CN102068698A - Nanometer vaccine and preparation method thereof - Google Patents
Nanometer vaccine and preparation method thereof Download PDFInfo
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- CN102068698A CN102068698A CN2009101895408A CN200910189540A CN102068698A CN 102068698 A CN102068698 A CN 102068698A CN 2009101895408 A CN2009101895408 A CN 2009101895408A CN 200910189540 A CN200910189540 A CN 200910189540A CN 102068698 A CN102068698 A CN 102068698A
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Abstract
The invention provides a nanometer vaccine and a preparation method thereof. A new mannose glycosylated-cationic liposome complex can be used as a vaccine vector and can be applied to the vaccine. In the new liposome complex, neutral phospholipid and mannose are mixed into cationic lipid, so the immunologic adjuvant effect and the antigen-presenting cell targeting property of a liposome are improved, and the cytotoxic effect of the liposome is reduced obviously. The invention provides a novel high-efficiency vaccine vector and an adjuvant which do not have side effects and can improve the immunologic effect of the vaccine obviously.
Description
[technical field]
The present invention relates to a kind of vaccine and preparation method thereof, particularly a kind of liposome that adopts is as vaccine of carrier and preparation method thereof.
The present invention relates to a kind of vaccine carrier again.
The invention still further relates to a kind of liposome complex that can be applicable to vaccine carrier.
[background technology]
Vaccine is the important means that prevention and control infectious disease take place and develop.Immunological adjuvant (claiming the nonspecific immunity proliferant agent again) then is constituent indispensable in the vaccine, can promote vaccine-induced specific immune response effectively.Along with the develop rapidly of vaccine research, novel recombinant vaccine just is being widely used in the development of vaccine.This vaccine has advantages such as purity height, high specificity, but because its molecule is little, and immunogenicity is relatively poor relatively, presses in conjunction with effective immunological adjuvant, to improve the immune efficacy of vaccine.The recent decades in past, people find and have developed various novel immunological adjuvants under study for action, yet factors such as potential safety and stability have restricted promoting the use of of immunological adjuvant greatly.
Elaioplast nanometer particle is the spheric entity that is formed by phospholipid bilayer shell parcel aqueous core, and its similar biomembrane is a kind of good biocompatibility and nontoxic nano material.It can seal water solublity and fat-soluble medicine, has to reduce drug dose, slow release, and advantage such as targeting release medicine, thereby is widely used in the exploitation of nanometer antitumor drug.In addition; nanometer liposome also is a kind of good antigen vectors, not only can wrap up the antigen and the immunological adjuvant of series of physicochemical different in kind, and the protected protein polypeptide antigen is not degraded; can also promote antigen-presenting cell to engulf and present, improve the specific immune response of body antigenic.Based on above these advantages, elaioplast nanometer particle is used for developments such as bacterial vaccine, viral vaccine, parasiticide vaccine and anti-tumor vaccine just gradually as a kind of novel vaccine carrier.
At present the most frequently used liposome protein vaccine carrier is to be main component with neutral phospholipid and cholesterol.Though the neutral elaioplast nanometer particle of this class is a kind of good protein carrier, can not directly promote immunoreation.In addition, not modified liposome targeting is poor, can not be absorbed by antigen-presenting cell (as dendritic cell and mononuclear phagocyte) effectively.
It is carrier that existing liposome bacterin adopts the neutral fat plastid more, yet because neutral fat plastid itself can not directly promote immunoreation and targeting poor, has therefore restricted the immune efficient of vaccine.Can promote immunoreation and antigen-presenting cell targeting though adopt cation lipid to replace neutral phospholipid, too high surface charge makes it have significant cytotoxicity.
In sum, existing liposome is applied to vaccine carrier, and disappearance is difficult to satisfy the demands to some extent.
[summary of the invention]
In view of this, be necessary defective at existing liposome bacterin carrier, a kind of cation lipid nanocrystal composition of new mannose groupization is proposed, can be used as vaccine carrier and be applied to vaccine, this new liposome complex mixes neutral phospholipid and mannose in cation lipid, the immunoadjuvant function and the antigen-presenting cell targeting of liposome have not only been improved, and obviously lower its cytotoxicity, a kind of novel vaccine carrier that efficiently has no side effect and adjuvant are provided, can have significantly strengthened the immune effect of vaccine.
In order to realize above-mentioned goal of the invention, technical scheme of the present invention is as follows:
The invention provides a kind of cation lipid nanocrystal composition of mannose groupization, comprise the cationic lipid that is mixed in proportion, neutral phospholipid and mannoside, the mol ratio of cationic lipid and neutral phospholipid is 19: 1 to 2: 18, mannoside with comprise that the mol ratio of the liposome of cationic lipid and neutral phospholipid is 2: 98 to 20: 80.
Preferably, cationic lipid is to have the positive charge amphiphatic molecule, comprise the head and the hydrophobic tail that have positive charge, positively charged polar head contains amine groups such as amino, quaternary ammonium salt and polyamines, and hydrophobic tail comprises saturated or unsaturated fatty acid chain or cholesterol ring.
Preferably; cationic lipid comprises two ten alkyl dimethyl ammonium bromide (Didecyldimethylammoniumbromide; be called for short DDAB); two oleoyl trimethyl ammonium propane (ioleoyltrimethylammoniumpropane; be called for short DOTAP); two oleoyl propyl group chlorination trimethylammonium (dioleoylpropyltrimethylammonium; be called for short DOTMA); dimethylaminoethyl carbamyl-cholesterol (3-(N-(N '; the carbamoyl of N '-Dimethylaminoethane)) cholesterol; be called for short DC-Chol) and dioleoyl phospholipid phatidylcholine ether (dioleyl ether phosphatidylcholine, abbreviation DOEPC) in one or more combination.
Preferably, neutral phospholipid is amphiphatic molecule, comprises the hydrophilic head of substituted radical (containing ammonia alkali or the alcohols) formation that phosphoric acid links to each other and the hydrophobic tail that fatty acid chain constitutes, and the surface electrostatic lotus is substantially near 0.
Preferably, neutral phospholipid comprises dioleoyl phospholipid phatidylcholine (Dioleoylphatidylcholine, be called for short DOPC), two myristoyl phosphatidylcholine (dimyristoylphosphatidylcholine, be called for short DMPC), dilinoleoylphosphatidylcholine (dilinoleoylphosphatidylcholine, be called for short DLPC), dipalmitoyl phosphatidyl choline (Dipalmitoylphosphatidylcholine, be called for short DPPC), distearoyl phosphatidylcholine (distearoylphosphatidylcholine, be called for short DSPC), two myristoyl phosphoethanolamine (Dimyristoylphosphatidylethanolamine, DMPE), two palmityl PHOSPHATIDYL ETHANOLAMINE (Dipalmitoylphosphatidylethanolamine, be called for short DPPE) and DSPE (distearoylphosphatidylethanolamine, abbreviation DSPE) in one or more combination.
Preferably, mannoside comprises (Methyl-α-D-mannopyranoside), 4-nitrobenzophenone-α-D-mannopyranose glycosides (4-Nitrophenyl-α-D-mannopyranoside), 4-methyl umbelliferone base-α-D-mannopyranose glycosides (4-Methylumbelliferyl-α-D-mannopyranoside) and the 4-aminophenyl-D-mannoside (combination of one or more among the 4-Aminophenyl α-D-mannopyranoside) of methyl D-mannoside.
The present invention provides a kind of vaccine carrier with immunological adjuvant effect again, the parcel vaccine antigen, vaccine carrier is the cation lipid nanocrystal composition of mannose groupization, comprise the cationic lipid that is mixed in proportion, neutral phospholipid and mannoside, the mol ratio of cationic lipid and neutral phospholipid is 19: 1 to 2: 18, mannoside with comprise that the mol ratio of the liposome of cationic lipid and neutral phospholipid is 2: 98 to 20: 80.
Preferably, cationic lipid is to have the positive charge amphiphatic molecule, comprise the head and the hydrophobic tail that have positive charge, positively charged polar head contains amine groups such as amino, quaternary ammonium salt and polyamines, and hydrophobic tail comprises saturated or unsaturated fatty acid chain or cholesterol ring.
Preferably, cationic lipid comprises one or more the combination among DDAB, DOTAP, DODAP, DOTMA and the DOEPC.
Preferably, neutral phospholipid is amphiphatic molecule, comprises the hydrophilic head of substituted radical (containing ammonia alkali or the alcohols) formation that phosphoric acid links to each other and the hydrophobic tail that fatty acid chain constitutes, and the surface electrostatic lotus is substantially near 0.
Preferably, neutral phospholipid comprises one or more the combination among DOPC, DMPC, DLPC, DPPC, DSPC, DMPE, DPPE and the DSPE.
Preferably, mannoside comprises one or more the combination in methyl D-mannoside, 4-nitrobenzophenone-α-D-mannopyranose glycosides, 4-methyl umbelliferone base-α-D-mannopyranose glycosides and the 4-aminophenyl-D-mannoside.
The present invention provides a kind of nano vaccine again, the liposome that comprises vaccine antigen and parcel vaccine antigen, liposome adopts the cation lipid nanocrystal composition of mannose groupization, comprise the cationic lipid that is mixed in proportion, neutral phospholipid and mannoside, the mol ratio of cationic lipid and neutral phospholipid is 19: 1 to 2: 18, mannoside with comprise that the mol ratio of the liposome of cationic lipid and neutral phospholipid is 2: 98 to 20: 80.
Preferably, cationic lipid is to have the positive charge amphiphatic molecule, comprise the head and the hydrophobic tail that have positive charge, positively charged polar head contains amine groups such as amino, quaternary ammonium salt and polyamines, and hydrophobic tail comprises saturated or unsaturated fatty acid chain or cholesterol ring.
Preferably, cationic lipid comprises one or more the combination among DDAB, DOTAP, DODAP, DOTMA and the DOEPC.
Preferably, described neutral phospholipid is amphiphatic molecule, comprises the hydrophilic head of substituted radical (containing ammonia alkali or the alcohols) formation that phosphoric acid links to each other and the hydrophobic tail that fatty acid chain constitutes, and the surface electrostatic lotus is substantially near 0.
Preferably, neutral phospholipid comprises one or more the combination among DOPC, DMPC, DLPC, DPPC, DSPC, DMPE, DPPE and the DSPE.
Preferably, mannoside comprises one or more the combination in methyl D-mannoside, 4-nitrobenzophenone-α-D-mannopyranose glycosides, 4-methyl umbelliferone base-α-D-mannopyranose glycosides and the 4-aminophenyl-D-mannoside.
Preferably, antigen is each albuminoid, polypeptide, polysaccharide, DNA or RNA, and described antigen comes from virus, antibacterial, other microorganisms, tumor or engineered protein product.
Preferably, the immunization ways of vaccine is subcutaneous injection or intramuscular injection.
The present invention also provides a kind of manufacture method with vaccine carrier of immunological adjuvant effect, comprises the steps: to get cationic lipid, neutral phospholipid and mannoside and is dissolved in chloroform-methanol respectively, is mixed in proportion then to be placed in the container; Mixture in the container is dried up into the layer of even thin film, then carry out vacuum drying treatment; Add and contain antigenic buffer, carry out aquation then and handle and the water-bath supersound process, pushed polycarbonate membrane, cold preservation is placed standby.
Preferably, the volume ratio of chloroform and methanol is 2: 1 in the chloroform-methanol.
Preferably, the mixture in the container dries up with stable nitrogen current rotation.
Preferably, vacuum drying treatment is that vacuum drying spends the night in vacuum drying oven, and adding in second day contains antigenic buffer.
Preferably, add contain antigenic phosphate (Phosphate buffered saline is called for short PBS) buffer after, be positioned over 4 ℃ of aquations 12 hours, ultrasonic 10 minutes of water-bath, it is standby to push twice, 4 ℃ of placement of polycarbonate membrane.
Because adopt above-mentioned technical scheme, beneficial effect of the present invention is as follows:
The cation lipid nanocrystal composition of mannose groupization of the present invention is added a certain proportion of neutral phospholipid and mannose in cationic lipid, synthetic surface carries the cation lipid nanocrystal composition of positive charge and mannose group, not only improve the liposome targeting antigen greatly and be the ability of delivery cell, and significantly strengthened the immunoadjuvant function of liposome.With traditional neutral lipid bulk phase ratio, very big improvement has been arranged on function, cation lipid nanocrystal composition of the present invention not only has powerful immunological adjuvant effect, can also promote immunocyte that antigenic picked-up is presented more effectively, promotes the immune effect of vaccine.Mix the cytotoxicity that the neutral phospholipid composition of certain proportion can also obviously lower cationic lipid in addition, make vaccine safer.
The cation lipid nanocrystal composition of mannose groupization of the present invention is applied to vaccine carrier, can wrap up one or more antigen simultaneously, not only can promote antigenic targeted delivery, and significantly improve the immunoreation of antigen induction, strengthen the immune efficacy of vaccine, and safe without toxic side effect.
Vaccine of the present invention as vaccine carrier, improves the immune efficacy of vaccine with a kind of novel mannose group cation lipid nanocrystal composition, makes up nano vaccine safely and efficiently.
[description of drawings]
For further understanding feature of the present invention and technology contents, see also following relevant accompanying drawing of the present invention, yet appended graphic reference and the explanation usefulness of only providing not is to be used for the present invention is limited.
Fig. 1 is that the cation lipid nanocrystal composition with mannose groupization of the present invention is the structural representation of the nano vaccine of carrier;
Fig. 2 is the flow chart of the preparation method of nano vaccine of the present invention;
Fig. 3 is that cation lipid nanocrystal composition of the present invention is induced monocyte activation and expressed CD86 molecule comparison diagram;
Fig. 4 is that cation lipid nanocrystal composition of the present invention induces dendritic cell to express the CD83 comparison diagram;
Fig. 5 is that cation lipid nanocrystal composition of the present invention promotes the picked-up comparison diagram of macrophage to antigen (BSA-FITC);
Fig. 6 is the influence comparison diagram of cation lipid nanocrystal composition to the monocyte survival rate.
[specific embodiment]
Below in conjunction with accompanying drawing,, will make technical scheme of the present invention and other beneficial effects apparent by the specific embodiment of the present invention is described in detail.
Embodiment 1: the cation lipid nanocrystal composition of mannose groupization
The cation lipid nanocrystal composition that the present invention proposes to adopt mannose groupization makes up nano vaccine safely and efficiently as novel vaccine carrier.
The structure of the cation lipid nanocrystal composition of mannose groupization is referring to Fig. 1, comprise the cationic lipid that is mixed in proportion, neutral phospholipid and mannoside, the mol ratio of cationic lipid and neutral phospholipid is 19: 1 to 2: 18, mannoside with comprise that the mol ratio of the liposome of cationic lipid and neutral phospholipid is 2: 98 to 20: 80.
Cationic lipid is to have the positive charge amphiphatic molecule, comprise the head and the hydrophobic tail that have positive charge, positively charged polar head contains amine groups such as amino, quaternary ammonium salt and polyamines, and hydrophobic tail comprises saturated or unsaturated fatty acid chain or cholesterol ring, molecular backbone is a glycerol, second hydroxyl of glycerol is positively charged quaternary ammonium salt, and two hydroxyls are by saturated or unsaturated fatty acid esterization in addition; Cationic lipid comprises one or more the combination among DDAB, DOTAP, DODAP, DOTMA and the DOEPC.
Neutral phospholipid is amphiphatic molecule, comprise the hydrophilic head of substituted radical (containing ammonia alkali or the alcohols) formation that phosphoric acid links to each other and the hydrophobic tail that fatty acid chain constitutes, the surface electrostatic lotus is substantially near 0, molecular backbone is a glycerol, the 3rd hydroxyl of glycerol is by Phosphation, two other hydroxyl is by saturated or unsaturated fatty acid esterization, and phosphate group links to each other with choline or cholamine group again; Neutral phospholipid comprises one or more the combination among DOPC, DMPC, DLPC, DPPC, DSPC, DMPE, DPPE and the DSPE.
Mannoside comprises one or more the combination in methyl D-mannoside, 4-nitrobenzophenone-α-D-mannopyranose glycosides, 4-methyl umbelliferone base-α-D-mannopyranose glycosides and the 4-aminophenyl-D-mannoside.
Embodiment 2: the vaccine carrier with immunological adjuvant effect
Vaccine carrier parcel vaccine antigen with immunological adjuvant effect of the present invention, vaccine carrier is the cation lipid nanocrystal composition of mannose groupization.
The structure of the cation lipid nanocrystal composition of mannose groupization is referring to Fig. 1, comprise the cationic lipid that is mixed in proportion, neutral phospholipid and mannoside, the mol ratio of cationic lipid and neutral phospholipid is 19: 1 to 2: 18, mannoside with comprise that the mol ratio of the liposome of cationic lipid and neutral phospholipid is 2: 98 to 20: 80.Cationic lipid is to have the positive charge amphiphatic molecule, comprise the head and the hydrophobic tail that have positive charge, positively charged polar head contains amine groups such as amino, quaternary ammonium salt and polyamines, and hydrophobic tail comprises saturated or unsaturated fatty acid chain or cholesterol ring, it is the amphiphatic molecule that positive charge is carried on the surface, molecular backbone is a glycerol, and second hydroxyl of glycerol is positively charged quaternary ammonium salt, and two hydroxyls are by saturated or unsaturated fatty acid esterization in addition; Cationic lipid comprises one or more the combination among DDAB, DOTAP, DODAP, DOTMA and the DOEPC.Neutral phospholipid is amphiphatic molecule, comprise the hydrophilic head of substituted radical (containing ammonia alkali or the alcohols) formation that phosphoric acid links to each other and the hydrophobic tail that fatty acid chain constitutes, the surface electrostatic lotus is substantially near 0, molecular backbone is a glycerol, the 3rd hydroxyl of glycerol is by Phosphation, two other hydroxyl is by saturated or unsaturated fatty acid esterization, and phosphate group links to each other with choline or cholamine group again; Neutral phospholipid comprises one or more the combination among DOPC, DMPC, DLPC, DPPC, DSPC, DMPE, DPPE and the DSPE.Mannoside comprises one or more the combination in methyl D-mannoside, 4-nitrobenzophenone-α-D-mannopyranose glycosides, 4-methyl umbelliferone base-α-D-mannopyranose glycosides and the 4-aminophenyl-D-mannoside.
Embodiment 3: nano vaccine
Nano vaccine of the present invention, the liposome that comprises vaccine antigen and parcel vaccine antigen, liposome adopts the cation lipid nanocrystal composition of mannose groupization, antigen is each albuminoid, polypeptide, polysaccharide, DNA or RNA, and antigen comes from virus, antibacterial, other microorganisms, tumor or engineered protein product.
The structure of the cation lipid nanocrystal composition of mannose groupization is referring to Fig. 1, comprise the cationic lipid that is mixed in proportion, neutral phospholipid and mannoside, the mol ratio of cationic lipid and neutral phospholipid is 19: 1 to 2: 18, mannoside with comprise that the mol ratio of the liposome of cationic lipid and neutral phospholipid is 2: 98 to 20: 80.Cationic lipid is to have the positive charge amphiphatic molecule, comprise the head and the hydrophobic tail that have positive charge, positively charged polar head contains amine groups such as amino, quaternary ammonium salt and polyamines, and hydrophobic tail comprises saturated or unsaturated fatty acid chain or cholesterol ring, it is the amphiphatic molecule that positive charge is carried on the surface, molecular backbone is a glycerol, and second hydroxyl of glycerol is positively charged quaternary ammonium salt, and two hydroxyls are by saturated or unsaturated fatty acid esterization in addition; Cationic lipid comprises one or more the combination among DDAB, DOTAP, DODAP, DOTMA and the DOEPC.Neutral phospholipid is amphiphatic molecule, comprise the hydrophilic head of substituted radical (containing ammonia alkali or the alcohols) formation that phosphoric acid links to each other and the hydrophobic tail that fatty acid chain constitutes, the surface electrostatic lotus is substantially near 0, molecular backbone is a glycerol, the 3rd hydroxyl of glycerol is by Phosphation, two other hydroxyl is by saturated or unsaturated fatty acid esterization, and phosphate group links to each other with choline or cholamine group again; Neutral phospholipid comprises one or more the combination among DOPC, DMPC, DLPC, DPPC, DSPC, DMPE, DPPE and the DSPE.Mannoside comprises one or more the combination in methyl D-mannoside, 4-nitrobenzophenone-α-D-mannopyranose glycosides, 4-methyl umbelliferone base-α-D-mannopyranose glycosides and the 4-aminophenyl-D-mannoside.
This vaccine based on the cation lipid nanocrystal composition can adopt methods such as subcutaneous injection and intramuscular injection that body is carried out immunity.
Embodiment 4: the preparation method of nano vaccine
Referring to Fig. 2, adopt concrete preparation method as follows:
Take by weighing a certain amount of cationic lipid, neutral phospholipid and mannoside and be dissolved in chloroform-methanol (2: 1) respectively, mix by a certain percentage then, place round-bottomed flask.Dry up with stable nitrogen current rotation, make it into the layer of even thin film, vacuum drying spends the night in the vacuum drying oven and be placed on.Adding in second day contains antigenic PBS buffer, is positioned over 4 ℃ of aquations 12 hours.After ultrasonic 10 minutes of water-bath, it is standby to push twice, 4 ℃ of placement of polycarbonate membrane.
Experiment shows, the cation lipid nanocrystal composition can significantly be induced monocytes CD86 (a kind of costimulatory molecules), point out this complex to promote monocytic activation, and the neutral fat plastid does not have obvious influence to monocytic activity, referring to Fig. 3, three kinds of embodiment of the present invention shown in the figure, promptly the mol ratio of cationic lipid and neutral phospholipid is respectively 19: 1,10: 10 and 2: 18 o'clock the experimental data and the contrast of contrasting data.
The cation lipid nanocrystal composition also significantly induces dendritic cell to express CD83, and the maturation of pointing out this complex to promote dendritic cell is referring to Fig. 4.
In addition, the cation lipid nanocrystal composition is the carrier of antigen (BSA-FITC), significantly improved antigen-presenting cell (mouse macrophage) to antigenic picked-up, referring to Fig. 5, three kinds of embodiment of the present invention shown in the figure, promptly the mol ratio of cationic lipid and neutral phospholipid is respectively 19: 1,10: 10 and 2: 18 o'clock the experimental data and the contrast of contrasting data.Compare with free antigen, up to more than 10 times, this result shows that the cation lipid nanocrystal composition is a kind of antigen vectors efficiently to cation lipid nanocrystal composition raising macrophage to antigenic phagocytic rate.
The result of Fig. 6 shows that simple use cationic lipid obviously reduces monocytic survival rate, point out it to have certain cytotoxicity, three kinds of embodiment of the present invention shown in the figure, promptly the mol ratio of cationic lipid and neutral phospholipid is respectively 19: 1,10: 10 and 2: 18 o'clock the experimental data and the contrast of contrasting data.In cationic lipid, mix neutral phospholipid and make the cytotoxicity that the cation lipid nanocrystal composition then alleviates cationic lipid greatly, monocytic survival rate is not had obvious influence.
The cation lipid nanocrystal composition of mannose groupization of the present invention not only can be used as vaccine carrier, can also improve the immunologic enhancement of adjuvant as the carrier of immunological adjuvant, reduces the side effect that adjuvant brings.The cation lipid nanocrystal composition of mannose groupization of the present invention not only can coating antigen, can also unite other adjuvants of parcel, with the immune effect of further raising vaccine.
Be understandable that, for those of ordinary skills, can be equal to replacement or change according to technical scheme of the present invention and inventive concept thereof, and all these changes or replacement all should belong to the protection domain of the appended claim of the present invention.
Claims (24)
1. the cation lipid nanocrystal composition of a mannose groupization, it is characterized in that, comprise the cationic lipid that is mixed in proportion, neutral phospholipid and mannoside, the mol ratio of cationic lipid and neutral phospholipid is 19: 1 to 2: 18, mannoside with comprise that the mol ratio of the liposome of cationic lipid and neutral phospholipid is 2: 98 to 20: 80.
2. the cation lipid nanocrystal composition of mannose groupization according to claim 1, it is characterized in that, described cationic lipid is to have the positive charge amphiphatic molecule, comprise the head and the hydrophobic tail that have positive charge, positively charged polar head contains amine groups such as amino, quaternary ammonium salt and polyamines, and hydrophobic tail comprises saturated or unsaturated fatty acid chain or cholesterol ring.
3. the cation lipid nanocrystal composition of mannose groupization according to claim 2; it is characterized in that described cationic lipid comprises one or more the combination in two ten alkyl dimethyl ammonium bromide, two oleoyl trimethyl ammonium propane, two oleoyl propyl group chlorination trimethylammoniums, dimethylaminoethyl carbamyl-cholesterol and the dioleoyl phospholipid phatidylcholine ether.
4. according to the cation lipid nanocrystal composition of each described mannose groupization in the claim 1 to 3, it is characterized in that, described neutral phospholipid is amphiphatic molecule, comprise hydrophilic head that contains ammonia alkali or alcohols substituted radical formation that phosphoric acid links to each other and the hydrophobic tail that fatty acid chain constitutes, the surface electrostatic lotus is substantially near 0.
5. the cation lipid nanocrystal composition of mannose groupization according to claim 4, it is characterized in that described neutral phospholipid comprises one or more the combination in dioleoyl phospholipid phatidylcholine, two myristoyl phosphatidylcholines, dilinoleoylphosphatidylcholine, dipalmitoyl phosphatidyl choline, distearoyl phosphatidylcholine, two myristoyl phosphoethanolamines, two palmityl PHOSPHATIDYL ETHANOLAMINE and the DSPE.
6. the cation lipid nanocrystal composition of mannose groupization according to claim 4, it is characterized in that described mannoside comprises one or more the combination in methyl D-mannoside, 4-nitrobenzophenone-α-D-mannopyranose glycosides, 4-methyl umbelliferone base-α-D-mannopyranose glycosides and the 4-aminophenyl-D-mannoside.
7. vaccine carrier with immunological adjuvant effect, the parcel vaccine antigen, it is characterized in that, described vaccine carrier is the cation lipid nanocrystal composition of mannose groupization, comprise the cationic lipid that is mixed in proportion, neutral phospholipid and mannoside, the mol ratio of cationic lipid and neutral phospholipid is 19: 1 to 2: 18, mannoside with comprise that the mol ratio of the liposome of cationic lipid and neutral phospholipid is 2: 98 to 20: 80.
8. the vaccine carrier with immunological adjuvant effect according to claim 7, it is characterized in that, described cationic lipid is to have the positive charge amphiphatic molecule, comprise the head and the hydrophobic tail that have positive charge, positively charged polar head contains amine groups such as amino, quaternary ammonium salt and polyamines, and hydrophobic tail comprises saturated or unsaturated fatty acid chain or cholesterol ring.
9. the vaccine carrier with immunological adjuvant effect according to claim 8; it is characterized in that described cationic lipid comprises one or more the combination in two ten alkyl dimethyl ammonium bromide, two oleoyl trimethyl ammonium propane, two oleoyl propyl group chlorination trimethylammoniums, dimethylaminoethyl carbamyl-cholesterol and the dioleoyl phospholipid phatidylcholine ether.
10. according to each described vaccine carrier in the claim 7 to 9 with immunological adjuvant effect, it is characterized in that, described neutral phospholipid is amphiphatic molecule, comprise hydrophilic head that contains ammonia alkali or alcohols substituted radical formation that phosphoric acid links to each other and the hydrophobic tail that fatty acid chain constitutes, the surface electrostatic lotus is substantially near 0.
11. the vaccine carrier with immunological adjuvant effect according to claim 10, it is characterized in that described neutral phospholipid comprises one or more the combination in dioleoyl phospholipid phatidylcholine, two myristoyl phosphatidylcholines, dilinoleoylphosphatidylcholine, dipalmitoyl phosphatidyl choline, distearoyl phosphatidylcholine, two myristoyl phosphoethanolamines, two palmityl PHOSPHATIDYL ETHANOLAMINE and the DSPE.
12. the vaccine carrier with immunological adjuvant effect according to claim 10, it is characterized in that described mannoside comprises one or more the combination in methyl D-mannoside, 4-nitrobenzophenone-α-D-mannopyranose glycosides, 4-methyl umbelliferone base-α-D-mannopyranose glycosides and the 4-aminophenyl-D-mannoside.
13. nano vaccine, the liposome that comprises vaccine antigen and parcel vaccine antigen, it is characterized in that, described liposome adopts the cation lipid nanocrystal composition of mannose groupization, comprise the cationic lipid that is mixed in proportion, neutral phospholipid and mannoside, the mol ratio of cationic lipid and neutral phospholipid is 19: 1 to 2: 18, mannoside with comprise that the mol ratio of the liposome of cationic lipid and neutral phospholipid is 2: 98 to 20: 80.
14. nano vaccine according to claim 13, it is characterized in that, described cationic lipid is to have the positive charge amphiphatic molecule, comprise the head and the hydrophobic tail that have positive charge, positively charged polar head contains amine groups such as amino, quaternary ammonium salt and polyamines, and hydrophobic tail comprises saturated or unsaturated fatty acid chain or cholesterol ring.
15. nano vaccine according to claim 14; it is characterized in that described positive cations fat comprises one or more the combination in two ten alkyl dimethyl ammonium bromide, two oleoyl trimethyl ammonium propane, two oleoyl propyl group chlorination trimethylammoniums, dimethylaminoethyl carbamyl-cholesterol and the dioleoyl phospholipid phatidylcholine ether.
16. according to each described nano vaccine in the claim 13 to 15, it is characterized in that, described neutral phospholipid is amphiphatic molecule, comprises hydrophilic head that contains ammonia alkali or alcohols substituted radical formation that phosphoric acid links to each other and the hydrophobic tail that fatty acid chain constitutes, and the surface electrostatic lotus is substantially near 0.
17. nano vaccine according to claim 16, it is characterized in that described neutral phospholipid comprises one or more the combination in dioleoyl phospholipid phatidylcholine, two myristoyl phosphatidylcholines, dilinoleoylphosphatidylcholine, dipalmitoyl phosphatidyl choline, distearoyl phosphatidylcholine, two myristoyl phosphoethanolamines, two palmityl PHOSPHATIDYL ETHANOLAMINE and the DSPE.
18. nano vaccine according to claim 16, it is characterized in that described mannoside comprises one or more the combination in methyl D-mannoside, 4-nitrobenzophenone-α-D-mannopyranose glycosides, 4-methyl umbelliferone base-α-D-mannopyranose glycosides and the 4-aminophenyl-D-mannoside.
19. nano vaccine according to claim 16 is characterized in that, described antigen is each albuminoid, polypeptide, polysaccharide, DNA or RNA, and described antigen comes from virus, antibacterial, other microorganisms, tumor or engineered protein product.
20. the preparation method of nano vaccine according to claim 13 comprises the steps:
Get cationic lipid, neutral phospholipid and mannoside and be dissolved in chloroform-methanol respectively, be mixed in proportion then and be placed in the container;
Mixture in the container is dried up into the layer of even thin film, then carry out vacuum drying treatment;
Add and contain antigenic buffer, carry out aquation then and handle and the water-bath supersound process, pushed polycarbonate membrane, place standby.
21. method according to claim 20 is characterized in that, the volume ratio of chloroform and methanol is 2: 1 in the described chloroform-methanol.
22. the method according to claim 20 is characterized in that, the mixture in the described container dries up with stable nitrogen current rotation.
23. the method according to claim 20 is characterized in that, vacuum drying treatment is that vacuum drying spends the night in vacuum drying oven, and adding in second day contains antigenic PBS buffer.
24. the method according to claim 20 is characterized in that, add contain antigenic phosphate buffer after, be positioned over 4 ℃ of aquations 12 hours, ultrasonic 10 minutes of water-bath, it is standby to push twice, 4 ℃ of placement of polycarbonate membrane.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102973506A (en) * | 2011-09-05 | 2013-03-20 | 中国科学院深圳先进技术研究院 | Cationic liposome and preparation method thereof |
| CN103405386A (en) * | 2012-09-21 | 2013-11-27 | 上海泽润生物科技有限公司 | Liposome preparation method and method for preparing liposome adjuvant |
| CN105106116A (en) * | 2015-09-21 | 2015-12-02 | 中国科学院过程工程研究所 | Lipidosome nucleic acid vaccine adjuvant as well as preparation method and application thereof |
| CN107236052A (en) * | 2012-03-15 | 2017-10-10 | 方济各安吉利克化学联合股份有限公司 | Glycogen base cationic polymer |
| CN109125740A (en) * | 2017-06-28 | 2019-01-04 | 四川大学 | A kind of novel tumor vaccine and application thereof |
| CN111298128A (en) * | 2019-12-25 | 2020-06-19 | 中国科学院长春应用化学研究所 | Efficient targeting nano vaccine carrier and preparation method thereof, and targeting nano vaccine and preparation method thereof |
| WO2021169484A1 (en) * | 2020-02-26 | 2021-09-02 | 浙江大学 | Nanovaccine and preparation method therefor |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102973506A (en) * | 2011-09-05 | 2013-03-20 | 中国科学院深圳先进技术研究院 | Cationic liposome and preparation method thereof |
| CN107236052A (en) * | 2012-03-15 | 2017-10-10 | 方济各安吉利克化学联合股份有限公司 | Glycogen base cationic polymer |
| CN103405386A (en) * | 2012-09-21 | 2013-11-27 | 上海泽润生物科技有限公司 | Liposome preparation method and method for preparing liposome adjuvant |
| CN103405386B (en) * | 2012-09-21 | 2016-05-18 | 上海泽润生物科技有限公司 | A kind of preparation method of liposome and the method for making Liposome Adjuvant |
| CN105106116A (en) * | 2015-09-21 | 2015-12-02 | 中国科学院过程工程研究所 | Lipidosome nucleic acid vaccine adjuvant as well as preparation method and application thereof |
| CN109125740A (en) * | 2017-06-28 | 2019-01-04 | 四川大学 | A kind of novel tumor vaccine and application thereof |
| CN111298128A (en) * | 2019-12-25 | 2020-06-19 | 中国科学院长春应用化学研究所 | Efficient targeting nano vaccine carrier and preparation method thereof, and targeting nano vaccine and preparation method thereof |
| CN111298128B (en) * | 2019-12-25 | 2021-07-02 | 中国科学院长春应用化学研究所 | Efficient targeting nano vaccine carrier and preparation method thereof, and targeting nano vaccine and preparation method thereof |
| WO2021169484A1 (en) * | 2020-02-26 | 2021-09-02 | 浙江大学 | Nanovaccine and preparation method therefor |
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