CN103372217B - Polymer nano carrier preparation as well as preparation method and application thereof - Google Patents
Polymer nano carrier preparation as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a polymer nano carrier preparation as well as a preparation method and an application thereof. The nano carrier preparation employs a nano carrier micelle of a three-layer structure to carry DNA (Deoxyribonucleic Acid) antigen, protein antigen, polypeptide antigen and immunologic adjuvant. The polymer nano carrier preparation is good in stability and immunogenicity, and can not only prompt ingestion of antigen presentation cells such as dendritic cells for the antigen, but also remarkably induce organisms to make specific immune response.
Description
[technical field]
The present invention relates to nanosecond medical science field, relate in particular to a kind of polymer nanoparticle drug carriers preparation and its preparation method and application.
[background technology]
Vaccine is prevention and controls the important means that infectious disease occurs.Immunological adjuvant (claiming again nonspecific immunity proliferant agent) is constituent indispensable in vaccine, and it can promote vaccine-induced specific immune response effectively.Along with the develop rapidly of vaccine research, novel recombinant vaccine just progressively replaces traditional attenuated live vaccine and inactivated vaccine.Compare with traditional vaccine, the advantage such as recombinant vaccine has that purity is high, high specificity, safety are good, however because its immunogenicity is generally poor, in the urgent need in conjunction with effective immunological adjuvant, thereby improve the immune efficacy of vaccine.
Along with the development of nanotechnology, the development that the degradable nano material of a series of safety (as liposome, polymer nano granules, nanogel etc.) is vaccine and immunological adjuvant provides brand-new thinking.The effect of nano material mainly comprises following several respects: 1, the different antigen of the multiple physicochemical property of load, and protection antigen is not degraded; 2, promote the antigen-presenting cells such as dendritic cell (dendritic cell, DC) to the picked-up of antigen vaccine and present; 3, regulation antigen transporting and discharging in cell and tissue; 4, part nano material can also be induced the antigen-presenting cell activation such as DC.Therefore, the novel vaccine carrier system based on nano material will become the important technical of breakthrough bottleneck, promotes development and the innovation of tumor vaccine and immunotherapy of tumors.
But traditional nano vaccine carrier still exists many deficiencies, a little less than as low in antigen load rate, less stable, immunogenicity, the existence of these shortcomings has limited the extensive use of nano vaccine carrier to a great extent.
[summary of the invention]
Based on this, be necessary to provide a kind of can each albuminoid of high-efficient carrier or antigen nucleic acid, there is good stability and immunogenic polymer nanocomposite vaccine carrier preparation and preparation method thereof.
A polymer nanoparticle drug carriers preparation, comprises each components of following parts by weight:
DNA antigen 0-20 part,
Proteantigen 0-20 part,
Polypeptide antigen 0-20 part,
Immunological adjuvant be greater than zero be less than or equal to 10 parts and
Nano-carrier micelle 1-100 part;
And the gross weight umber of described DNA antigen, described proteantigen and described polypeptide antigen is greater than zero;
Described nano-carrier micelle is the complex with three-decker being formed by amphipathic three block copolymer, and described amphipathic three block copolymer is for comprising polyethyleneglycol derivative, polylysine and poly-leucic linear polymer; Wherein, one end of described polylysine is connected with described polyethyleneglycol derivative by amido link, the other end is connected with described poly-leucine by peptide bond, and the molecular weight ranges of described polyethyleneglycol derivative is 500-10000, the degree of polymerization of described polylysine is 10-200, and the described poly-leucic degree of polymerization is 10-200; Described poly-leucine forms the internal layer of described complex, and described polylysine forms the intermediate layer of described complex, and described polyethyleneglycol derivative forms the skin of described complex; Described DNA antigen, described proteantigen, described polypeptide antigen and immunological adjuvant are dispersed in the described polylysine in intermediate layer.
In one embodiment, described DNA antigen is the coding gene order of one or more antigens or the plasmid vector of described gene order.
In one embodiment, described proteantigen is the proteantigen that derives from microorganism or histiocytic proteantigen extract or restructuring.
In one embodiment, described polypeptide antigen is restructuring polypeptide antigen.
In one embodiment, described immunological adjuvant is polyinosinic acid-polycytidylicacid, Monophosphoryl lipid A or cytidylic acid guanosine oligodeoxynucleotide.
In one embodiment, described polyethyleneglycol derivative is the amino Polyethylene Glycol of monomethyl ether or amino polyethylene carboxylic acid.
In one embodiment, the dosage form of described polymer nanoparticle drug carriers preparation is lyophilized injectable powder or aqueous solution injection, and the particle size range of described nano-carrier micelle is 10-200nm, and the Zeta potential of described aqueous solution injection is 10mV-50mV.
A preparation method for polymer nanoparticle drug carriers preparation, comprises the steps:
The polyethyleneglycol derivative that is 500-10000 by molecular weight ranges is dissolved in organic solvent, and concentration range is 1-100mg/mL; Under nitrogen protection, above-mentioned solution is heated to 30-50 ℃, then the lysine cyclic acid anhydride monomer that adds side chain protected, the lysine cyclic acid anhydride monomer of described side chain protected and the mol ratio of described polyethyleneglycol derivative are 10-200: 1, isothermal reaction 24-120 hour under nitrogen protection; Add leucine cyclic acid anhydride monomer, the mol ratio of described leucine cyclic acid anhydride monomer and described polyethyleneglycol derivative is 10-200: 1, under nitrogen protection, continue isothermal reaction 24-120 hour, reaction adds after finishing 5-50 doubly to the ether sedimentation of reaction system volume, filtration, the dry polylysine and the poly-leucic triblock copolymer that comprise polyethyleneglycol derivative, side chain protected of obtaining, wherein, one end of the polylysine of described side chain protected is connected with described polyethyleneglycol derivative by amido link, and the other end is connected with described poly-leucine by peptide bond; The described polylysine that comprises polyethyleneglycol derivative, side chain protected and poly-leucic triblock copolymer are dissolved in the trifluoroacetic acid of 0 ℃, adding HBr volume fraction is 30% HBr/HAc solution, wherein, the mole of HBr be lysine side chain protected group mole 2-4 doubly, react after 2-4 hour, then add 5-50 doubly to the ether of reaction system volume, to precipitate, filter; And solid product after filtration is dissolved in polar organic solvent, use bag filter that molecular cut off the is 2000-8000 12-96 hour that dialyses in water, every 2-6 hour changes water-dialyzing once, lyophilizing subsequently, obtain comprising amphipathic three block copolymer described in polyethyleneglycol derivative, polylysine and poly-leucine, wherein, one end of described polylysine is connected with described polyethyleneglycol derivative by amido link, and the other end is connected with described poly-leucine by peptide bond;
According to following parts by weight, take each component:
DNA antigen 0-20 part,
Proteantigen 0-20 part,
Polypeptide antigen 0-20 part,
Immunological adjuvant be greater than zero be less than or equal to 10 parts,
Amphipathic three block copolymer 1-100 part and
Organic solvent 20-500 part;
And the gross weight umber of described DNA antigen, described proteantigen and described polypeptide antigen is greater than zero;
Described amphipathic three block copolymer is dissolved in described organic solvent and forms uniform solution, described solution is carried out to dialysis treatment and remove organic solvent, obtain nano-carrier micelle;
Described nano-carrier micelle is soluble in water, add described DNA antigen, described proteantigen, described polypeptide antigen and immunological adjuvant, the standing 0.5-2 hour at 37 ℃ that vibrates after 0.5-2 hour, the solution obtaining after standing or dry after the pressed powder that obtains be described polymer nanoparticle drug carriers preparation.
In one embodiment, described organic solvent is selected from least one in acetone, methanol, ethanol, oxolane, DMF, N,N-dimethylacetamide, dimethyl sulfoxide and N-Methyl pyrrolidone.
Above-mentioned polymer nanoparticle drug carriers preparation carrys out load DNA antigen with the three-decker complex that the amphipathic three block copolymer with good biological affinity forms, proteantigen, polypeptide antigen and immunological adjuvant, corresponding antigen and immunological adjuvant can be dispersed in the polylysine in intermediate layer, and amphipathic three block copolymer combines the advantage of Polyethylene Glycol and polyamino acid, can in aqueous solution, self assembly form the nano-carrier with three-decker, wherein the poly-leucine segment of innermost layer is assembled formation hydrophobic cores, the polylysine in intermediate layer forms positive charge layer, outermost PEG derivant shields.The cation nanometer carrier that polyethyleneglycol derivative-polylysine-poly-leucine triblock copolymer is prepared from as basis of take has formed a positive charge layer because its special structure makes polylysine positive charge obtain assembling, greatly Zeng Qiang the ability of the electronegative species such as load gene and protein, the secondary structure that simultaneously has alpha-helix due to poly-leucine is increased cation nanometer carrier stability, because this cation nanometer carrier has hydrophobic cores and positive charge layer simultaneously, therefore can the different DNA antigen of high-efficient carrier physicochemical property, proteantigen, polypeptide antigen and immunological adjuvant.Polymer nanoparticle drug carriers preparation not only promotes the picked-up of the antigen-presenting cells such as dendritic cell to antigen, also induces significantly body to produce specific immunoreation.
By using synthetic this amphipathic three block copolymer of method of NCA ring-opening polymerisation, again this amphipathic three block copolymer is prepared to polymer nanoparticle drug carriers preparation by nanoassemble technology formation nano-carrier micelle load DNA antigen, proteantigen, polypeptide antigen and immunological adjuvant, process is simple, and combined coefficient is high.
Above-mentioned amphipathic three block copolymer and hydrophobic drug can self assembly form nano-carrier micelle in aqueous solution, the positively charged polylysine in micelle intermediate layer is DNA antigen, proteantigen, polypeptide antigen and the immunological adjuvant of adsorption band negative charge easily, utilize the preparation process of polymer nanoparticle drug carriers preparation of this characteristic simple, can wide popularization and application in the preparation field of immune vaccine and dendritic cell vaccine.This polymer nanoparticle drug carriers preparation can be used as novel nano vaccine, adopts the methods such as subcutaneous injection and intramuscular injection to carry out immunity to body, for prevention or treatment infectious disease and malignant tumor.
This polymer nanoparticle drug carriers preparation also can be used for dendritic cell vaccine and must prepare.As a kind of dendritic cell vaccine, it is at 37 ℃, 5%CO by above-mentioned polymer nanoparticle drug carriers preparation and human dendritic cell
2under environment, hatch altogether 30 minutes-24 hours, then add maturation promoting factor to continue to hatch to prepare for 24 hours.This dendritic cell vaccine can be widely used in the fields such as prevention and treatment infectious disease and malignant tumor.
[accompanying drawing explanation]
Fig. 1 is the structural representation of the amphipathic three block copolymer of an embodiment, and in figure, line weight only shows difference different material;
Fig. 2 is the structural representation that amphipathic three block copolymer forms the nano-carrier of three-decker in Fig. 1;
Fig. 3 is the dsl data figure of polymer nanoparticle drug carriers preparation paclitaxel loaded in embodiment 5;
Fig. 4 is the dsl data figure of the polymer nanoparticle drug carriers preparation of load amycin in embodiment 6;
Fig. 5 is the dsl data figure of the polymer nanoparticle drug carriers preparation of embodiment 7 load P 53 genes;
Fig. 6 is the dsl data figure of the polymer nanoparticle drug carriers preparation of embodiment 8 load SiRNA genes;
Fig. 7 is the dsl data figure of the polymer nanoparticle drug carriers preparation of embodiment 9 load BSA;
Fig. 8 be in embodiment 10 simultaneously load the SEM figure of polymer nanoparticle drug carriers preparation of paclitaxel, SiRNA;
Fig. 9 be in embodiment 10 load the dsl data figure of polymer nanoparticle drug carriers preparation of paclitaxel, SiRNA;
Figure 10 is the Electronic Speculum figure of the cell of GFP in embodiment 11 (green fluorescent protein) labelling;
Figure 11 is the impact of polymer nanoparticle drug carriers preparation on GFP gene expression in embodiment 11;
Figure 12 is the impact of polymer nanoparticle drug carriers preparation on dendritic cell picked-up OVA antigen in embodiment 12;
Figure 13 is the impact of polymer nanoparticle drug carriers preparation on the anti-OVA antibody of mice in embodiment 13;
Figure 14 is the design sketch of self assembly polypeptide nano micelle-antigen-adjuvant combination vaccine (NP/PIC/OVA) treatment B16-OVA.
[specific embodiment]
Mainly in conjunction with the drawings and the specific embodiments polymer nanoparticle drug carriers preparation and corresponding preparation method and application are described in further detail below.
As shown in Figure 1, the amphipathic three block copolymer of an embodiment, it is for comprising polyethyleneglycol derivative, polylysine and poly-leucic linear polymer (polyethyleneglycol derivative-polylysine-poly-leucine).Molecular weight ranges is preferably at 3000-60000.Wherein, polylysine one end is connected with polyethyleneglycol derivative by amido link, and the other end is connected with poly-leucine by peptide bond.And the molecular weight ranges of this polyethyleneglycol derivative is 500-10000, the degree of polymerization of polylysine is 10-200, and the poly-leucic degree of polymerization is 10-200.
Preferably, the polyethyleneglycol derivative of present embodiment is the amino Polyethylene Glycol (CH of monomethyl ether
3o-PEG-NH
2) or amino polyethylene carboxylic acid (NH
2-PEG-COOH).
Polyethylene Glycol (PEG) has good biocompatibility, biodegradability and catabolite and there is no toxicity, obtains the approval of FDA in the U.S., and is widely used in technical field of biological material and nanosecond medical science field.Nano material, after PEG finishing, can reduce its cell adhesion, reduces the adsorptivity of material in serum, reduce the rejection of macrophage simultaneously and engulf, so the nano material after PEG modification can significantly increase the time of body-internal-circulation.This amphipathic three block copolymer; it is polyethyleneglycol derivative-polylysine-poly-leucine triblock copolymer (PEG-PLys-PLeu); combine the advantage of Polyethylene Glycol and polyamino acid; can in aqueous solution, self assembly form the nano-carrier with three-decker; as shown in Figure 2; wherein the poly-leucine segment of innermost layer is assembled formation hydrophobic cores, and the polylysine in intermediate layer forms positive charge layer, and outermost PEG derivant shields.The cation nanometer carrier that polyethyleneglycol derivative-polylysine-poly-leucine triblock copolymer is prepared from as basis of take has formed a positive charge layer because its special structure makes polylysine positive charge obtain assembling, greatly Zeng Qiang the ability of the electronegative species such as load gene and protein, the secondary structure that simultaneously has alpha-helix due to poly-leucine is increased cation nanometer carrier stability, because this cation nanometer carrier has hydrophobic cores and positive charge layer simultaneously, therefore can be simultaneously effective load micromolecule hydrophobic drug, genetic stew and protein or polypeptide, it is a kind of multifunctional nano carrier.
Present embodiment also provides a kind of preparation method of amphipathic three block copolymer, comprises the steps:
Step S110: using polyethyleneglycol derivative as initiator; by the synthetic polylysine that comprises polyethyleneglycol derivative, side chain protected of method and the poly-leucic linear three block copolymer of NCA ring-opening polymerisation; wherein; polylysine one end of side chain protected is connected with polyethyleneglycol derivative by amido link; the other end is connected with poly-leucine by peptide bond; and the molecular weight ranges of polyethyleneglycol derivative is 500-10000; the degree of polymerization of the polylysine of side chain protected is 10-200, and the poly-leucic degree of polymerization is 10-200.
Specific as follows: the polyethyleneglycol derivative that is 500-10000 by molecular weight ranges is dissolved in organic solvent, concentration range is 1-100mg/mL, under nitrogen protection, be heated to add after 30-50 ℃ the LysZ-NCA (cyclic acid anhydride of benzyloxycarbonyl group lysine, a kind of lysine cyclic acid anhydride of side chain protected) monomer, the mol ratio of LysZ-NCA monomer and polyethyleneglycol derivative is 10-200: 1, under nitrogen protection, after isothermal reaction 24-120 hour, add Leu-NCA (leucic cyclic acid anhydride) monomer, the mol ratio of Leu-NCA monomer and polyethyleneglycol derivative is 10-200: 1, under nitrogen protection, continue isothermal reaction 24-120 hour, after finishing, reaction add 5-50 doubly to the ether sedimentation of reaction system volume, filter, be dried and obtain comprising polyethyleneglycol derivative, the polylysine of side chain protected and poly-leucic triblock copolymer.
Step S120: slough the side chain protected group of the polylysine of side chain protected, obtain comprising polyethyleneglycol derivative, polylysine and poly-leucine and molecular weight at the amphipathic three block copolymer of 3000-60000.
Specific as follows: the polylysine that comprises polyethyleneglycol derivative, side chain protected and poly-leucic triblock copolymer to be dissolved in the trifluoroacetic acid of 0 ℃, add HBr/HAc solution reaction that a certain amount of HBr volumetric concentration is 30% after 2-4 hour, add again 5-50 doubly to the ether of reaction system volume, to precipitate, filter, wherein, the mole of the HBr adding is 2-4 times of lysine side-chain blocking group mole; Solid product after filtration is dissolved in polar organic solvent, uses bag filter that molecular cut off the is 2000-8000 12-96 hour that dialyses in water, every 2-6 hour changes water-dialyzing once, and lyophilizing subsequently, obtains amphipathic three block copolymer.
By using synthetic this amphipathic three block copolymer of method of NCA ring-opening polymerisation, preparation process is simple, and combined coefficient is high.
Meanwhile, present embodiment also provides polymer nanoparticle drug carriers preparation of a kind of load hydrophobic drug, genetic stew, protein or polypeptide and preparation method thereof.
This polymer nanoparticle drug carriers preparation, comprises each components of following parts by weight:
Hydrophobic drug 0-20 part;
Genetic stew 0-20 part;
Protein or polypeptide 0-20 part; And
Nano-carrier micelle 1-100 part;
Wherein, the gross weight umber of hydrophobic drug, genetic stew and protein or polypeptide is greater than 0.
This nano-carrier micelle is the complex with three-decker being formed by amphipathic three block copolymer, and wherein, poly-leucine forms internal layer, and polylysine forms intermediate layer, and polyethyleneglycol derivative forms outer; Hydrophobic drug is dispersed in poly-leucine internal layer.Genetic stew, protein or polypeptide are dispersed in positively charged polylysine intermediate layer.
Preferably, the hydrophobic drug of present embodiment is selected from least one in amycin, paclitaxel, cisplatin, fluorouracil, methotrexate and camptothecine.Genetic stew is selected from least one in antioncogene, suicide gene, SiRNA, mRNA and antisensenucleic acids.Protein or polypeptide are at least one in serum albumin, recombiant protein, polypeptide vaccine, pharmaceutical protein molecule, antigen, treatment peptide, somatomedin and monoclonal antibody.Organic solvent is selected from least one in acetone, methanol, ethanol, oxolane, DMF, N,N-dimethylacetamide, dimethyl sulfoxide and N-Methyl pyrrolidone.
This polymer nanoparticle drug carriers preparation can be lyophilized injectable powder or aqueous solution injection.Wherein, the particle size range of nano-carrier micelle is 10-200nm.If aqueous solution injection, its Zeta potential is 10mV-50mV.
The preparation method of this polymer nanoparticle drug carriers preparation, comprises the steps:
Step S210: prepare amphipathic three block copolymer: using polyethyleneglycol derivative as initiator, by the synthetic polylysine that comprises Polyethylene Glycol, side chain protected of method and the poly-leucic triblock copolymer of NCA ring-opening polymerisation, wherein, polylysine one end of side chain protected is connected with polyethyleneglycol derivative by amido link, the other end is connected with poly-leucine by peptide bond, and the molecular weight ranges of polyethyleneglycol derivative is 500-10000, the degree of polymerization of the polylysine of side chain protected is 10-200, and the poly-leucic degree of polymerization is 10-200; Slough the side chain protected group of the polylysine of side chain protected, obtain comprising polyethyleneglycol derivative, polylysine and poly-leucine and molecular weight at the amphipathic three block copolymer of 3000-60000, wherein poly-leucine is kernel, polylysine is intermediate layer, and polyethyleneglycol derivative is outer;
Step S220: get each component according to following parts by weight:
Hydrophobic drug 0-20 part,
Genetic stew 0-20 part,
Protein or polypeptide 0-20 part,
Amphipathic three block copolymer 1-100 part,
Organic solvent 20-500 part,
And the gross weight umber of hydrophobic drug, genetic stew and protein or polypeptide is greater than 0.
Step S230: amphipathic three block copolymer and hydrophobic drug are dissolved in and form uniform solution in organic solvent, solution is placed in to bag filter, at 10-500, doubly in the water of reaction system volume, dialyse and within 12-96 hour, remove organic solvent, this amphipathic three block copolymer self assembly forms the nano-carrier micelle with three-decker, under the condition that has hydrophobic drug to exist, hydrophobic drug can be wrapped in the poly-leucine of internal layer automatically.In dialysis procedure, every 2-6 hour changes water once.
Step S240: nano-carrier micelle is made to corresponding preparation, obtain polymer nanoparticle drug carriers preparation; Or
Nano-carrier micelle is soluble in water, add genetic stew, protein or polypeptide, the standing 0.5-2 hour at 37 ℃ that vibrates after 1-6 hour, then the preparation that the solution obtaining after standing is made required dosage form is obtained to polymer nanoparticle drug carriers preparation.
The process of load hydrophobic small molecules medicine can add hydrophobic small molecules medicine together in micelle formation process, and self assembly forms nano-medicament carrier.The process of load gene is first synthetic polyethylene glycol-lysine-poly-leucine triblock copolymer to be formed to cation nanometer micelle, then adds by a certain percentage after DNA or RNA and vibrate and mix.The process of load protein is first synthetic polyethylene glycol-lysine-poly-leucine triblock copolymer to be formed to cation nanometer micelle, then adds by a certain percentage after electronegative protein and vibrate and mix.The process of the hydrophobic small molecules of load simultaneously medicine, gene and protein is first to prepare nano-medicament carrier, then adds by a certain percentage gene and protein vibration to mix.
As: the preparation method of the polymer nanoparticle drug carriers preparation of load hydrophobic drug can be with reference to as follows:
Take PEG-PLys-PLeu triblock copolymer 1-100 part, hydrophobic drug 1-20 part, organic solvent 20-500 part, is dissolved in proportion organic solvent completely by medicine and polymer and forms homogeneous, transparent solution; The solution making is placed in to bag filter, and then, at the 10-500 times of 12-96 hour that dialyses in the water of reaction system volume, every 2-6 hour changes water once; After dialysis finishes, the carrier micelle aqueous solution forming in bag filter is made to the preparation of required dosage form.
The preparation method of the polymer nanoparticle drug carriers preparation of load genetic stew can be with reference to as follows:
Take PEG-PLys-PLeu triblock copolymer 1-100 part, organic solvent 20-500 part, is dissolved in organic solvent completely by polymer and forms homogeneous, transparent solution; The solution making is placed in to bag filter, and then, at the 10-500 times of 12-96 hour that dialyses in the water of reaction system volume, every 2-6 hour changes water once; After dialysis finishes, obtain micelle water-soluble, take genetic stew 1-20 part and join micellar aqueous solution mesoscale eddies vibration 1-6 hour, 37 ℃ of conditions are made required preparation by gained solution after transferring and setting to 0 .5-2 hour.
The preparation method of the nano-carrier preparation of load protein can be with reference to as follows:
Take PEG-PLys-PLeu triblock copolymer 1-100 part, organic solvent 20-500 part, is dissolved in organic solvent completely by polymer and forms homogeneous, transparent solution; The solution making is placed in to bag filter, and then, at the 10-500 times of 12-96 hour that dialyses in the water of reaction system volume, every 2-6 hour changes water once; After dialysis finishes, obtain micelle water-soluble, take protein 1-20 part and join micellar aqueous solution mesoscale eddies vibration 1-6 hour, 37 ℃ of conditions are made required preparation by gained solution after transferring and setting to 0 .5-2 hour.
Simultaneously the preparation method of the nano-carrier preparation of load hydrophobic drug, genetic stew and protein or polypeptide can be with reference to as follows:
Take PEG-PLys-PLeu triblock copolymer 1-100 part medicine 1-20 part, organic solvent 20-500 part, is dissolved in proportion organic solvent completely by medicine and polymer and forms homogeneous, transparent solution; The solution making is placed in to bag filter, and then, at the 10-500 times of 12-96 hour that dialyses in the water of reaction system volume, every 2-6 hour changes water once, after dialysis finishes, obtains carrier micelle aqueous solution; Take protein 1-20 part and join carrier micelle aqueous solution mesoscale eddies vibration 1-6 hour, 37 ℃ of conditions are made required preparation by gained solution after transferring and setting to 0 .5-2 hour.
This polymer nanoparticle drug carriers preparation load simultaneously micromolecule hydrophobic drug, genetic stew, protein or polypeptide, and load efficiency is higher, nano-carrier preparation in vivo can be degradable, and participate in metabolism, can avoid immune removing, than being easier to arrive focus.
Above-mentioned amphipathic three block copolymer and hydrophobic drug can be in aqueous solution self assembly form the micelle of nano-carrier, hydrophobic drug is dispersed in poly-leucine hydrophobic inner core, and the nano-micelle obtaining easily genetic stew, protein or the polypeptide of adsorption band negative charge to positively charged intermediate layer, utilize the preparation process of polymer nanoparticle drug carriers preparation of this characteristic simple, can wide popularization and application.
In addition, present embodiment also provides polymer nanoparticle drug carriers preparation of a kind of Antigen and immunological adjuvant and preparation method thereof.
The polymer nanoparticle drug carriers preparation of this Antigen and immunological adjuvant, comprises each components of following parts by weight:
DNA antigen 0-20 part,
Proteantigen 0-20 part,
Polypeptide antigen 0-20 part,
Immunological adjuvant be greater than zero be less than or equal to 10 parts and
Nano-carrier micelle 1-100 part;
And the gross weight umber of DNA antigen, proteantigen and polypeptide antigen is greater than zero;
Nano-carrier micelle is the complex with three-decker being formed by amphipathic three block copolymer, and amphipathic three block copolymer is for comprising polyethyleneglycol derivative, polylysine and poly-leucic linear polymer; Wherein, one end of polylysine is connected with polyethyleneglycol derivative by amido link, and the other end is connected with poly-leucine by peptide bond, and the molecular weight ranges of polyethyleneglycol derivative is 500-10000, the degree of polymerization of polylysine is 10-200, and the poly-leucic degree of polymerization is 10-200; Poly-leucine forms the internal layer of complex, and polylysine forms the intermediate layer of complex, and polyethyleneglycol derivative forms the skin of complex; DNA antigen, proteantigen, polypeptide antigen and immunological adjuvant are dispersed in the polylysine in intermediate layer.
Wherein, DNA antigen is the coding gene order of one or more antigens or the plasmid vector of gene order.Proteantigen is the proteantigen that derives from microorganism or histiocytic proteantigen extract or restructuring.Polypeptide antigen is restructuring polypeptide antigen.Immunological adjuvant is polyinosinic acid-polycytidylicacid (Polyinosinic:polycytidylic acid, Poly IC), Monophosphoryl lipid A (monophosphoryl lipid A, MPLA) or cytidylic acid guanosine oligodeoxynucleotide (CpGODN).The dosage form of the polymer nanoparticle drug carriers preparation of this Antigen and immunological adjuvant is lyophilized injectable powder or aqueous solution injection, and the particle size range of nano-carrier micelle is 10-200nm, and the Zeta potential of aqueous solution injection is 10mV-50mV.
The preparation method of the polymer nanoparticle drug carriers preparation of this Antigen and immunological adjuvant, comprises the steps:
Prepare amphipathic three block copolymer: the same;
According to following parts by weight, take each component:
DNA antigen 0-20 part,
Proteantigen 0-20 part,
Polypeptide antigen 0-20 part,
Immunological adjuvant be greater than zero be less than or equal to 10 parts,
Amphipathic three block copolymer 1-100 part and
Organic solvent 20-500 part;
And the gross weight umber of DNA antigen, proteantigen and polypeptide antigen is greater than zero;
Amphipathic three block copolymer is dissolved in organic solvent and forms uniform solution, solution is carried out to dialysis treatment and remove organic solvent, obtain nano-carrier micelle;
Nano-carrier micelle is soluble in water, add DNA antigen, proteantigen, polypeptide antigen and immunological adjuvant, the standing 0.5-2 hour at 37 ℃ that vibrates after 0.5-2 hour, the solution obtaining after standing or dry after the pressed powder that obtains be polymer nanoparticle drug carriers preparation.
Above-mentioned polymer nanoparticle drug carriers preparation carrys out load DNA antigen with the three-decker complex that the amphipathic three block copolymer with good biological affinity forms, proteantigen, polypeptide antigen and immunological adjuvant, corresponding antigen and immunological adjuvant can be dispersed in the polylysine in intermediate layer, and amphipathic three block copolymer combines the advantage of Polyethylene Glycol and polyamino acid, can in aqueous solution, self assembly form the nano-carrier with three-decker, wherein the poly-leucine segment of innermost layer is assembled formation hydrophobic cores, the polylysine in intermediate layer forms positive charge layer, outermost PEG derivant shields.The cation nanometer carrier that polyethyleneglycol derivative-polylysine-poly-leucine triblock copolymer is prepared from as basis of take has formed a positive charge layer because its special structure makes polylysine positive charge obtain assembling, greatly Zeng Qiang the ability of the electronegative species such as load gene and protein, the secondary structure that simultaneously has alpha-helix due to poly-leucine is increased cation nanometer carrier stability, because this cation nanometer carrier has hydrophobic cores and positive charge layer simultaneously, therefore can all kinds of DNA antigen of payload, proteantigen, polypeptide antigen and immunological adjuvant.Polymer nanoparticle drug carriers preparation has good stability and immunogenicity, not only promotes the picked-up of the antigen-presenting cells such as dendritic cell to antigen, also induces significantly body to produce specific immunoreation.
By using synthetic this amphipathic three block copolymer of method of NCA ring-opening polymerisation, again this amphipathic three block copolymer is prepared to polymer nanoparticle drug carriers preparation by nanoassemble technology formation nano-carrier micelle load DNA antigen, proteantigen, polypeptide antigen and immunological adjuvant, process is simple, and combined coefficient is high.
Above-mentioned amphipathic three block copolymer and hydrophobic drug can self assembly form nano-carrier micelle in aqueous solution, the positively charged polylysine in micelle intermediate layer is DNA antigen, proteantigen, polypeptide antigen and the immunological adjuvant of adsorption band negative charge easily, utilize the preparation process of polymer nanoparticle drug carriers preparation of this characteristic simple, can wide popularization and application in the preparation field of immune vaccine and dendritic cell vaccine.
As a kind of dendritic cell vaccine, it is at 37 ℃, 5%CO by above-mentioned polymer nanoparticle drug carriers preparation and human dendritic cell
2under environment, hatch altogether 30 minutes-24 hours, then add maturation promoting factor to continue to hatch to prepare for 24 hours.This dendritic cell vaccine can be widely used in the fields such as prevention and treatment infectious disease and malignant tumor.
Be below specific embodiment part:
The preparation of example 1, poly glycol monomethyl ether-polylysine-poly-leucine triblock copolymer
By inflated with nitrogen protection after polymerization pipe evacuation, the CH that is 500 by 1g molecular weight
3o-PEG-NH
2after dissolving with 20mLDMF, join in polymerization pipe, by LysZ-NCA monomer and CH
3o-PEG-NH
2mol ratio be 10: 1 ratio adds LysZ-NCA monomer, under nitrogen protection, isothermal reaction is 24 hours, then presses Leu-NCA monomer and CH
3o-PEG-NH
2mol ratio be 10: 1 ratio adds Leu-NCA monomer, under nitrogen protection, continue isothermal reaction 24 hours, after reaction finishes, add 50 times of ether sedimentations to reaction system volume, filtration, the dry PEG-PLysZ-PLeu triblock copolymer that obtains.
The PEG-PLysZ-PLeu polymer of above-mentioned gained is dissolved in the trifluoroacetic acid of 0 ℃, after when to add r volumetric concentration be 30% HBr/HAc solution reaction 2, add 10 times of ether sedimentations to reaction system volume, filtration, wherein, the mole of HBr is 2 times of lysine side-chain blocking group mole; Products therefrom is dissolved in to DMF, uses the bag filter that molecular cut off is 2000 in water, to dialyse 48 hours, within every 2 hours, change water-dialyzing once, lyophilizing subsequently obtains PEG-PLys-PLeu triblock copolymer, and mean molecule quantity is 3000.
The preparation of example 2, carboxy polyethylene glycol-polylysine-poly-leucine triblock copolymer
By inflated with nitrogen protection after polymerization pipe evacuation, the NH that is 2000 by 0.5g molecular weight
2-PEG-COOH joins in polymerization pipe after dissolving with 20mLDMF, by LysZ-NCA monomer and CH
3o-PEG-NH
2mol ratio be 20: 1 ratio adds LysZ-NCA monomer, under nitrogen protection, isothermal reaction is 24 hours, then presses Leu-NCA monomer and CH
3o-PEG-NH
2mol ratio be 40: 1 ratio adds Leu-NCA monomer, nitrogen, protection is lower continues isothermal reaction 48 hours, adds 20 times of ether sedimentations to reaction system volume, filtration, the dry PEG-PLysZ-PLeu triblock copolymer that obtains after reaction finishes.
Above gained PEG-PLysZ-PLeu polymer is dissolved in the trifluoroacetic acid of 0 ℃, after when to add HBr volumetric concentration be 30% HBr/HAc solution reaction 2, add 15 times of ether sedimentations to reaction system volume, filtration, wherein, the mole of HBr is 2 times of lysine side-chain blocking group mole; Products therefrom is dissolved in to DMF, uses the bag filter that molecular cut off is 3500 in water, to dialyse 12 hours, within every 2 hours, change water-dialyzing once, lyophilizing subsequently obtains PEG-PLys-PLeu triblock copolymer, and mean molecule quantity is 8300.
The preparation of example 3, carboxy polyethylene glycol-polylysine-poly-leucine triblock copolymer
By inflated with nitrogen protection after polymerization pipe evacuation, the NH that is 5000 by 0.2g molecular weight
2-PEG-COOH joins in polymerization pipe after dissolving with 20mLDMF, by LysZ-NCA monomer and CH
3o-PEG-NH
2mol ratio be 200: 1 ratio adds LysZ-NCA monomer, under nitrogen protection, isothermal reaction is 24 hours, then presses Leu-NCA monomer and CH
3o-PEG-NH
2mol ratio be 100: 1 ratio adds Leu-NCA monomer, nitrogen, protection is lower continues isothermal reaction 96 hours, adds 30 times of ether sedimentations to reaction system volume, filtration, the dry PEG-PLysZ-PLeu triblock copolymer that obtains after reaction finishes.
Above gained PEG-PLysZ-PLeu polymer is dissolved in the trifluoroacetic acid of 0 ℃, after when to add HBr volumetric concentration be 30% HBr/HAc solution reaction 4, add 30 times of ether sedimentations to reaction system volume, filtration, wherein, the mole of HBr is 4 times of lysine side-chain blocking group mole; Products therefrom is dissolved in to dimethyl sulfoxide, uses the bag filter that molecular cut off is 8000 in water, to dialyse 48 hours, within every 4 hours, change water-dialyzing once, lyophilizing subsequently obtains PEG-PLys-PLeu triblock copolymer, and mean molecule quantity is 40000.
The preparation of example 4, poly glycol monomethyl ether-polylysine-poly-leucine triblock copolymer
By inflated with nitrogen protection after polymerization pipe evacuation, the CH that is 10000 by 0.1g molecular weight
3o-PEG-NH
2after dissolving with 20mLDMF, join in polymerization pipe, by LysZ-NCA monomer and CH
3o-PEG-NH
2mol ratio be 200: 1 ratio adds LysZ-NCA monomer, under nitrogen protection, isothermal reaction is 24 hours, then presses Leu-NCA monomer and CH
3o-PEG-NH
2mol ratio be 200: 1 ratio adds Leu-NCA monomer, nitrogen, protection is lower continues isothermal reaction 120 hours, adds 50 times of ether sedimentations to reaction system volume, filtration, the dry PEG-PLysZ-PLeu triblock copolymer that obtains after reaction finishes.
Above gained PEG-PLysZ-PLeu polymer is dissolved in to the trifluoroacetic acid of 0 ℃, after when to add HBr volumetric concentration be 30% HBr/HAc solution reaction 4, add 50 times of ether sedimentations to reaction system volume, filtration, wherein, the mole of HBr is 4 times of lysine side-chain blocking group mole; Products therefrom is dissolved in to dimethyl sulfoxide, uses the bag filter that molecular cut off is 8000 in water, to dialyse 96 hours, within every 6 hours, change water-dialyzing once, lyophilizing subsequently obtains PEG-PLys-PLeu triblock copolymer, and mean molecule quantity is 54600.
The preparation of example 5, paclitaxel loaded polymer nanoparticle drug carriers preparation
Take the PEG-PLys-PLeu triblock copolymer 10mg that embodiment 1 makes, paclitaxel 2mg, uses 10mL dmso solution, and ultrasonic 10min under room temperature fully dissolves medicine and polymer, forms homogeneous, transparent organic phase solution; It is 2000 bag filters that the dimethyl sulphoxide solution making is placed in to molecular cut off, then in 1L water, dialyses 48 hours, within every 2 hours, changes water once; After dialysis finishes, collect the carrier micelle aqueous solution forming in bag filter, as shown in Figure 3, DSL records size and divides 60nm, and TEM photo shows that the mean diameter of carrier micelle nano-particle is 60nm left and right, and particle is spherical in shape, disperses more even.
The preparation of the polymer nanoparticle drug carriers preparation of example 6, load amycin
Take the PEG-PLys-PLeu triblock copolymer 80mg that embodiment 2 makes, paclitaxel 5mg, with 100mLN, dinethylformamide dissolves, and ultrasonic 10min under room temperature, fully dissolves medicine and polymer, forms homogeneous, transparent organic phase solution; By the N making, it is 2000 bag filters that N-dimethyl formyl solution is placed in molecular cut off, then in 2L water, dialyses 60 hours, within every 4 hours, changes water once; After dialysis finishes, collect the carrier micelle aqueous solution forming in bag filter, then lyophilization obtains pulverulent solids.As shown in Figure 4, DSL records size and divides 85nm, and TEM photo shows that the mean diameter of carrier micelle nano-particle is 80nm left and right, and particle is spherical in shape, disperses more even.
The preparation of the polymer nanoparticle drug carriers preparation of example 7, load P 53 genes
Take the PEG-PLys-PLeu triblock copolymer 1mg that embodiment 1 makes, with 4mLN, dinethylformamide dissolves, and ultrasonic 10min under room temperature, fully dissolves polymer, by the N making, it is 2000 bag filters that N-dimethyl formyl solution is placed in molecular cut off, then in 1.5L water, dialyses 60 hours, within every 6 hours, changes water once, after dialysis finishes, collect the polymer micelle aqueous solution forming in bag filter; Measure P53 gene 0.05mg and join the polymer micelle aqueous solution mesoscale eddies vibration that makes 2 hours, under 37 ℃ of conditions, place the nano-carrier preparation of 1 hour back loading P53 gene.As shown in Figure 5, DSL records size and divides 156nm, disperses more even.
The preparation of the polymer nanoparticle drug carriers preparation of example 8, load SiRNA gene
Take the PEG-PLys-PLeu triblock copolymer 10mg that embodiment 1 makes, use 5mL dmso solution, ultrasonic 10min under room temperature, polymer is fully dissolved, and it is 2000 bag filters that the dimethyl sulphoxide solution making is placed in to molecular cut off, then in 1.5L water, dialyses 60 hours, within every 6 hours, change water once, after dialysis finishes, collect the polymer micelle aqueous solution forming in bag filter, be diluted to 1mg/mL.Measure SiRNA5 μ g and be placed in centrifuge tube, then enter to the polymer micelle aqueous solution 100 μ L vortex vibration making 2 hours, under 37 ℃ of conditions, place the nano-carrier preparation of 1 hour back loading SiRNA gene.As shown in Figure 6, DSL records size and divides 50nm, disperses more even.
The preparation of the polymer nanoparticle drug carriers preparation of example 9, load BSA
Take the PEG-PLys-PLeu triblock copolymer 1mg that embodiment 1 makes, with 4mLN, dinethylformamide dissolves, and ultrasonic 10min under room temperature, fully dissolves polymer, by the N making, it is 2000 bag filters that N-dimethyl formyl solution is placed in molecular cut off, then in 1.5L water, dialyses 60 hours, within every 6 hours, changes water once, after dialysis finishes, collect the polymer micelle aqueous solution forming in bag filter; Measure BSA albumen 0.05mg and join the polymer micelle aqueous solution mesoscale eddies vibration that makes 2 hours, under 37 ℃ of conditions, place the nano-carrier preparation of 1 hour back loading BSA albumen.As shown in Figure 7, DSL records size and divides 95nm, disperses more even.
Embodiment 10, simultaneously load the preparation of polymer nanoparticle drug carriers preparation of paclitaxel, SiRNA
Take the PEG-PLys-PLeu triblock copolymer 10mg that embodiment 1 makes, paclitaxel 2mg, use 5mL dmso solution, ultrasonic 10min under room temperature, fully dissolves polymer, it is 2000 bag filters that the dimethyl sulphoxide solution making is placed in to molecular cut off, then in 1.5L water, dialyse 60 hours, within every 6 hours, change water once, after dialysis finishes, collect the polymer medicament carrying micelle aqueous solution forming in bag filter, be diluted to 1mg/mL.Measure SiRNA5 μ g and be placed in centrifuge tube, then enter to the polymer micelle aqueous solution 100 μ L vortex vibration making 2 hours, under 37 ℃ of conditions, place the nano-carrier preparation of 1 hour back loading SiRNA gene, as shown in Figure 8.As shown in Figure 9, DSL records size and divides 56nm, disperses more even.
Embodiment 11
The plasmid DNA that polymer nanoparticle drug carriers preparation (NP) load that adopts embodiment 1 to make contains GFP (green fluorescent protein), preparation method, with reference to previous embodiment, is observed GFP at the expression of 293T cell.Result shows that nano-particle has significantly improved the protein expression of GFP, as shown in Figure 10 and Figure 11.
Embodiment 12
Polymer nanoparticle drug carriers preparation (NP) the coating antigen albumen (OVA-FITC) that adopts embodiment 1 to make, preparation method, with reference to previous embodiment, is observed the mouse dcs picked-up to antigen in vitro.Result shows that nano-particle has significantly improved the picked-up of dendritic cell to antigen, as shown in figure 12.
Embodiment 13
The polymer nanoparticle drug carriers preparation (NP) that employing embodiment 1 makes is as vaccine carrier, and parcel ovalbumin (Ovalbumin, OVA) is prepared nano vaccine, and mice is carried out to subcutaneous injection immunity, and preparation method is with reference to previous embodiment.Result shows that nano-carrier has improved anti-OVAIgG antibody significantly, as shown in figure 13.
Embodiment 14
The polymer nanoparticle drug carriers preparation (NP) that employing embodiment 1 makes is as vaccine carrier, and parcel ovalbumin (Ovalbumin, OVA) and Poly IC prepare composite nano vaccine, adopts intraperitoneal administration to verify its antitumous effect.Result shows that it can suppress the growth of B16-OVA tumor significantly, and extends the life cycle of tumor-bearing mice, as shown in figure 14, and abscissa express time, unit: day (day); Vertical coordinate represents gross tumor volume.
Comprehensive embodiment 11-14 is known, and the polymer nanoparticle drug carriers preparation of present embodiment is a kind of good vaccine carrier, utilizes it to carry vaccine and imports target tissue, can effectively improve the immune efficacy of vaccine.
The above embodiment has only expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (10)
1. a polymer nanoparticle drug carriers preparation, is characterized in that, comprises each component of following parts by weight:
And the gross weight umber of described DNA antigen, described proteantigen and described polypeptide antigen is greater than zero;
Described nano-carrier micelle is the complex with three-decker being formed by amphipathic three block copolymer, and described amphipathic three block copolymer is for comprising polyethyleneglycol derivative, polylysine and poly-leucic linear polymer; Wherein, one end of described polylysine is connected with described polyethyleneglycol derivative by amido link, the other end is connected with described poly-leucine by peptide bond, and the molecular weight ranges of described polyethyleneglycol derivative is 500-10000, the degree of polymerization of described polylysine is 10-200, and the described poly-leucic degree of polymerization is 10-200; Described poly-leucine forms the internal layer of described complex, and described polylysine forms the intermediate layer of described complex, and described polyethyleneglycol derivative forms the skin of described complex; Described DNA antigen, described proteantigen, described polypeptide antigen and immunological adjuvant are dispersed in the described polylysine in intermediate layer.
2. polymer nanoparticle drug carriers preparation as claimed in claim 1, is characterized in that, described DNA antigen is the coding gene order of one or more antigens or the plasmid vector of described gene order.
3. polymer nanoparticle drug carriers preparation as claimed in claim 1, is characterized in that, described proteantigen is the proteantigen that derives from microorganism or histiocytic proteantigen extract or restructuring.
4. polymer nanoparticle drug carriers preparation as claimed in claim 1, is characterized in that, described polypeptide antigen is restructuring polypeptide antigen.
5. polymer nanoparticle drug carriers preparation as claimed in claim 1, is characterized in that, described immunological adjuvant is polyinosinic acid-polycytidylicacid, Monophosphoryl lipid A or cytidylic acid guanosine oligodeoxynucleotide.
6. polymer nanoparticle drug carriers preparation as claimed in claim 1, is characterized in that, described polyethyleneglycol derivative is the amino Polyethylene Glycol of monomethyl ether or amino polyethylene carboxylic acid.
7. polymer nanoparticle drug carriers preparation as claimed in claim 1, it is characterized in that, the dosage form of described polymer nanoparticle drug carriers preparation is lyophilized injectable powder or aqueous solution injection, and the particle size range of described nano-carrier micelle is 10-200nm, and the Zeta potential of described aqueous solution injection is 10mV-50mV.
8. a preparation method for polymer nanoparticle drug carriers preparation, is characterized in that, comprises the steps:
The polyethyleneglycol derivative that is 500-10000 by molecular weight ranges is dissolved in organic solvent, and concentration range is 1-100mg/mL;
Under nitrogen protection, above-mentioned solution is heated to 30-50 ℃, then the lysine cyclic acid anhydride monomer that adds side chain protected, the lysine cyclic acid anhydride monomer of described side chain protected and the mol ratio of described polyethyleneglycol derivative are 10-200:1, isothermal reaction 24-120 hour under nitrogen protection;
Add leucine cyclic acid anhydride monomer, the mol ratio of described leucine cyclic acid anhydride monomer and described polyethyleneglycol derivative is 10-200:1, under nitrogen protection, continue isothermal reaction 24-120 hour, reaction adds after finishing 5-50 doubly to the ether sedimentation of reaction system volume, filtration, the dry polylysine and the poly-leucic triblock copolymer that comprise polyethyleneglycol derivative, side chain protected of obtaining, wherein, one end of the polylysine of described side chain protected is connected with described polyethyleneglycol derivative by amido link, and the other end is connected with described poly-leucine by peptide bond;
The described polylysine that comprises polyethyleneglycol derivative, side chain protected and poly-leucic triblock copolymer are dissolved in the trifluoroacetic acid of 0 ℃, adding HBr volume fraction is 30% HBr/HAc solution, wherein, the mole of HBr be lysine side chain protected group mole 2-4 doubly, react after 2-4 hour, then add 5-50 doubly to the ether of reaction system volume, to precipitate, filter; And
Solid product after filtration is dissolved in polar organic solvent, use bag filter that molecular cut off the is 2000-8000 12-96 hour that dialyses in water, every 2-6 hour changes water-dialyzing once, lyophilizing subsequently, obtain comprising amphipathic three block copolymer described in polyethyleneglycol derivative, polylysine and poly-leucine, wherein, one end of described polylysine is connected with described polyethyleneglycol derivative by amido link, and the other end is connected with described poly-leucine by peptide bond;
According to following parts by weight, take each component:
And the gross weight umber of described DNA antigen, described proteantigen and described polypeptide antigen is greater than zero;
Described amphipathic three block copolymer is dissolved in described organic solvent and forms uniform solution, described solution is carried out to dialysis treatment and remove organic solvent, obtain nano-carrier micelle;
Described nano-carrier micelle is soluble in water, add described DNA antigen, described proteantigen, described polypeptide antigen and immunological adjuvant, the standing 0.5-2 hour at 37 ℃ that vibrates after 0.5-2 hour, the solution obtaining after standing or the solution obtaining is dry after the pressed powder that obtains be described polymer nanoparticle drug carriers preparation.
9. the preparation method of polymer nanoparticle drug carriers preparation as claimed in claim 8, it is characterized in that, described organic solvent is selected from least one in acetone, methanol, ethanol, oxolane, DMF, N,N-dimethylacetamide, dimethyl sulfoxide and N-Methyl pyrrolidone.
10. a dendritic cell vaccine, is characterized in that, the polymer nanoparticle drug carriers preparation in claim 1-7 described in any one and human dendritic cell are at 37 ℃, 5%CO
2under environment, hatch altogether 30 minutes-24 hours, then add maturation promoting factor to continue to hatch to obtain for 24 hours.
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| CN104288758B (en) * | 2014-10-22 | 2018-04-03 | 中国科学院生物物理研究所 | Pegylated phospholipids are the micella vaccine of carrier |
| CN107496915A (en) * | 2015-01-23 | 2017-12-22 | 四川大学 | A kind of vaccine carrier based on aluminium hydrate nano grain |
| CN105194663B (en) * | 2015-10-19 | 2018-11-02 | 上海天汇化学制药有限公司 | Pegylated phospholipids are the micella vaccine of carrier |
| CN106729681B (en) * | 2016-12-21 | 2020-02-21 | 上海交通大学医学院附属新华医院 | Nanocarrier protein platform for improving antigen immunogenicity |
| CN107050463B (en) * | 2017-04-19 | 2021-01-15 | 国家纳米科学中心 | Medicine and preparation method and application thereof |
| CN108938568B (en) * | 2018-08-07 | 2021-01-05 | 中国医学科学院生物医学工程研究所 | Phospholipid hybrid polymer vesicle (DCs) vaccine based on co-entrapped antigen and double immune agonists as well as preparation method and application of DCs vaccine |
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