bilayer membrane, can prevent to swallow well
Cell recognition and intake liposome are conducive to drug and enter lesion tissue, greatly improve to extend liposome circulation time
Therapeutic index and curative effect.
PEG is also applied in the preparation of vaccine, as immediate patent: patent pegylated phospholipids are to carry
The micella vaccine (ZL201410570624.7) of body prepares micella using pegylated phospholipids, and polypeptide vaccine is wrapped in
In micella, the mentality of designing of the patent mainly utilizes solubilization of the pegylated phospholipids to polypeptide.The poly- second of patent
Glycol-inactivated vaccine mucous membrane immunizing agent (ZL200410017527.1) uses polyethylene glycol as in vaccinogen liquid production link
A kind of material, and be retained in final vaccine product, to enhance immunization.
Through inquiring, before the application proposition, there is not yet technical solution same as the present application.
Bibliography:
[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 influenza
vaccines 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].MMWR
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 seasonal
influenza vaccine for adults and children in preventing laboratory-confirmed
influenza 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 future
trends[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] immunologic mechanism in She Zhennan, Zhai Wenjun, Deng Yihui " blood flow is accelerated to remove " phenomenon analyzes the Shenyang [J] medicine
Section's college journal, 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.
Summary of the invention:
The object of the present invention is to provide a kind of polyethyleneglycol modified influenza vaccines liposomes, while disclosing a kind of simple easy
It is capable, quality controllable, can industrialized production preparation method.
The purpose of the present invention is achieved through the following technical solutions: polyethyleneglycol modified influenza vaccines liposome, by influenza
The composition such as virolysis inactivated vaccine, phosphatide, cholesterol and polyethylene glycol；Wherein the quality of influenza virus cracking inactivated vaccine with
HA calculates the mass ratio of Shi Qiyu phosphatide as 1:60~1:200, and the mass ratio of cholesterol and phosphatide is 1:2~1:5, polyethylene glycol
Mass ratio with cholesterol is 1:1~1:3.
Preferably, polyethyleneglycol modified influenza vaccines liposome of the present invention, wherein influenza virus cracking inactivates epidemic disease
The quality of seedling is 1:60~1:100 with the mass ratio that HA calculates Shi Qiyu phosphatide, and the mass ratio of cholesterol and phosphatide is 1:3~1:
4, the mass ratio of polyethylene glycol and cholesterol is 1:1~1:3；More preferably, the mass ratio of the polyethylene glycol and cholesterol
For 1:2.
As most preferably, polyethyleneglycol modified influenza vaccines liposome of the present invention, wherein influenza virus cracking goes out
The quality of live vaccine is 1:70~1:80 with the mass ratio that HA calculates Shi Qiyu phosphatide, and the mass ratio of cholesterol and phosphatide is 1:
3.75, the mass ratio of polyethylene glycol and cholesterol is 1:2.
Preferably, the molecular weight of polyethylene glycol of the present invention is 1000~10000.
Preferably, the polyethylene glycol is polyethylene glycol-1000, Polyethylene glycol-2000, polyethylene glycol -3000, poly- second two
One of alcohol -4000, polyethylene glycol-6000, polyethylene glycol-8 000, polyethylene glycol-1000 0 or a variety of mixing.As more
It is preferred that the polyethylene glycol is Polyethylene glycol-2000, polyethylene glycol -3000, polyethylene glycol-4000, polyethylene glycol-6000, gathers
One of ethylene glycol -8000 or a variety of mixing.
Preferably, phosphatide of the present invention be selected from soybean lecithin, hydrogenated soy phosphatidyl choline, egg phosphatide, phosphatidyl choline,
One of phosphatidyl-ethanolamine or a variety of mixing.More preferably, the phosphatide is selected from soybean lecithin.
The present invention also provides a kind of preparation method of polyethyleneglycol modified influenza vaccines liposome simultaneously, technique include with
Lower step:
(a) phosphatide and cholesterol are dissolved in 50~60 DEG C with ethyl alcohol, ethanol consumption is with can be complete by phosphatide and cholesterol
Subject to fully dissolved, 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 10mg/mL, and is added and gathers by recipe quantity
Ethylene glycol carries out rotation aquation at 50-60 DEG C later to get liposome first product.Preferably, the pH value of phosphate buffer
It is 7.0 ± 0.2, hydration time is 40~50min.
(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, in -40 DEG C to 25
Multigelation 2~4 times between DEG C；Later in -40 DEG C or so 8~12h of low temperature pre-freeze；Finally on freeze drier with -52 DEG C,
28h or more is lyophilized under the conditions of 0.06mbar, obtains white loose shape freeze-drying powder, as polyethyleneglycol modified influenza vaccines lipid
Body sets 4 DEG C of storages.
Compared with the prior art, the invention has the following advantages:
(1) compared to the vaccinogen liquid for not adding adjuvant, or the vaccine and conventional vaccine rouge of conventional aluminium adjuvant are added
Plastid, polyethyleneglycol modified influenza vaccines liposome cell immunogenicity is stronger, can induce body and generates stronger cellular immunity.
(2) compared to the vaccinogen liquid liquid drugs injection or conventional vaccine lipidosome freeze-dried powder for not adding adjuvant, polyethylene glycol
It is more preferable to modify influenza vaccines lipidosome freeze-dried powder storage stability.
(3) polyethyleneglycol modified influenza vaccines liposome preparation simple process provided by the invention, preparation process do not contact
Toxic organic solvent, without high temperature and acutely, operation, the self structure that influenza vaccines can be effectively protected are not destroyed overall process.
(4) the application prepares liposome with cholesterol and phosphatide, adds polyethylene glycol later and modifies as water-wetted surface
Object finally prepares polyethyleneglycol modified influenza vaccines liposome using freeze thawing-desivac.With described in CN201410570624.7
Technical solution is compared, and the present invention wraps up step without using expensive pegylated phospholipids, and without micella, prepares work
Skill is simpler, and process is more controllable.Compared with CN200410017527.1, the present invention is by antigen (vaccine), liposome, poly- second
Glycol combines, and can preferably increase the Immunestimulatory effect of antigen, while simple process, be easy to industrialization.
Specific embodiment:
For a clearer understanding of the present invention, completed below in conjunction with the technical solution under this invention that inventor provides
The present invention is described in further detail for embodiment.The invention is not limited to these embodiments, appoint to what the present invention was done
What formal accommodation and/or change falls within the scope of the present invention.
In the present invention, all equipment or raw material etc. can be obtained from market or the industry is common.Wherein, popular
Sexuality emits employing virus cracking liquid and is provided by Jiangsu Watson Biotechnology Ltd.；Standard Bovine serum albumin BSA purchase is certainly
U.S. Sigma；Soybean lecithin, hydrogenated soy phosphatidyl choline, egg phosphatide, hydrogenated yolk lecithin, phosphatidyl choline, phosphatidyl second
Hydramine is bought from Beijing Mei Yasi phosphatide technology company；Cholesterol purchase is public from Beijing ancient cooking vessel state prosperity biotechnology Limited Liability
Department；Polyethylene glycol is bought from Shanghai Mike's woods biochemical technology Co., Ltd.
The abbreviation of each ingredient used is as follows in following example:
SPC: soybean lecithin
EPC: egg yolk lecithin
HSPC: hydrogenated soy phosphatidyl choline
DSPC: Distearoyl Phosphatidylcholine
DPPC: dipalmitoylcholine
DSPE: Distearoyl Phosphatidylethanolamine
DPPE: dipalmitoylphosphatidylethanolamine
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-8 000
PEG-10000: polyethylene glycol-1000 0
PBS buffer solution: phosphate buffer
HA: hemagglutinin
The influenza vaccines liposome preparation prescription 1 of embodiment 1PEG modification:
Prescription 2:
Prescription 3:
Prescription 4:
Prescription 5:
Prescription 6:
Prescription 7:
Prescription 8:
Prescription 9:
Prescription 10:
Prescription 11:
Prescription 12:
Prescription 13:
Prescription 14:
Prescription 15:
Prescription 16:
Prescription 17:
Prescription 18:
In the present embodiment prescription 1 to prescription 18 polyethyleneglycol modified influenza liposome bacterin the preparation method is as follows:
(1) it disperses cholesterol and phosphatide in 18~22mL dehydrated alcohol, keeps it sufficiently molten under 50~60 DEG C of water-baths
Solution.
(2) mixed solution of above-mentioned dissolution is depressurized to rotary evaporation under the conditions of 30~40 DEG C and removes ethyl alcohol, revolving speed 40
~50r/min, until film forming.
(3) lipid film that the PBS buffer solution of the pH 7.0 ± 0.2 of about 25~35mL is formed in cholesterol and phosphatide is added
In, the PEG of recipe quantity is added, carries out rotation 40~50min of aquation at 50~60 DEG C later to get liposome first product.
(4) liposome first product is processed using Probe Ultrasonic Searching technique: by ultrasonic probe be put into liposome first product into
The intermittent ultrasonic treatment of row (ultrasonic 30s suspends 30s, supersonic frequency 40~50%), is ultrasonically treated total time 2min, ultrasound knot
Shu Hou continues to place 1~2h in 50~60 DEG C of water-baths, obtains milky liposome turbid liquor.
(5) the influenza vaccines stoste of suspension obtained above and corrresponding quality (with HA Mass Calculation) is pressed into suitable proportion
It mixes well, is sub-packed in ampoule bottle, 2ml/ bottles；The ampoule bottle 1 that drug-loaded liposome is housed is rapidly frozen in -40 DEG C of low temperature refrigerators
After~2h, slowly melt (1 number of freezing and thawing) under room temperature environment, freezes 8~12h in -40 DEG C of low temperature refrigerators again after thawing.
(6) drug-loaded liposome of pre-freeze is put on freeze drier (0.06mbar, -52 DEG C) plug-in freeze-drying 28h or more,
Can be obtained freeze-dried powder, by freeze-dried powder sealing be placed in 4 DEG C under the conditions of storage to get.
Obtained freeze-drying prods appearance is loose, white or close white.Partial size is 1~2 μm after redissolution.
Embodiment 2 is without modification influenza vaccines liposome preparation
Prescription 19:
Prescription 19 is consistent in embodiment 1 without modification influenza liposome bacterin preparation method in the present embodiment, only
The step of PEG is added is not included in whole preparation process.What is be prepared will be used for animal without modification influenza liposome bacterin
Immunogenicity experiments and estimation of stability.
3 animal immune originality experimental study of embodiment
1, animal packet and immune
Mouse is randomly divided into polyethyleneglycol modified influenza vaccines liposome group, PBS blank control group, vaccinogen liquid group,
Without modification influenza vaccines liposome group, every group 3.Wherein PBS is negative blank control group, and vaccinogen liquid is positive controls.
Vaccine administration group dosage is calculated as 6 μ g/ only with HA., intraperitoneal administration immune using pulmonary administration after anesthesia it is one of immune in
0d is immunized, 7d later, 14d, and 28d carries out Immune Indexes detection.Wherein polyethyleneglycol modified influenza vaccines lipid
Vaccine used in body group is prepared according to prescription 2 in embodiment 1 and prescription 8；Without epidemic disease used in modification stream influenza vaccines liposome group
Seedling is prepared according to prescription 19 in embodiment 2, and vaccinogen liquid group and PBS group are administered after being directly formulated as solution.
2, spleen lymphocyte proliferation is tested
Under aseptic condition, 10~15% fetal calf serums of the splenic lymphocytes suspension 3mL of the mouse prepared suspend
Cell, adjustment cell number is about 3.0 × 106/ mL, experimental port and blank control wells into 96 well culture plates are separately added into preparation
The final concentration of cells in 100 hole μ L/ of splenic lymphocytes suspension of mouse out and every hole is 3.0 × 106/mL；Again in experimental port
The con A solution (ConA solution) of 20 μ g/mL is added, it is 100 μ that RPMI-1640 culture medium, which is added, in blank control wells
The hole L/, wherein experimental group and blank group are all provided with 3 multiple holes.In 37 DEG C, 5%CO after dosing2It is cultivated 48 hours in cell incubator
Afterwards, it takes out, in being protected from light 20 μ L/ hole MTT of addition in aseptic superclean bench, (5mg/mL is mixed gently, and is put into cell incubator
Continue to terminate reaction after cultivating 4h, 100 μ L/ Kong Sanlian liquid are added and dissolve purple crystal.It is tested after 8~12h in microplate reader 570nm
Wavelength and 630nm reference wavelength measure each hole OD value, are with the every hole mouse ConA mean OD value/blank control wells mean OD value
Stimulus index (SI) judges that the proliferation of mouse spleen lymphocyte is horizontal.
3, flow cytomery T lymphocyte surface markers are tested
Under aseptic condition, the splenic lymphocytes suspension 3mLPBS suspension cell of the mouse prepared adjusts cell number
About 3 × 106/ mL takes 1mL cell suspension in EP pipe, and points 4 groups, the 1st group of cell suspension is that blank control is not added dyestuff, and the 2nd
Anti-mouse CD4 PE (L3T4,0.2mg/mL, the eBioscience of 1 μ L are added in group cell suspensionTM,
Thermofisher it) mixes, Anti-mouse CD8a FITC (Ly-2, the 0.5mg/ of 1 μ L is added in the 3rd group of cell suspension
ML, eBioscienceTM, thermofisher) and it mixes, the Anti-mouse CD4 PE of 1 μ L is added in the 4th group of cell suspension
It is mixed with the Anti-mouse CD8a FITC of 1 μ L, flow cytometer detects after being protected from light label 30min with masking foil, uses
SS and FS divide group, are analyzed with Beckman Coulter CXP.Flow cytomery CD4+/CD8+ ratio is investigated its cell and is exempted from
Epidemic focus.
4, experimental result
Show labelling experiment by spleen lymphocyte proliferation experiment and T lymphocyte to verify.Increased using splenic lymphocytes
Grow experiment (table 1) and T lymphocyte surface markers experiment (table 2), the results showed that, polyethyleneglycol modified influenza vaccines liposome tool
Play the role of enhancing cellular immunity, and effect is better than without modification influenza vaccines liposome group.Polyethyleneglycol modified influenza vaccines rouge
The cell immunogenicity of plastid is stronger, can induce body and generates stronger cellular immunity.Compare vaccinogen liquid, no modification influenza epidemic disease
Seedling liposome also enhances the cell immunogenicity of body.
1 spleen lymphocyte proliferation experimental result of table
2 T lymphocyte of table shows labelling experiment result 7d, 14d, 28d (n=3)
The experiment of 4 influenza vaccines liposome stability of embodiment
Polyethyleneglycol modified influenza vaccines liposome turbid liquor and freeze-dried powder are prepared according to prescription 2 in embodiment 1, according to place
19 preparation of side is without modification influenza vaccines lipidosome freeze-dried powder.
The polyethyleneglycol modified influenza vaccines liposome turbid liquor of preparation, polyethyleneglycol modified influenza vaccines liposome are frozen
Dry powder and without modification influenza vaccines lipidosome freeze-dried powder be individually positioned in different temperature (37 ± 2 DEG C, 25 ± 2 DEG C and 4 DEG C) into
Trip temperature Acceleration study samples at the time point (0,5,10,15,30d) of setting, after redissolving lipidosome freeze-dried powder with PBS, respectively
In 4 DEG C, 45000r/min high speed centrifugation 1h, protein content is measured with Lowry method, to calculate its encapsulation rate.Investigate poly- second two
Alcohol modifies influenza vaccines liposome turbid liquor, polyethyleneglycol modified influenza vaccines lipidosome freeze-dried powder and without modification influenza vaccines rouge
The situation of change of the encapsulation rate of plastid freeze-dried powder in different time points, encapsulation rate result are indicated with (X ± SD) %.
To be placed on 40 ± 2 DEG C without modification influenza vaccines lipidosome freeze-dried powder in 10d sample, be placed at 25 ± 2 DEG C
Without modification influenza vaccines lipidosome freeze-dried powder in 30d sample, using Microhemagglutination inhibit (HI) method measurement latter timing is immunized
Between mice serum, with H1NIType influenza vaccines standard antigen measures corresponding antibody, investigates and freezes without modification influenza vaccines liposome
Dry powder place at high temperature after immunogenicity.
Measurement result is shown in Table 3, table 4, table 5 and table 6 respectively.According to result it is found that as time went on, compared to without
The polyethyleneglycol modified influenza vaccines liposome turbid liquor of freeze-drying, the polyethyleneglycol modified influenza vaccines after freeze-drying are lipidosome freeze-dried
Powder and without modification influenza vaccines lipidosome freeze-dried powder have higher encapsulation rate.Illustrate either by it is polyethyleneglycol modified still
Physical stability dramatically increases before being relatively lyophilized after literalness influenza vaccines are lipidosome freeze-dried.From table 4,0-15 days encapsulation rate drops
Low numerical value it can be seen that polyethyleneglycol modified influenza vaccines liposome no matter be lyophilized before with to be expected to reach mitigation after freeze-drying cold
The purpose requirement of chain transport pressure.By table 6 without modification influenza vaccines liposome biological stability data it can be seen that influenza epidemic disease
Seedling lipidosome freeze-dried powder room temperature 30 days places 10 days immune rear and antibody before deimmunisation titre ratios for 40 ± 2 DEG C and is all larger than 4, still
It is so immune effective.
3 each group vaccine liposome stability test result (37 ± 2 DEG C) of table
4 each group vaccine liposome stability test result (25 ± 2 DEG C) of table
5 each group vaccine liposome stability test result (4 ± 2 DEG C) of table
Table 6 is without modification influenza vaccines liposome biological stability test result
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
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.
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