CN108452299A - A kind of preparation method of liposome and the method for making Liposome Adjuvant - Google Patents

Abstract

The present invention relates to a kind of preparation method of liposome and its as the application of vaccine immunity adjuvant, a kind of method for preparing lipidosome provided includes the following steps：1) hydrogenated soya phosphatide HSPC, cholesterol CHOL, 1,2 dioleoyl oxygroup trimethyl ammonium chloride DOTAP Cl, vitamin V E are weighed, mixture is made in the ratio of mass fraction 70~90: 0~30: 10~20: 0.5；2) mixture described in step 1 is dissolved in obtained in chloroform/methanol solvent lipoid organic solution it is spare, the chloroform/methanol solvent by volume ratio be 24: 72: 4 5.1 chloroform, methanol and ultra-pure water form；3) water-soluble powder shape solid support is weighed to be placed in the round-bottomed flask of 250ml, add the organic solution of above-mentioned lipoid, it stirs evenly, it is placed on Rotary Evaporators rotation under conditions of ice water bath constant temperature and boils off organic solvent, the quality of the wherein described solid support is 5 times of the mixture quality, includes the one or more of lactose, sucrose, mannitol, sodium chloride.The preparation method simple process and low cost is suitable for mass propgation liposome and is applied to vaccine immunity adjuvant.

Description

A kind of preparation method of liposome and the method for making Liposome Adjuvant

Technical field

It is helped the present invention relates to the preparation method of biomedicine field more particularly to a kind of liposome and its as vaccine immunity The application of agent.

Background technology

Vaccine inoculation disease preventing and treating is a kind of very effective method, but certain traditional vaccines can cause different degrees of pair Effect.In recent years, with immunological investigation deepen continuously and the rapid development of technique for gene engineering, live vector vaccine, DNA epidemic diseases The research of the new generation vaccines such as seedling and protein vaccine makes encouraging progress.These new generation vaccine purity height, high specificities, but Immunogenicity is weak, and the immune response that induction body generates is not strong enough, therefore, enhances vaccine with both safe and effective adjuvant Effect has become current problem anxious to be resolved.

Currently the only be formally approved for the adjuvant of human vaccination is that (main component is aluminium hydroxide or phosphoric acid to aluminium adjuvant Aluminium).Although the aluminium adjuvant that facts have proved for many years is substantially safely and effectively that it also has significant limitations, and this The adjuvant of single type cannot be satisfied different needs of a variety of vaccines to multiple types adjuvant.To meet the development of vaccine industry, It is imperative with some new means and method exploitation novel adjuvant.Hot spot is had become in the research and development of Overseas New adjuvant, and Though domestic research is more, it is still in the starting stage.

Nineteen sixty-five finds phosphatide in water first by the Bangham of Britain can spontaneously form liposome (liposomes) Since, it is increasingly extensive to its experimental study.This structure of liposome can carry various hydrophilic, hydrophobic and both sexes Substance, they are typically entrapped within liposome interior water phase, or are inserted into class lipid bilayer, or absorption, the surface for being connected in liposome. As simulation cell membrane and pharmaceutical carrier, the research and application of liposome are just increasingly deeply and extensive.Originally it is contemplated that, by drug Be wrapped in liposome interior, can escape or mitigate host immune response generated to drug, but Allison in 1974 and Gregoriadis has found that liposome has apparent immunoadjuvant function to mixing diphtheria toxoid wherein, is opened from this The prelude that liposome is studied as immunopotentiator.With the further accumulation of people's liposome bacterin data, liposome is expected Become after aluminium adjuvant another in the near future and is approved for the novel adjuvant of human vaccination.

Liposome has the advantages that many uniquenesses as adjuvant：(1) antigen can enhancement antigen with liposome simple mixing Immunogenicity, if package or if being fixed on liposome it is stronger；(2) there is natural targeting, antigen can be targeted in netted Dermal system, to preferentially be absorbed by antigen presenting cell (APC)；(3) Small side effects, mild degree, no general toxic reaction, can The phenomenon that biodegradation, non-toxic and immunogenicity itself, the not yet reaction of discovery liposome bacterin induced metamorphosis and autoantibody； (4) warehouse effect, liposome have slow releasing function to the antigen of package, and body is made to keep high titre antibody for a long time, reduce antigen Dosage and inoculation times；(5) can be changed immune response type and mode, can simultaneously induction body fluid and cellular immunity, hence it is evident that Better than the aluminium adjuvant that cellular immunity induces force difference, when enhancing humoral immunity shows that the hypotype conversion of antibody, titre increase, continue Between it is long, can also generate immunological memory；(6) single phosphatidyl lipid A (MPL), interferon, interleukins can be packed in liposome 2 and cell factor etc. play synergistic effect, stimulation and promotion APC phase interactions between the processing of antigen, submission and immunocyte With；Humoral and cellular immune response is further enhanced, while reducing the dosage of standard adjuvant, the dosage of toxicity and antigen；(7) fat Plastid has antigen presentation, and high concentration antigen is also in no APC and major histocompatibility complex molecule, in liposome Certain T cells can be activated.

Continued to bring out although as new generation vaccines such as DNA recombinant vaccines, synthetic peptide vaccines, immunologic adjuvant research increasingly by The concern of people.But since the reaction of adjuvant booster immunization is an extremely complex process, the suitable assistant of each antigen Agent prescription needs to screen again, how to develop and is applicable in the immunoadjuvant system of HPV viruse vaccine protein and studies at present less, does not also have There is the report seen about liposome as HPV viruse vaccine immunity adjuvant.This vaccine research and development department determines to open with nanotechnology Novel lipide adjuvant products are sent out, follow up vaccine product is can be used not only for, makes contributions for the listing of specific vaccine kind, more exists In being made that breakthrough, academic significance and foreground market are larger for China field.

Liposome is recognized by more and more people as a kind of advanced drug delivery system, advantage.At present It is dedicated to the important collection of Research Emphasis of domestic and international technology platform and the product of exploitation used by developing the company of liposome medicament In 3 aspect：A wraps up antitumor chemotherapeutics, especially adriamycin class, vincristine class and camptothecine.B is used Make the carrier of nucleic acid drug.C is used as the carrier of protein-polypeptide vaccine and DNA vaccination.

Liposome is a kind of effective immunologic adjuvant, and it is a good direction that the carrier as vaccine, which carries out immunization therapy, The adverse reaction of the adjuvants such as aluminium adjuvant or viral vector can be reduced.The therapeutic index for improving drug enhances the immune of body Reaction.But the company of each exploit person liposome bacterin class drug is still in development phase mostly, makes substantial progress Only BernaBiotech companies and Biomira companies.BernaBiotech companies use the technology platform of Virosomes, use In transmission antigen, DNA-RNA or curative drug.But it is mainly used as the carrier of vaccine at present.Using Virosomes technologies, BernaBiotech has developed 2 products, and one is influenza vaccines (Inflexal.V), and one is Aimmugen (Epaxal.).The standby liposome medicament of Biomira corporations is that this artificial antigen and adjuvant incorporation enter, obtains liposome Vaccine.The said firm's product being developed is that BLP25 liposome bacterins are currently in clinical trial, which can make machine Body generates tumor associated antigen mucin MUC1 the cell immune response of specificity.And at home by liposome for vaccine There is not been reported in field.

Invention content

A kind of method for preparing lipidosome of this offer, this method is easy to operate, is easy to be prepared on a large scale, avoids organic solvent pair Bioactive substance activity influence.Obtained lipid adjuvant is relatively stable in preparation and storing process, is applied to public affairs It takes charge of the prevention researched and developed and preferable albumen can be obtained on the treatment relevant disease vaccine of HPV viruse and contain ability and immune response water It is flat.

Method for preparing lipidosome provided by the invention includes the following steps：1) hydrogenated soya phosphatide HSPC, cholesterol are weighed CHOL, 1,2- dioleoyl oxygroup trimethyl ammonium chlorides DOTAP-Cl, vitamin V E, by mass fraction 70~90: 0~30: 10~ 20: 0.5 ratio is made mixture, 2) mixture described in step 1 is dissolved in chloroform/methanol solvent and obtains the organic of lipoid Solution for standby, the chloroform/methanol solvent are made of chloroform, methanol and the ultra-pure water that volume ratio is 24: 72: 4-5.1；3) it weighs Water-soluble powder shape solid support is placed in the round-bottomed flask of 250ml, adds the organic solution of above-mentioned lipoid, and stirring is equal It is even, it is placed on Rotary Evaporators rotation under conditions of ice water bath constant temperature and boils off organic solvent, wherein the matter of the solid support Amount is 5 times of the mixture quality, includes the one or more of lactose, sucrose, mannitol, sodium chloride；4) round bottom burning is removed Bottle takes out solid sample, is placed at -18 DEG C and is sealed.

The present invention additionally provides a kind of method for preparing lipidosome, include the following steps：1) weigh hydrogenated soya phosphatide HSPC, Cholesterol CHOL, 1,2- distearoyl phosphatidyl glycerol sodium salts DSPG-Na, vitamin V E, by mass fraction 70~90: 0~30: Mixture is made in 10~20: 0.5 ratio；2) mixture described in step 1 is dissolved in chloroform/methanol solvent and obtains lipoid Organic solution is spare, and the chloroform/methanol solvent is made of chloroform, methanol and the ultra-pure water that volume ratio is 24: 72: 4-5.1；3) It weighs water-soluble powder shape solid support to be placed in the round-bottomed flask of 250ml, adds the organic solution of above-mentioned lipoid, stir Uniformly, it is placed on Rotary Evaporators rotation under conditions of ice water bath constant temperature and boils off organic solvent, wherein the solid support Quality is 5 times of the mixture quality, includes the one or more of lactose, sucrose, mannitol, sodium chloride；4) round bottom is removed Flask takes out solid sample, is placed at -18 DEG C and is sealed.

The present invention additionally provides a kind of methods using above-mentioned liposome preparation Liposome Adjuvant, weigh according to the method described above Proliposome obtained is added in conical flask, is added in suitable ultra-pure water or buffer solution, and gently oscillation is allowed to disperse, and obtains To the thick suspension of blank liposome；The thick suspension of blank liposome is added in high pressure homogenizer and carries out high speed shear processing and high pressure Homogenization continues to increase pressure to 300bar homogeneous 3-5 times to get to liposome sample through homogeneous under 0bar pressure 3-5 times Moist heat sterilization under the conditions of 115 DEG C of the liposomal samples progress of preparation, 30min is obtained final Liposome Adjuvant by product；Then It is preserved under the conditions of 4 DEG C.

Description of the drawings

Fig. 1 shows the liposome solutions of different cholesterol levels；

Fig. 2 shows be cholesterol level be 0% liposome solutions graininess；

Fig. 3 shows that cholesterol level is the graininess of 10% liposome solutions；

Fig. 4 shows that cholesterol level is the graininess of 20% liposome solutions；

Fig. 5 shows that cholesterol level is the graininess of 30% liposome solutions；

Fig. 6 shows the particle diameter distribution of liposome in the liposome solutions of different cholesterol levels；

Fig. 7 shows situation of change of the liposome to Z-Average grain sizes before and after the absorption of HPV 16L1 albumen；

Fig. 8 shows situation of change of the liposome to Zeta potential before and after the absorption of HPV 16L1 albumen；

Fig. 9 shows situation of change of the liposome to Z-Average grain sizes before and after the absorption of VR111 albumen；

Figure 10 shows influence of the liposome solutions of different cholesterol levels to mouse tumor formation rate；

Figure 11 shows influence of the liposome solutions of different cholesterol levels to mouse mean tumour volume；

Figure 12 shows influence of the liposome solutions of different cholesterol levels to tumour inhibiting rate in Mice Body；

Figure 13 shows influence of the liposome solutions to mouse survival rate of different cholesterol levels.

Specific implementation mode

Weigh hydrogenated soya phosphatide HSPC, cholesterol CHOL, 1,2- dioleoyl oxygroup trimethyl ammonium chlorides DOTAP-Cl, dimension Mixture is made in the ratio of mass fraction 70~90: 0~30: 10~20: 0.5 in raw element VE, 2) by mixture described in step 1 Be dissolved in obtained in chloroform/methanol solvent lipoid organic solution it is spare, the chloroform/methanol solvent by volume ratio be 24: 72: Chloroform, methanol and the ultra-pure water composition of 4-5.1；；3) round-bottomed flask that water-soluble powder shape solid support is placed in 250ml is weighed In, the organic solution of above-mentioned lipoid is added, is stirred evenly, is placed on Rotary Evaporators and is rotated under conditions of ice water bath constant temperature Organic solvent is boiled off, wherein the quality of the solid support is 5 times of the mixture quality, including lactose, sucrose, sweet dew The one or more of alcohol, sodium chloride；4) round-bottomed flask is removed, solid sample is taken out to get proliposome, is placed at -18 DEG C It is sealed.

It weighs proliposome obtained according to the method described above to be added in conical flask, suitable ultra-pure water or buffering is added In liquid, gently oscillation is allowed to disperse, and obtains the thick suspension of blank liposome；The thick suspension of blank liposome is added to high pressure homogenizer Middle progress high speed shear processing and high-pressure homogeneous processing, homogeneous 3-5 times under 0bar pressure continue to increase pressure to 300bar homogeneous 3-5 times to get to liposomal samples, by the liposomal samples of preparation under the conditions of 115 DEG C moist heat sterilization 30 minutes；Then 4 DEG C Under the conditions of preserve.

Conclusion is provided to the various aspects of performance of Liposome Adjuvant below by experiment.

(1) appearance for the liposome that Liposome Adjuvant ingredient is prepared under each ratio, average grain are investigated according to experiment The indexs such as form under diameter and its distribution, transmission electron microscope, in this, as the foundation for selecting best adjuvant prescription.

1, appearance character

The different Liposome Adjuvant of cholesterol level is matched using ultra-pure water or other oroteins solution buffer solution for solvent It is set to a concentration of 2.0mg/ml solution and is sealed in cillin bottle, observe the appearance character of Liposome Adjuvant solution.As a result see attached drawing 1.

In Fig. 1, cholesterol level is 0% in the Liposome Adjuvant solution in the first from left bottle, the Liposome Adjuvant in the second from left bottle Cholesterol level is 10% in solution, and cholesterol level is 20% in the Liposome Adjuvant solution in three bottles of a left side, in four bottles left Cholesterol cholesterol level in raw mixture is 30% in Liposome Adjuvant solution.Liposome Adjuvant solution as shown in Figure 1 Be the solution with white opalescence, through a long time is placed to be precipitated without solid sample, solution colour with cholesterol level increase And deepen.When wherein cholesterol level is below 10%, clarity is preferable.

2, particle shape

By the different Liposome Adjuvant solution example of above-mentioned four bottles of cholesterol levels after being diluted to 2mg/ml, it is added dropwise to phosphorus Wolframic acid dyes, and room temperature volatilizes, in the particle shape of transmissioning electric mirror determining sample.As a result see 2~attached drawing of attached drawing 5.

Compare Electronic Speculum result, it is possible to find in the different Liposome Adjuvant solution of cholesterol level, liposome particles are most Spherical in shape or elliposoidal structure, dispersibility is preferably.The size of liposome particles with the increase of cholesterol ratio in liposome and Increase.When wherein cholesterol level is below 10%, granular size is more uniform.

3, average grain diameter and distribution

After the different Liposome Adjuvant solution example of above-mentioned four bottles of cholesterol levels is diluted to 2mg/ml, using Marven The particle diameter and polydispersity coefficient (PDI) of the Zetasizer Nano ZS determination samples of company.As a result see attached drawing 6 and attached Table 1.

Liposome title	Average grain diameter (Z-Average)	Polydispersity coefficient (PDI)
			0% liposome of CHOL contents	95.30nm	0.252
10% liposome of CHOL contents	109.90nm	0.235
			20% liposome of CHOL contents	118.00nm	0.237
30% liposome of CHOL contents	201.50nm	0.178

The Z-Average grain sizes and its polydispersity coefficient of 1 liposome of table

By Z-Average grain sizes and the particle diameter distribution result of liposome it is found that the Z-Average grain sizes of liposome generally exist 200nm hereinafter, be uniformly dispersed PDI about 0.25 hereinafter, have apparent nanostructure, particle is more uniform, liposome particles it is big The small increase with cholesterol ratio in liposome and increase.The experimental result is consistent with Electronic Speculum testing result in summary every As a result, cholesterol level is more excellent formula at 10% when in liposome.

4, the Zeta potential (surface potential) of ionic liposome

Buffer solution I final concentrations：0.486mol/L sodium chloride, 0.01mol/L histidines, 0.01% Tween 80, pH6.2；

Buffer solution II final concentrations：0.154mol/L sodium chloride, 0.01mol/L histidines, 0.01% Tween 80, pH6.2；

Buffer solution III final concentrations：0.041mol/L sodium dihydrogen phosphates, 0.009mol/L disodium hydrogen phosphates, pH 6.2；

Influence of the charged phospholipids component to liposome Zeta potential：It is surveyed using the Zetasizer NanoZS of Marven companies Determine using cationic-liposome of the lactose as solid support and containing the vitamin V E for being 0.5% with total lipid body mass ratio (HSPC80/DOTAP 10/CHOL 10/VE0.5, HSPC70/DOTAP20/CHOL 10/VE0.5) and anionic liposome The Zeta potential of (HSPC80/DSPG 10/CHOL 10/VE0.5, HSPC70/DSPG20/CHOL 10/VE0.5), the above lipid The solution system of body assist agent solution is buffer solution I.As a result 2 are seen attached list.

The Z-Average grain sizes and its polydispersity coefficient of 2 liposome of table

Influence of the solution system to liposome Zeta potential：For HSPC70/DSPG20/CHOL10/VE0.5, use The Zetasizer Nano ZS of Marven companies measure the liposome with lactose (or sodium chloride) for solid support respectively, Zeta potential in ultra-pure water, buffer solution I, buffer solution II, buffer solution III, 8% lactose or 8% sodium chloride.Knot Fruit sees attached list 3.

Zeta potential of 3 liposome of table in different solutions

The experimental results showed that phospholipid species, solid support type, buffer solution type, ionic strength etc. have relationship. According to project needs, different types of electrically charged liposome vectors are selected, liposome and oppositely charged protein molecular are passed through Between electrostatic attraction carry out adhesion protein.Fat is adjusted by solid support, buffer solution type, the ionic strength etc. of screening liposome The height of the Zeta potential of plastid, come influence the stability of liposome in the solution height and Liposome Adjuvant to protein adsorption The power of ability.

5, accelerated stability

By taking HSPC70/DSPG20/CHOL10/VE0.5 as an example, liposome aqueous solution is respectively placed in 37 ± 2 DEG C and 4 ± 2 DEG C stable condition under, respectively at 0 day, 7 days, 14 days, 21 days sampling using Marven companies ZetasizerNanoZS measure The Z-Average grain sizes of liposome and the variation of Zeta potential.As a result 4 and subordinate list 5 are seen attached list.

4 temperature of table, influence of the holding time to liposome Z-Average grain sizes

5 temperature of table, influence of the holding time to liposome Zeta potential

The aqueous solution of the liposome is under 4 DEG C and 37 DEG C of storage temperatures, for the clear solution with white opalescence, experiment Samples taken is without apparent solid Precipitation in the process.The Z-Average grain sizes of liposome do not obviously increase, zeta current potentials Also relatively stable, have apparent elecrtonegativity, liposome stability preferable.

6, the binding ability of liposome and protein vaccine

By taking HSPC70/DSPG20/CHOL 10/VE0.5 as an example, binding ability of the liposome to HPV 16L1 albumen is measured. Each concentration liposome is mixed with HPV 16L1 albumen at 1: 1 by volume respectively, is adsorbed 2 hours.Lipid before and after measurement adhesion protein The Z-Average grain sizes and zeta current potentials of body, and liposome is estimated according to the variation tendency of the two, ability is contained to vaccine. Wherein the ratio of Liposome Adjuvant and HPV16L1 albumen be respectively 100: 1,50: 1,25: 1,12.5: 1,6.25: 1,3.13: 1, 1.56∶1、0.78∶1.As a result 6, attached drawing 7 and attached drawing 8 are seen attached list.

6 liposome of table is to the Z-Average grain sizes and Zeta potential before and after HPV 16L1 protein adsorptions

By taking HSPC70/DSPG20/CHOL10/VE0.5 as an example, binding ability of the liposome to VR111 albumen is measured.It is each dense Degree liposome is mixed with the VR111 albumen of a concentration of 0.02mg/ml at 1: 1 by volume respectively, is adsorbed 2 hours.Measure absorption egg The Z-AVerage grain sizes of liposome after Cynanchum glaucescens, and liposome is estimated according to the variation tendency of the two, ability is contained to vaccine. Wherein the ratio of Liposome Adjuvant and HPV 16L1 albumen be respectively 200: 1,100: 1,50: 1,25: 1,12.5: 1,6.25: 1, 3.13∶1、1.56∶1.As a result 7, attached drawing 9 is seen attached list.

Absorption of 7 liposome of table to VR111 albumen

Experiment shows the absorption that the albumen that electrically charged liposome is conducive to oppositely charged is added, when negatively charged The ratio of HSPC70/DSPG20/CHOL 10/VE0.5 Liposome Adjuvants and positively charged HPV16L1 albumen has when being more than 3 times Preferable absorption result.And HSPC70/DSPG20/CHOL 10/VE0.5 Liposome Adjuvants and same electronegative VR111 albumen Ratio need be more than 10 times when just have preferable absorption result.

(2) liposome enhances ability to the humoral immunity of vaccine

Experiment purpose：It investigates homemade Liposome Adjuvant, liposome/aluminum phosphate and combines adjuvant to example Protein reconstitution human milk The effect of the humoral immunity humidification of head 18 type protein vaccine of tumor virus (18 L1 of HPV).

Experimental animal：Bal/C mouse, 6-8 weeks, female, 20.0 ± 2.0g.

Immune sample：18 L1 eggs of HPV are adsorbed with Liposome Adjuvant, liposome/aluminum phosphate joint adjuvant, Aluminium phosphate adjuvant Prepared by white stoste is immunized sample, and composition such as table 8, solution buffer system is 0.01M L-His, 0.01%Tween-80, pH 6.2：

The composition of 8 each 18 L1 vaccines of adjuvant type HPV of table

Immunization method：It is primary that above-mentioned sample gives Bal/C mouse immunes, and immunization route is 5 points of injections of subcutaneous abdomen, every It is inoculated with 0.5ml.After mouse immune 28 days, blood, separation serum is taken to carry out the detection of Conversion rate using eyeball is plucked.Experimental result is shown in Table 9。

Grouping	Serum antibody OD average values	Antibody titer
			A groups	1.164	117376.5(592000)
B groups	1.529	256000(288000)
			C groups	0.431	\
D groups	0.194	\
			E groups	0.809	82997.73(118000)
F groups	0.008	\

9 animal experiment testing result of table

Such as can be obtained preferably the results show that Liposome Adjuvant is used alone for serum antibody OD values in table and antibody titer Immunoenhancement result can reduce the spine or inoculation times of antigen protein；And liposome has good synergistic effect with aluminium adjuvant, These adjuvants are used in conjunction with to the humidification of 18 L1 vaccine proteins institute induction body fluid immune responses of HPV better than commercialization phosphoric acid Aluminium adjuvant Adju-phos.

(3) liposome enhances ability to the cellular immunity of vaccine

Experiment purpose：Homemade Liposome Adjuvant, liposome/aluminium hydroxide joint adjuvant is investigated to use example protein for treatment The effect of the cellular immunity humidification of 16 type E7 amalgamation protein vaccines (VR111) of recombinant human papilloma virus.

Experiment material and experimental animal：Human TNF-a ELISA KIT：mabtech cat：3510-1H-6；PMA (phorbol 12-myristate 13-acetate) SIGMA P8139-1MG, are dissolved with DMSO, packing；C57BL/6 mouse, 6-8 weeks, female, 20.0 ± 2.0g；With TC-1 cell inoculation C57BL/6 mouse, E7 tumour In vivo models are established.

Immune sample：VR111 eggs are adsorbed with Liposome Adjuvant, liposome/aluminium hydroxide joint adjuvant, aluminum hydroxide adjuvant Prepared by white stoste is immunized sample, and composition such as table 10, solution buffer system is 0.008mol/L sodium dihydrogen phosphates, 0.042mol/L Disodium hydrogen phosphate, pH7.5.

The composition of 10 each adjuvant type VR111 vaccines of table

The immune sample prepared is subjected to cell in vitro immunostimulatory activity potency, Mice Body inner cell Immune-enhancing effect And the tumor inhibitory effect in animal model, investigate liposome enhances ability to the drug effect of VR111 protein vaccines.

Immunization method：

Method one：Cell in vitro is immune

It is diluted with the culture medium containing PMA (phorbol ester) is added.Sample does 82 times and is serially diluted.The sample of various concentration is added Product C and sample E, while setting 50 holes μ l/ of negative control and PMA controls.THP-1 cells are collected, appropriate cell kind is in 96 orifice plates. 37 degree are cultivated 2-3 days.Cell conditioned medium is taken, TNF-α content is detected.Testing result is shown in Table 11.

Immune sample	Operating reference product	Sample group C	Sample group E
				External relative effectivenes	\	1.075	0.927

The outer relative effectivenes of 11 protein vaccine primary liquid of table

The experimental results showed that VR111 protein sample cell in vitro immunostimulatory activities after liposome, which are added, is higher than non-stuffing The sample activity of plastid.

Method two, Mice Body inner cell are immune

Immune sample A and sample B is serially diluted.Initial concentration is 60 μ g/ml, does 63 times of dilutions.Mouse peritoneal is noted 0.5ml is penetrated, is immunized in 0 day, 14 days, takes spleen lymphocyte within 28 days, the spot of IFN-r is secreted with Elispot methods detection cell Points.Positive criterion：Spot number is more than 20, and the spot number of the background for not adding stimulant more than 4 times.Testing result It is shown in Table 12.

Albumen dosage (μ g)	Operating reference product	Sample group A	Sample group B
				30	100%	100%	100%
10	87.5%	87.5%	87.5%
				3.3333	75.00%	62.50%	50.00%
1.1111	0.00%	12.50%	25.00%
				0.3704	0%	0%	0%
0.1235	0%	0%	0%
				ED50 values (μ g)	3.070	2.960	2.916
ED50 values operating reference product/sample	\	1.037	1.053

Cell-mediated Immunity in 12 VR111 vaccine bodies of table

Increase the experimental results showed that liposome/aluminum hydroxide adjuvant type VR111 vaccine finished product sample Mice Body inner cells are immune Epistasis is not less than the sample activity of aluminum hydroxide adjuvant type VR111 vaccine finished products.

Method three, the tumor inhibitory effect in animal model

Injecting immune sample A~D gives the hypodermic injection of mouse nape part.Inoculation TC-1 cells after 48h for the first time be immunized to Medicine reinforces administration in 16 days.Every 0.5ml of injection dosage observes animal tumor growing state, different time after record inoculation daily There is the number of animals of tumour, calculates tumor formation rate, that is, the ratio between bearing animals number and every group of practical number of animals occur；Tumour is accessible Afterwards, the vernier caliper measurement tumour line of apsides is used 2 times a week, calculates gross tumor volume, i.e. gross tumor volume=(major diameter × minor axis 2)/2； Record lead for animal tumor life span.Experimental result is shown in 10~attached drawings of attached drawing 13.

Compare Mice Body in tumor formation rate, gross tumor volume, tumor killing effect, survival rate experimental result it is found that liposome is single Adjuvant and liposome/aluminium hydroxide joint adjuvant inhibit VR111 vaccine finished product sample mouse interior tumors to be not less than hydrogen The sample activity of aluminum adjuvant type VR111 vaccine finished products.

In summary three experimental results are it is found that the single adjuvant of liposome, liposome/aluminium hydroxide joint adjuvant can be improved The Study On Cellular Immune of VR111 can reduce the spine or inoculation times of antigen protein；The single adjuvant of liposome and liposome/hydrogen Aluminium oxide combines the immunoenhancement result of adjuvant not less than the effect of aluminum hydroxide adjuvant, can be used as the replacement assistant of aluminium adjuvant Agent.

Liposome Adjuvant prepared by the present invention can be used for preparing preventative and therapeutic type HPV vaccines, be follow up vaccine product Technical support is provided.Liposome itself has preferable immunoenhancement result, and liposome has with aluminium adjuvant and cooperates with work well With being used in conjunction with these adjuvants, immunological effect can be improved.Liposome is common as the replacement adjuvant of aluminium adjuvant or with aluminium adjuvant It uses, new thinking is provided for the exploitation and application of new generation vaccine.Preparation method provided by the invention has easy to operate, easy In being prepared on a large scale, avoid the advantages that organic solvent is to bioactive substance activity influence.The stability of liposome is preferable, can root According to needing project needs to optimize, it is such as suitably added the lipid composition for carrying opposite charges with vaccine, improves the absorption of vaccine Rate.Liposome itself has preferable immunoenhancement result, and liposome has good synergistic effect with aluminium adjuvant, is used in conjunction with These adjuvants can improve immunological effect.

The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention With within principle, any modification, equivalent substitution, improvement and etc. done should be included within the scope of protection of the invention god.

Claims (8)

1. a kind of method for preparing lipidosome, includes the following steps：

1) hydrogenated soya phosphatide HSPC, cholesterol CHOL, 1,2- dioleoyl oxygroup trimethyl ammonium chlorides DOTAP-Cl, dimension life are weighed Mixture is made in the ratio of mass fraction 70~90: 0~30: 10~20: 0.5 in plain VE；

2) mixture described in step 1 is dissolved in obtained in chloroform/methanol solvent lipoid organic solution it is spare, the chloroform/ Methanol solvate is made of chloroform, methanol and the ultra-pure water that volume ratio is 24: 72: 4-5.1；

3) it weighs water-soluble powder shape solid support to be placed in the round-bottomed flask of 250ml, adds the organic molten of above-mentioned lipoid Liquid stirs evenly, and is placed on Rotary Evaporators rotation under conditions of ice water bath constant temperature and boils off organic solvent, wherein the solid The quality of support is 5 times of the mixture quality, includes the one or more of lactose, sucrose, mannitol, sodium chloride；

4) round-bottomed flask is removed, solid sample is taken out, is placed at -18 DEG C and is sealed.

2. the method as described in claim 1, which is characterized in that in the mixture mass percentage of cholesterol be 0%, 10%, 20% or 30%.

3. a kind of method for preparing lipidosome, includes the following steps：

1) hydrogenated soya phosphatide HSPC, cholesterol CHOL, 1,2- distearoyl phosphatidyl glycerol sodium salts DSPG-Na, dimension life are weighed Mixture is made in the ratio of mass fraction 70~90: 0~30: 10~20: 0.5 in plain VE；

2) mixture described in step 1 is dissolved in obtained in chloroform/methanol solvent lipoid organic solution it is spare, the chloroform/ Methanol solvate is made of chloroform, methanol and the ultra-pure water that volume ratio is 24: 72: 4-5.1；

3) it weighs water-soluble powder shape solid support to be placed in the round-bottomed flask of 250ml, adds the organic molten of above-mentioned lipoid Liquid stirs evenly, and is placed on Rotary Evaporators rotation under conditions of ice water bath constant temperature and boils off organic solvent, wherein the solid The quality of support is 5 times of the mixture quality, includes the one or more of lactose, sucrose, mannitol, sodium chloride；

4) round-bottomed flask is removed, solid sample is taken out, is placed at -18 DEG C and is sealed.

4. method as claimed in claim 3, which is characterized in that in the mixture mass percentage of cholesterol be 0%, 10%, 20% or 30%.

5. liposome prepared by a kind of method in 1-4 using claim is the method for preparing Liposome Adjuvant, including as follows Step：

1) it weighs proliposome obtained according to the method described above to be added in conical flask, suitable ultra-pure water or buffer solution is added In, gently oscillation is allowed to disperse, and obtains the thick suspension of blank liposome；

2) the thick suspension of blank liposome is added in high pressure homogenizer and carries out high speed shear processing and high-pressure homogeneous processing, warp Homogeneous 3-5 times under 0bar pressure continues to increase pressure to 300bar homogeneous 3-5 times to get to liposomal samples；

3) moist heat sterilization under the conditions of 115 DEG C of the liposomal samples of preparation progress, 30min is obtained into final Liposome Adjuvant.

6. method as claimed in claim 5, wherein the buffer Final concentration is 0.486mol/L sodium chloride, 0.01mol/L Histidine, 0.01% Tween 80, pH6.2.

7. method as claimed in claim 5, wherein the buffer Final concentration is 0.154mol/L sodium chloride, 0.01mol/L Histidine, 0.01% Tween 80, pH6.2.

8. method as claimed in claim 5, wherein the buffer Final concentration be 0.041mol/L sodium dihydrogen phosphates, 0.009mol/L disodium hydrogen phosphates, pH6.2.