CN114159583A - ZIF-8 polypeptide composite nano preparation and preparation method thereof - Google Patents
ZIF-8 polypeptide composite nano preparation and preparation method thereof Download PDFInfo
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- CN114159583A CN114159583A CN202111495287.6A CN202111495287A CN114159583A CN 114159583 A CN114159583 A CN 114159583A CN 202111495287 A CN202111495287 A CN 202111495287A CN 114159583 A CN114159583 A CN 114159583A
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 91
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 90
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 89
- 239000013154 zeolitic imidazolate framework-8 Substances 0.000 title claims abstract description 75
- MFLKDEMTKSVIBK-UHFFFAOYSA-N zinc;2-methylimidazol-3-ide Chemical compound [Zn+2].CC1=NC=C[N-]1.CC1=NC=C[N-]1 MFLKDEMTKSVIBK-UHFFFAOYSA-N 0.000 title claims abstract description 75
- 238000002360 preparation method Methods 0.000 title claims abstract description 41
- 239000002131 composite material Substances 0.000 title claims abstract description 34
- YSWBFLWKAIRHEI-UHFFFAOYSA-N 4,5-dimethyl-1h-imidazole Chemical compound CC=1N=CNC=1C YSWBFLWKAIRHEI-UHFFFAOYSA-N 0.000 claims abstract description 22
- 238000000034 method Methods 0.000 claims abstract description 12
- 150000003751 zinc Chemical class 0.000 claims abstract description 11
- 239000000243 solution Substances 0.000 claims description 34
- ONDPHDOFVYQSGI-UHFFFAOYSA-N zinc nitrate Chemical compound [Zn+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ONDPHDOFVYQSGI-UHFFFAOYSA-N 0.000 claims description 34
- 238000002156 mixing Methods 0.000 claims description 32
- 239000000203 mixture Substances 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 7
- 238000009472 formulation Methods 0.000 claims description 6
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 claims description 6
- RZLVQBNCHSJZPX-UHFFFAOYSA-L zinc sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Zn+2].[O-]S([O-])(=O)=O RZLVQBNCHSJZPX-UHFFFAOYSA-L 0.000 claims description 6
- 239000012266 salt solution Substances 0.000 claims description 4
- XIOUDVJTOYVRTB-UHFFFAOYSA-N 1-(1-adamantyl)-3-aminothiourea Chemical compound C1C(C2)CC3CC2CC1(NC(=S)NN)C3 XIOUDVJTOYVRTB-UHFFFAOYSA-N 0.000 claims description 3
- 229940057499 anhydrous zinc acetate Drugs 0.000 claims description 3
- 238000009792 diffusion process Methods 0.000 claims description 3
- 230000008014 freezing Effects 0.000 claims description 3
- 238000007710 freezing Methods 0.000 claims description 3
- 229940076153 heptahydrate zinc sulfate Drugs 0.000 claims description 3
- 238000003760 magnetic stirring Methods 0.000 claims description 3
- 238000010907 mechanical stirring Methods 0.000 claims description 3
- 238000003860 storage Methods 0.000 claims description 3
- DJWUNCQRNNEAKC-UHFFFAOYSA-L zinc acetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O DJWUNCQRNNEAKC-UHFFFAOYSA-L 0.000 claims description 3
- 239000011592 zinc chloride Substances 0.000 claims description 3
- 235000005074 zinc chloride Nutrition 0.000 claims description 3
- 229960001763 zinc sulfate Drugs 0.000 claims description 3
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 3
- BEAZKUGSCHFXIQ-UHFFFAOYSA-L zinc;diacetate;dihydrate Chemical compound O.O.[Zn+2].CC([O-])=O.CC([O-])=O BEAZKUGSCHFXIQ-UHFFFAOYSA-L 0.000 claims description 3
- RNZCSKGULNFAMC-UHFFFAOYSA-L zinc;hydrogen sulfate;hydroxide Chemical compound O.[Zn+2].[O-]S([O-])(=O)=O RNZCSKGULNFAMC-UHFFFAOYSA-L 0.000 claims description 3
- 239000002105 nanoparticle Substances 0.000 abstract description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 14
- 238000011068 loading method Methods 0.000 abstract description 9
- 239000002904 solvent Substances 0.000 abstract description 7
- 239000003960 organic solvent Substances 0.000 abstract description 6
- 230000004044 response Effects 0.000 abstract description 6
- 239000012452 mother liquor Substances 0.000 description 12
- 239000000427 antigen Substances 0.000 description 8
- 102000036639 antigens Human genes 0.000 description 8
- 108091007433 antigens Proteins 0.000 description 8
- 239000008367 deionised water Substances 0.000 description 8
- 229910021641 deionized water Inorganic materials 0.000 description 8
- 239000003446 ligand Substances 0.000 description 8
- 239000002245 particle Substances 0.000 description 7
- 239000000843 powder Substances 0.000 description 5
- 230000002378 acidificating effect Effects 0.000 description 4
- 210000000612 antigen-presenting cell Anatomy 0.000 description 4
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- 210000000805 cytoplasm Anatomy 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
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- 238000000634 powder X-ray diffraction Methods 0.000 description 3
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
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- 239000012621 metal-organic framework Substances 0.000 description 2
- 229920001606 poly(lactic acid-co-glycolic acid) Polymers 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- XXMFJKNOJSDQBM-UHFFFAOYSA-N 2,2,2-trifluoroacetic acid;hydrate Chemical compound [OH3+].[O-]C(=O)C(F)(F)F XXMFJKNOJSDQBM-UHFFFAOYSA-N 0.000 description 1
- IGRCWJPBLWGNPX-UHFFFAOYSA-N 3-(2-chlorophenyl)-n-(4-chlorophenyl)-n,5-dimethyl-1,2-oxazole-4-carboxamide Chemical compound C=1C=C(Cl)C=CC=1N(C)C(=O)C1=C(C)ON=C1C1=CC=CC=C1Cl IGRCWJPBLWGNPX-UHFFFAOYSA-N 0.000 description 1
- 238000003917 TEM image Methods 0.000 description 1
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- PMZXXNPJQYDFJX-UHFFFAOYSA-N acetonitrile;2,2,2-trifluoroacetic acid Chemical compound CC#N.OC(=O)C(F)(F)F PMZXXNPJQYDFJX-UHFFFAOYSA-N 0.000 description 1
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- 238000002619 cancer immunotherapy Methods 0.000 description 1
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- 238000000703 high-speed centrifugation Methods 0.000 description 1
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- 230000005847 immunogenicity Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
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- 229910021421 monocrystalline silicon Inorganic materials 0.000 description 1
- 239000013110 organic ligand Substances 0.000 description 1
- 229940023041 peptide vaccine Drugs 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000013557 residual solvent Substances 0.000 description 1
- 229940126586 small molecule drug Drugs 0.000 description 1
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- 238000010998 test method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- SWGJCIMEBVHMTA-UHFFFAOYSA-K trisodium;6-oxido-4-sulfo-5-[(4-sulfonatonaphthalen-1-yl)diazenyl]naphthalene-2-sulfonate Chemical compound [Na+].[Na+].[Na+].C1=CC=C2C(N=NC3=C4C(=CC(=CC4=CC=C3O)S([O-])(=O)=O)S([O-])(=O)=O)=CC=C(S([O-])(=O)=O)C2=C1 SWGJCIMEBVHMTA-UHFFFAOYSA-K 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
- A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
- A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/52—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an inorganic compound, e.g. an inorganic ion that is complexed with the active ingredient
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Abstract
The invention discloses a ZIF-8 polypeptide composite nano preparation, which comprises the following components in molar ratio: 0.05-0.06 part of polypeptide with low isoelectric point, 1 part of zinc salt and 35-70 parts of dimethyl imidazole; low isoelectric pH includes: 1-1.5, 1.5-2, 2-2.5, 2.5-3, 3-3.5, 3.5-4, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6.5-7, 7-7.5; the preparation only uses water as a solvent, does not need an organic solvent, and is environment-friendly and safe; the ZIF-8 polypeptide composite nanoparticles synthesized by the method have the advantages of uniform size and morphology, PH response and high polypeptide loading rate.
Description
Technical Field
The invention relates to the fields of biomedicine and pharmaceutics, in particular to a ZIF-8 polypeptide composite nano preparation and a preparation method thereof.
Background
The development and application of a personalized neoantigen (neoantigen) cancer vaccine has become a new approach for cancer immunotherapy. However, a single naked peptide vaccine can only produce low immunogenicity. This is because naked peptides are easily degraded and cleared in vivo, and are poorly phagocytosed and presented by antigen delivery presenting cells (APCs), such as Dendritic Cells (DCs).
ZIF-8 is a crystalline porous material commonly used in large families of Metal Organic Frameworks (MOFs), and is a three-dimensional network structure formed by zinc ions and dimethyl imidazole organic ligands through coordination bonds.
ZIF-8 belongs to a nano delivery carrier, the nano delivery carrier has good biocompatibility and unique physicochemical properties, can effectively deliver antigens, and can further activate antigen-specific cellular immune reaction on the basis of exciting humoral immunity of an organism by regulating and controlling the presenting way of the antigens in antigen presenting cells.
Common nano delivery vehicles include nanoliposomes, polymeric nanoparticles, inorganic nanoparticles, and the like. However, in the preparation process of these traditional nano delivery carriers, organic solvents are often required as solvents, for example, ethanol is required as a solvent for liposomes, acetone or chloroform is required as a solvent for polymer PLGA nanoparticles, and the biological safety, environmental friendliness and purification difficulty of organic solvents become indispensable considerations. In addition, high polypeptide loading rate of liposome or PLGA particles is difficult to achieve, which results in more waste of polypeptide drug and challenges for subsequent purification. Furthermore, when the nanoparticles are phagocytosed by APC cells, it is also a very challenging problem how to release the delivered antigenic molecules into the cytoplasm.
ZIF-8 has high porosity, high specific surface area and good biological safety, so that it has wide application in the biological field (including diagnosis, small molecule drug loading, etc.). However, organic solvents are generally required to be used for preparing the ZIF-8 nano delivery carrier in the prior art, and a method for wrapping polypeptide medicines by using ZIF-8 in an aqueous phase is not developed in the market, so that the problems are solved.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide the ZIF-8 polypeptide composite nano preparation and the preparation method thereof, only water is used as a solvent, no organic solvent is needed, and the preparation method is environment-friendly and safe; the synthesized ZIF-8 polypeptide composite nanoparticles have uniform size and morphology and have the advantages of PH response and high polypeptide loading rate.
In order to achieve the above object, the present invention adopts the following technical solutions:
a ZIF-8 polypeptide composite nano preparation comprises the following components in a molar ratio: 0.05-0.06 low isoelectric polypeptide: 1 zinc salt: 35-70 dimethylimidazole; low isoelectric pH includes: 1-1.5, 1.5-2, 2-2.5, 2.5-3, 3-3.5, 3.5-4, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6.5-7 and 7-7.5.
The ZIF-8 polypeptide composite nano preparation has a low isoelectric point pH value comprising: 3.5-4.5.
The ZIF-8 polypeptide composite nano-preparation comprises zinc salts: one or more of zinc nitrate hexahydrate, anhydrous zinc nitrate, anhydrous zinc acetate, dihydrate zinc acetate, zinc chloride, monohydrate zinc sulfate, heptahydrate zinc sulfate, and anhydrous zinc sulfate.
In the ZIF-8 polypeptide composite nano preparation, the sequence of the polypeptide with low isoelectric point comprises:
DDD-VNYIKGFRYELYCLARTARTPLK-DDD SEQID01, isoelectric point pH 4.18;
cy3-DDD-VNYIKGFRYELYCLARTARTPLK-DDD SEQ ID02, isoelectric point pH 4.18;
DDDDD-VNYIKGFRYELYCLARTARTPLK-DDDDD SEQID04, isoelectric pH 3.67.
A preparation method of a ZIF-8 polypeptide composite nano preparation comprises the following steps: uniformly mixing the polypeptide with the low isoelectric point and the concentration of 100-; low isoelectric pH includes: 1-1.5, 1.5-2, 2-2.5, 2.5-3, 3-3.5, 3.5-4, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6.5-7 and 7-7.5.
The preparation method of the ZIF-8 polypeptide composite nano preparation comprises the following steps: mixing the polypeptide with low isoelectric point and concentration of 100-1000 mu g/ml with the dimethyl imidazole solution with concentration of 350-700mM, adding the zinc salt solution with concentration of more than 10mM, uniformly mixing, standing at normal temperature for 4-6h, centrifuging to remove supernatant after reaction, re-dissolving, centrifuging again, and circulating for multiple times to obtain the purified ZIF-8/polypeptide composite nano preparation for freezing storage; low isoelectric pH includes: 1-1.5, 1.5-2, 2-2.5, 2.5-3, 3-3.5, 3.5-4, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6.5-7 and 7-7.5.
The preparation method of the ZIF-8 polypeptide composite nano preparation comprises the following mixing modes: magnetic stirring and mixing, mechanical stirring and mixing, shaking by hand, ultrasonic mixing, shaking table mixing, oscillator mixing, standing and diffusion mixing.
The invention has the advantages that:
the invention only uses water as solvent, is safe and environment-friendly;
the sample obtained by the synthetic method has the advantage of high polypeptide loading rate;
the ZIF-8 polypeptide composite nano-particles synthesized by the method have uniform size and appearance, and the size is about 260 nm;
the ZIF-8/polypeptide nano-particles synthesized by the method have the characteristic of acidic PH response, and are beneficial to the endosome escape of a nano-preparation in an antigen presenting cell, so that the antigen is delivered to cytoplasm.
Drawings
FIG. 1 is a schematic diagram of the synthesis of ZIF-8 polypeptide composite nanoparticles of the present invention;
FIG. 2 is a powder X-ray diffraction Pattern (PXRD) of ZIF-8 polypeptide composite nanoparticles obtained in example 2 of the present invention;
FIG. 3 is a field emission Scanning Electron Microscope (SEM) photograph of ZIF-8 polypeptide composite nanoparticles obtained in example 2 of the present invention, at a magnification of 9.5K;
FIG. 4 is a Transmission Electron Microscope (TEM) photograph of ZIF-8 polypeptide composite nanoparticles obtained in example 2 of the present invention, at a magnification of 40K;
FIG. 5 is a field emission Scanning Electron Microscope (SEM) photograph of ZIF-8 polypeptide composite nanoparticles obtained in example 3 of the present invention, at 18K magnification.
Detailed Description
The invention is described in detail below with reference to the figures and the embodiments.
A ZIF-8 polypeptide composite nano preparation comprises the following components in a molar ratio: 0.05-0.06 low isoelectric polypeptide: 1 zinc salt: 35-70 dimethylimidazole; low isoelectric pH includes: 1-1.5, 1.5-2, 2-2.5, 2.5-3, 3-3.5, 3.5-4, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6.5-7, 7-7.5; as a preference, the low isoelectric pH includes: 3.5-4.5.
As shown in fig. 1, the preparation method of the ZIF-8 polypeptide composite nano-formulation comprises: uniformly mixing the polypeptide with low isoelectric point and concentration of 100-1000 mu g/ml, the zinc salt solution with concentration of 10mM and the dimethyl imidazole solution with concentration of 350mM-700mM, standing for 4-6h at normal temperature, and purifying to obtain the ZIF-8/polypeptide composite nano preparation for freezing storage.
As a preferred method of purification: centrifuging to remove supernatant, re-dissolving, centrifuging again, and circulating for multiple times; the mixing mode of the polypeptide with low isoelectric point, the zinc salt solution and the dimethyl imidazole solution comprises the following steps: magnetic stirring and mixing, mechanical stirring and mixing, shaking by hand, ultrasonic mixing, shaking table mixing, oscillator mixing, standing and diffusion mixing.
As an example, zinc salts include: one or more of zinc nitrate hexahydrate, anhydrous zinc nitrate, anhydrous zinc acetate, dihydrate zinc acetate, zinc chloride, monohydrate zinc sulfate, heptahydrate zinc sulfate, and anhydrous zinc sulfate.
The technical effects of the present invention were verified by the ZIF-8 polypeptide composite nanoparticles obtained in the following examples.
Experimental materials:
antigen polypeptide: hangzhou peptide science and technology ltd, polypeptide sequence DDD-VNYIKGFRYELYCLARTARTPLK-DDD SEQ 01, (abbreviated as P1, isoelectric point pH 4.18), Cy3-DDD-VNYIKGFRYELYCLARTARTPLK-DDD SEQ 02 (abbreviated as Cy3-P1, isoelectric point pH 4.18), DVNYIKGFRYELYCLARTARTPLKD SEQ 03 (abbreviated as P2, isoelectric point pH 9.45), DDDDD-VNYIKGFRYELYCLARTARTPLK-DDDDD SEQ 04 (abbreviated as P3, isoelectric point pH 3.67).
Dimethyl imidazole: aladdin reagent (Shanghai) Co., Ltd.
Zinc nitrate: the zinc nitrate standard solution is measured by |0.01000mol/L | in the industry.
Water: the water for injection is prepared by self.
Example 1: the preparation method of the ZIF-8/P1 composite nano preparation specifically comprises the following steps:
preparing a solution:
preparing 10mM zinc nitrate mother liquor: 0.5ml of the solution was pipetted from a standard aqueous solution of zinc nitrate (10mM) using a pipette gun and added to a 1.5ml gauge centrifuge tube.
Preparing 350mM dimethyl imidazole mother liquor: 287.3mg of dimethylimidazole were weighed into a 15ml format centrifuge tube using a microanalytical balance and 10ml of deionized water was added to provide a 350mM ligand solution.
Preparing 1mg/ml of polypeptide mother liquor: dissolving 1mg of P1 in 1ml of PBS buffer solution to obtain 1mg/ml of P1 solution;
mu. l P1 solution was mixed with 500. mu.l of 350mM dimethylimidazole ligand solution, after which 500. mu.l of 10mM zinc nitrate solution was added. And (3) fully and uniformly mixing the mixed solution by using ultrasound, and then standing for 6 hours at normal temperature. After the reaction was completed, the nanoparticles were centrifuged with a high-speed centrifuge, and the supernatant was decanted. Then reconstituted with deionized water and centrifuged again, and this was repeated 3 times. The purified ZIF-8 polypeptide nanocomposite particle samples were stored at-20 ℃.
Example 2: the preparation method of the ZIF-8/Cy3-P1 composite nano preparation specifically comprises the following steps:
preparing a solution:
preparing 10mM zinc nitrate mother liquor: 0.5ml of the solution was pipetted from a standard aqueous solution of zinc nitrate (10mM) using a pipette gun and added to a 1.5ml gauge centrifuge tube.
Preparing 700mM dimethyl imidazole mother liquor: 574.5mg of dimethylimidazole were weighed into a 15ml format centrifuge tube using a microanalytical balance and 10ml of deionized water was added to provide a 700mM ligand solution.
Preparing 1mg/ml of polypeptide mother liquor: 1mg of Cy3-P1 was dissolved in 1ml of PBS buffer to obtain a 1mg/ml Cy3-P1 solution. Mu.l of Cy3-P1 solution was mixed with 500. mu.l of 700mM dimethylimidazole ligand solution, after which 500. mu.l of 10mM zinc nitrate solution was added. And (3) fully and uniformly mixing the mixed solution by using ultrasound, and then standing for 6 hours at normal temperature. After the reaction was completed, the nanoparticles were centrifuged with a high-speed centrifuge, and the supernatant was decanted. After this time, the mixture was reconstituted with deionized water and centrifuged again, and the cycle was repeated 3 times. The purified ZIF-8/polypeptide samples were stored at-20 ℃.
Example 3: the preparation method of the ZIF-8/P2 composite nano preparation specifically comprises the following steps:
preparing 10mM zinc nitrate mother liquor: 0.5ml of the solution was pipetted from a standard aqueous solution of zinc nitrate (10mM) using a pipette gun and added to a 1.5ml gauge centrifuge tube.
Preparing 700mM dimethyl imidazole mother liquor: 574.5mg of dimethylimidazole were weighed into a 15ml format centrifuge tube using a microanalytical balance and 10ml of deionized water was added to provide a 700mM ligand solution.
3) Preparing 1mg/ml of polypeptide mother liquor: 1mg of P2 was dissolved in 1ml of PBS buffer to give a 1mg/ml P2 solution. Mu. l P2 solution was mixed with 500. mu.l of 700mM dimethylimidazole ligand solution, after which 500. mu.l of 10mM zinc nitrate solution was added. And (3) fully and uniformly mixing the mixed solution by using ultrasound, and then standing for 6 hours at normal temperature. After the reaction was completed, the nanoparticles were centrifuged with a high-speed centrifuge, and the supernatant was decanted. After this time, the mixture was reconstituted with deionized water and centrifuged again, and the cycle was repeated 3 times. The purified ZIF-8/polypeptide samples were stored at-20 ℃.
Example 4: the preparation method of the ZIF-8/P3 composite nano preparation specifically comprises the following steps:
1) preparing 10mM zinc nitrate mother liquor: 0.5ml of the solution was pipetted from a standard aqueous solution of zinc nitrate (10mM) using a pipette gun and added to a 1.5ml gauge centrifuge tube.
2) Preparing 700mM dimethyl imidazole mother liquor: 574.5mg of dimethylimidazole were weighed into a 15ml format centrifuge tube using a microanalytical balance and 10ml of deionized water was added to provide a 700mM ligand solution.
3) Preparing 1mg/ml of polypeptide mother liquor: 1mg of P3 was dissolved in 1ml of PBS buffer to give a 1mg/ml P3 solution. Mu. l P3 solution was mixed with 500. mu.l of 700mM dimethylimidazole ligand solution, after which 500. mu.l of 10mM zinc nitrate solution was added. And (3) fully and uniformly mixing the mixed solution by using ultrasound, and then standing for 6 hours at normal temperature. After the reaction was completed, the nanoparticles were centrifuged with a high-speed centrifuge, and the supernatant was decanted. After this time, the mixture was reconstituted with deionized water and centrifuged again, and the cycle was repeated 3 times. The purified ZIF-8/polypeptide samples were stored at-20 ℃.
Experiment one: detecting the crystalline structure of the ZIF-8/polypeptide composite nanoparticle;
the ZIF-8 polypeptide nanocomposite powder obtained in example 2 was uniformly applied to a single-crystal silicon sample stage and subjected to a Brookfield D8X-ray diffractometer (light source Cu target, Ka wavelength)
) And scanning speed is 0.2s/step, and PXRD test is carried out on the ZIF-8/polypeptide powder sample. In addition, a CIF crystal structure file of ZIF-8 is downloaded from a Cambridge structure database CCDC, and a simulated diffraction pattern of ZIF-8 is obtained through conversion of software Jade 6.5. As can be observed from FIG. 2, the ZIF-8/polypeptide synthesized by the method has the same diffraction pattern as the simulated ZIF-8, and the ZIF-8/polypeptide composite nanoparticle is proved to have a good crystalline structure.
Removing residual solvent from the ZIF-8 polypeptide nano composite particle powder obtained in the embodiment 2 through a vacuum drying oven, smearing the powder on a conductive adhesive, and shooting the microscopic morphology of the powder through a JEOL JSM-7800F field emission scanning electron microscope. The ZIF-8/polypeptide can be observed to have a regular particle shape through a Scanning Electron Microscope (SEM) as shown in FIG. 3, and the size is about 260 nm.
Since the ZIF-8 used in the invention has positive charge, the ZIF-8 can be effectively combined with P1, Cy3-P1 or P3 with negative charge, and the polypeptide molecule can be effectively wrapped by the porous structure of the ZIF-8. To qualitatively verify the capture rate of the polypeptide, the original synthesized solution was centrifuged to see that the precipitate had a purple-red color, and the supernatant was almost colorless and transparent, which indicates that the fluorescence-labeled Cy3-P1 of example two can be effectively enriched by the method of the present patent.
Experiment two, polypeptide loading rate detection experiment:
to quantitatively characterize the capture rate of Cy3-P1 polypeptide molecules, we performed HPLC detection on the samples.
The specific process is as follows: the polypeptide was dissolved in 0.1% aqueous trifluoroacetic acid to prepare standard solutions of 250. mu.g/ml, 125. mu.g/ml, 62.5. mu.g/ml, 31.25. mu.g/ml and 15.625. mu.g/ml, respectively. The spectra were separately tested using a liquid chromatography model agilent 1260 (mobile phase a 0.1% TFA water, mobile phase B0.1% TFA acetonitrile, 30% -40% mobile phase B step 20 min test method) and standard curves were plotted. Then, the solution of ZIF-8/Cy3-P1 of the second example was subjected to high-speed centrifugation at 12000rpm to obtain a supernatant, which was then subjected to HPLC analysis. The enrichment rate of the Cy3-P1 polypeptide quantitatively obtained by high performance liquid chromatography was 90%, as shown in Table 1:
TABLE 1
| Sample name | Isoelectric point of polypeptide | Loading rate of polypeptide | Particle size |
| ZIF-8/Cy3-P1 | 4.18 | ～90％ | 260nm |
| ZIF-8/P2 | 9.45 | ～22％ | 280nm |
| ZIF-8/P3 | 3.67 | ～90％ | NA |
In addition, from FIG. 4 on a transmission electron micrograph, we can clearly find that P1 polypeptide molecules are adsorbed on the smooth ZIF-8 particle surface, which shows that the higher polypeptide loading rate effect of the invention is derived from the fact that the polypeptide is wrapped by ZIF-8 in situ on one hand, and is derived from surface electrostatic adsorption on the other hand. Compared with P1, Cy3-P1 or P3 with low isoelectric point, the size of ZIF-8/P2 (example 3 as a comparative example) obtained by P2 polypeptide (9.45) with high isoelectric point (FIG. 5) is about 280nm, which is close to that of ZIF-8/Cys-P1(260nm), however, the capture rate of ZIF-8/P2 polypeptide obtained by HPLC detection method is 22%, which is significantly lower than that of ZIF-8/Cy3-P1 sample in example two, which also shows that the method of the patent has unexpected technical effect by using low isoelectric point polypeptide for compounding.
Experiment three: detecting the acidic PH response of the ZIF-8/polypeptide nanoparticles;
the sample of ZIF-8/P1 polypeptide obtained in example 1 was dispersed in acetate buffer at PH 5.5, and ZIF-8 was completely decomposed to release the encapsulated polypeptide molecules. Therefore, the ZIF-8/polypeptide nanoparticle has the characteristic of acidic PH response and has the potential characteristic of facilitating the 'endosome escape' of the nano preparation in an antigen presenting cell, so that the antigen is delivered to cytoplasm.
In conclusion, the invention only needs water as a solvent, does not need an organic solvent, and is safe and environment-friendly; the ZIF-8/polypeptide nano composite particles obtained by the method have the advantages of uniform size and morphology and high polypeptide loading rate; and the nano-particle has the characteristic of acidic PH response, and is favorable for the 'endosome escape' of the nano-preparation in an antigen presenting cell, so that the antigen is delivered to cytoplasm.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It should be understood by those skilled in the art that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the scope of the present invention.
Claims (7)
1. A ZIF-8 polypeptide composite nano preparation is characterized by comprising the following components in a molar ratio: 0.05-0.06 low isoelectric polypeptide: 1 zinc salt: 35-70 dimethylimidazole; the low isoelectric pH includes: 1-1.5, 1.5-2, 2-2.5, 2.5-3, 3-3.5, 3.5-4, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6.5-7 and 7-7.5.
2. The ZIF-8 polypeptide complex nano-formulation of claim 1, wherein the low isoelectric pH comprises: 3.5-4.5.
3. The ZIF-8 polypeptide complex nano-formulation of claim 1, wherein the zinc salt comprises: one or more of zinc nitrate hexahydrate, anhydrous zinc nitrate, anhydrous zinc acetate, dihydrate zinc acetate, zinc chloride, monohydrate zinc sulfate, heptahydrate zinc sulfate, and anhydrous zinc sulfate.
4. The ZIF-8 polypeptide complex nano-formulation of claim 1, wherein the sequence of the low isoelectric polypeptide comprises:
DDD-VNYIKGFRYELYCLARTARTPLK-DDD SEQID01, isoelectric point pH 4.18;
cy3-DDD-VNYIKGFRYELYCLARTARTPLK-DDD SEQ ID02, isoelectric point pH 4.18;
DDDDD-VNYIKGFRYELYCLARTARTPLK-DDDDD SEQID04, isoelectric pH 3.67.
5. A preparation method of a ZIF-8 polypeptide composite nano preparation is characterized by comprising the following steps: uniformly mixing the polypeptide with the low isoelectric point and the concentration of 100-; the low isoelectric pH includes: 1-1.5, 1.5-2, 2-2.5, 2.5-3, 3-3.5, 3.5-4, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6.5-7 and 7-7.5.
6. The method of preparing a ZIF-8 polypeptide complex nano-formulation according to claim 5, comprising: mixing the polypeptide with the low isoelectric point and the concentration of 100-700 mu g/ml with the dimethyl imidazole solution with the concentration of 350-700mM, adding the zinc salt solution with the concentration of 10mM, uniformly mixing, standing at normal temperature for 4-6h, centrifuging to remove supernatant after the reaction is finished, re-dissolving, centrifuging again, and circulating for multiple times to obtain the purified ZIF-8/polypeptide composite nano preparation for freezing storage; the low isoelectric pH includes: 1-1.5, 1.5-2, 2-2.5, 2.5-3, 3-3.5, 3.5-4, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6.5-7 and 7-7.5.
7. The method of preparing a ZIF-8 polypeptide complex nano-formulation according to claim 6, wherein the mixing comprises: magnetic stirring and mixing, mechanical stirring and mixing, shaking by hand, ultrasonic mixing, shaking table mixing, oscillator mixing, standing and diffusion mixing.
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