CN114246943B - Polymer coupled polypeptide nanometer vaccine and preparation method thereof - Google Patents

Polymer coupled polypeptide nanometer vaccine and preparation method thereof Download PDF

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CN114246943B
CN114246943B CN202111500517.3A CN202111500517A CN114246943B CN 114246943 B CN114246943 B CN 114246943B CN 202111500517 A CN202111500517 A CN 202111500517A CN 114246943 B CN114246943 B CN 114246943B
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pam
arm peg
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陈枢青
王洪亮
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ZJU Hangzhou Global Scientific and Technological Innovation Center
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Abstract

The invention discloses a macromolecule coupled polypeptide nanometer vaccine and a preparation method thereof, wherein the vaccine comprises the following components in mole ratio: 1 a polymeric backbone having multiple reaction centers: 8.1 grafting molecules: 49167-213900 organic solvent: 170-657 organic base; the invention can control the particle size of the nano vaccine by regulating the stoichiometric ratio of the polypeptide and the Pam adjuvant molecule; the polypeptide nanometer vaccine obtained by the invention has a stable structure, is hardly affected by concentration, has no concentration limiting condition, and has the capability of stabilizing co-transport adjuvant molecules.

Description

Polymer coupled polypeptide nanometer vaccine and preparation method thereof
Technical Field
The invention relates to the field of biological medicine, in particular to a macromolecule coupled polypeptide nanometer vaccine and a preparation method thereof.
Background
The development and application of personalized new antigen (neoanti) cancer vaccines has become a new approach to cancer immunotherapy. However, since a single naked peptide vaccine can only produce a lower immunogenicity. This is due to the fact that naked peptides are readily cleaved and cleared in vivo and are inefficiently presented by antigen delivery presenting cells (APCs), such as Dendritic Cells (DCs), phagocytosis. Due to the diversity of the nature of the polypeptides, the design strategy of the amphiphilic polypeptides can simultaneously endow the polypeptide molecules with structural characteristics of containing hydrophobic fragments and hydrophilic fragments. The amphiphilic polypeptide can self-assemble in aqueous solution to form a supermolecule micelle structure, however, the precondition for forming the structure is that the self-assembled polypeptide molecule must reach the critical micelle concentration. Even if a high concentration (greater than the critical micelle concentration of the polypeptide) is injected into the organism, a portion of the self-assembled polypeptide micelles may be disintegrated due to the dilution of the body fluid, thereby losing the delivery advantage of the nanoparticle.
In addition, co-transporting the adjuvant molecule while delivering the antigen polypeptide can greatly enhance the immune activation ability of the nanovaccine, and a common strategy for co-transporting the adjuvant by self-assembling the polypeptide is to non-covalently bind the hydrophobic adjuvant molecule through the hydrophobic core of the self-assembly. However, this binding has on the one hand a large limitation on the kind of adjuvant molecule (which has to be hydrophobic) and the non-covalent binding is not strong, which may lead to leakage of the adjuvant molecule and thus to a reduced co-transport efficiency. Thus, the exact chemical composition of the nanovaccine is also difficult to determine.
The market needs to develop a preparation method for solving the concentration limitation condition of self-assembled polypeptide micelle and having the capability of stabilizing co-transport adjuvant molecules.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide a macromolecule coupled polypeptide nanometer vaccine and a preparation method thereof, wherein the macromolecule coupled self-assembled polypeptide is not limited by critical micelle concentration, and the macromolecule polypeptide vaccine obtained by the preparation method has a stable structure.
In order to achieve the above object, the present invention adopts the following technical scheme:
the high molecular coupled polypeptide nanometer vaccine comprises the following components in mole ratio: 1 a polymeric backbone having multiple reaction centers: 8.1 grafting molecules: 49167-213900 organic solvent: 170-657 organic base;
the grafted molecule is an organic molecule covalently coupled to a polymeric backbone, comprising: an antigenic polypeptide comprising a cysteine at the end and an adjuvant molecule comprising a cysteine at the end; the adjuvant molecule is an auxiliary substance which can enhance the immune response capability of an organism to the antigen or change the type of the immune response;
the polymer skeleton having multiple reaction centers includes: 8-Arm PEG-Acrylate (Acrylate modification), 8-Arm PEG-Mal (Maleimide modification), 8-Arm PEG-OPSS (Orthopyridyl Disulfide modification), 8-Arm PEG-VS (vinyl sulfate modification), 8-Arm PEG-epoxy (epoxy modification), 8-Arm PEG-Alkyne (Alkyne modification).
The polymer coupled polypeptide nanometer vaccine, wherein the antigen polypeptide comprises: C16-NLVPMVATVKKQYIKANSKFIGITELC, SEQ NO 01; NLVPMVATVKKQYIKANSKFIGITELC, SEQ NO 02; C16-NLVPMVATVKKQYIKANSKFIGITELKKK, SEQ NO 03.
The macromolecular conjugated polypeptide nano vaccine has an adjuvant molecule of Pam 2 CysSerLysLysLysLys-Cys、Pam 2 CysSer-Cys、Pam 3 CysSer-Cys、Pam 3 One of CysSerLysLysLysLys-Cys.
The polymer coupled polypeptide nanometer vaccine, wherein the organic solvent comprises: one or more of dimethyl sulfoxide, N-methylpyrrolidone, N-dimethylformamide or N, N-dimethylacetamide.
The polymer coupled polypeptide nano vaccine, wherein the organic base comprises: one or more of ethanolamine, methylamine, urea, dimethylamine, ethylenediamine, trimethylamine, triethylamine, propylamine, isopropylamine, 1, 3-propylenediamine, 1, 2-propylenediamine, tripropylamine, butylamine, isobutylamine, tert-butylamine.
The particle size of the polymer conjugate polypeptide nano vaccine obtained by using the antigen polypeptide modified by the C16 hydrophobic fragment is larger than that of the polymer conjugate polypeptide nano vaccine obtained by using the antigen polypeptide without the C16 hydrophobic fragment.
The molar ratio of the antigen polypeptide to the adjuvant molecule of the polymer coupled polypeptide nano vaccine influences the particle size of the polymer coupled polypeptide nano vaccine.
A preparation method of a macromolecule coupled polypeptide nanometer vaccine comprises the following steps:
step one, according to the mole ratio: 1 a polymeric backbone having multiple reaction centers: 8.1 grafting molecules: 49167-213900 organic solvent: 170-657 of organic base preparation material; dissolving a polymer skeleton with multiple reaction centers and grafted molecules in an organic solvent;
the grafted molecule is an organic molecule covalently coupled to a polymeric backbone, comprising: an antigenic polypeptide comprising a cysteine at the end and an adjuvant molecule comprising a cysteine at the end; the adjuvant molecule is an auxiliary substance which can enhance the immune response capability of an organism to the antigen or change the type of the immune response;
the polymer skeleton having multiple reaction centers includes: 8-Arm PEG-Acrylate (Acrylate modification), 8-Arm PEG-Mal (Maleimide modification), 8-Arm PEG-OPSS (Orthopyridyl Disulfide modification), 8-Arm PEG-VS (vinyl sulfate modification), 8-Arm PEG-epoxy (epoxy modification), 8-Arm PEG-Alkyne (Alkyne modification);
adding organic alkali, stirring, and performing light-shielding reaction at room temperature;
step three, adding cysteine with the concentration of 100mM to quench the reaction;
and step four, dialyzing and purifying to obtain a high molecular polypeptide coupled nano vaccine sample.
The preparation method of the macromolecule coupled polypeptide nanometer vaccine comprises the following step of reacting for 4-8 hours.
The preparation method of the macromolecule coupled polypeptide nanometer vaccine comprises the following specific steps of: and (3) dialyzing the reaction mixture obtained in the step (III) by using a dialysis bag with the molecular weight cut-off of 10000Da, removing unreacted substances, changing 200ml of water every 4 hours, and dialyzing for 16 hours.
The invention has the advantages that:
the invention utilizes the active reactive functional group on the polymer skeleton with multiple reaction centers to effectively couple with the sulfhydryl reactive functional group on the cysteine at the tail end of the polypeptide, and regulates and controls the chemical composition of the nanometer polymer polypeptide conjugate by accurately regulating and controlling the stoichiometric ratio of the grafted molecules; the size of the high molecular polypeptide conjugate is effectively regulated and controlled by coupling a polypeptide grafted molecule containing hydrophobic fragment modification;
the self-assembled polypeptide coupled with the polymer has a stable structure, so that the reaction is not influenced by the critical micelle concentration, the limiting condition of the micelle concentration of the self-assembled polypeptide is solved, and the self-assembled polypeptide has the capability of stabilizing co-transport adjuvant molecules.
Drawings
FIG. 1 is a schematic diagram of the reaction principle of the high molecular polypeptide conjugate nano vaccine of the invention;
FIG. 2 is a sample 8ARM-PEG-G1 obtained in example 1 of the present invention 8 Pam 0 Is frozen and transmitted by an electron microscope with a magnification of 73K;
FIG. 3 is sample 8ARM-PEG-G1 obtained in example 2 of the present invention 4 Pam 4 Is frozen and transmitted by an electron microscope with a magnification of 73K;
FIG. 4 is sample 8ARM-PEG-G2 obtained in example 3 of the present invention 2 Pam 6 Is frozen and transmitted by an electron microscope with a magnification of 73K;
FIG. 5 is sample 8ARM-PEG-G2 obtained in example 4 of the present invention 4 Pam 4 Is frozen and transmitted by an electron microscope with a magnification of 73K;
FIG. 6 is a sample 8ARM-PEG-G2 obtained in example 5 of the present invention 6 Pam 2 Is frozen and transmitted by an electron microscope with a magnification of 73K;
FIG. 7 is a graph of the relative fluorescence intensity of P1 polypeptides of the invention containing fluorescent probe molecules at different aqueous solution concentrations;
FIG. 8 is 8ARM-PEG-G1 of example 1 of the present invention at 10. Mu.g/ml 8 Pam 0 Frozen transmission electron microscope image, magnification 73K;
FIG. 9 is a diagram showing the chemical coupling mechanism of the multicenter polymer backbone of the present invention and a grafted molecule terminated with cysteine.
Detailed Description
The invention is described in detail below with reference to the drawings and the specific embodiments.
The high molecular coupled polypeptide nanometer vaccine comprises the following components in mole ratio: 1 a polymeric backbone having multiple reaction centers: 8.1 grafting molecules: 49167-213900 organic solvent: 170-657 organic base;
the grafted molecule is an organic molecule covalently coupled to a polymeric backbone, comprising: an antigenic polypeptide comprising a cysteine at the end and an adjuvant molecule comprising a cysteine at the end; the adjuvant molecule is an auxiliary substance which can enhance the immune response capability of an organism to the antigen or change the type of the immune response;
the polymer skeleton having multiple reaction centers includes: 8-Arm PEG-Acrylate (Acrylate modification), 8-Arm PEG-Mal (Maleimide modification), 8-Arm PEG-OPSS (Orthopyridyl Disulfide modification), 8-Arm PEG-VS (vinyl sulfate modification), 8-Arm PEG-epoxy (epoxy modification), 8-Arm PEG-Alkyne (Alkyne modification).
The antigen polypeptide comprises: C16-NLVPMVATVKKQYIKANSKFIGITELC, SEQ NO 01; NLVPMVATVKKQYIKANSKFIGITELC, SEQ NO 02; C16-NLVPMVATVKKQYIKANSKFIGITELKKK, SEQ NO 03.
Preferably, the adjuvant molecule is Pam 2 Cys-Cys; it should be noted that the adjuvant molecule is not limited, but may be Pam 2 CysSerLysLysLysLys-Cys、Pam 2 CysSer-Cys、Pam 3 CysSer-Cys、Pam 3 CysSerLysLysLysLys-Cys。
The organic solvents include: one or more of dimethyl sulfoxide, N-methylpyrrolidone, N-dimethylformamide or N, N-dimethylacetamide; it should be noted that the examples are not exhaustive, and the choice of organic solvent is not limited.
The organic base includes: one or more of ethanolamine, methylamine, urea, dimethylamine, ethylenediamine, trimethylamine, triethylamine, propylamine, isopropylamine, 1, 3-propylenediamine, 1, 2-propylenediamine, tripropylamine, butylamine, isobutylamine, tert-butylamine; it should be noted that the examples are not exhaustive, and the choice of organic solvent is not limited.
The reaction principle of the invention is shown in figure 1, the grafting molecule is connected to the multi-center polymer skeleton through chemical coupling reaction, the conjugate itself has hydrophilic area and hydrophobic area, and the conjugate can self-assemble in water solution to form polymer polypeptide coupled nano-particles under the drive of hydrophobic acting force. The coupling reaction of the polymer skeleton with multiple reaction centers and the grafting molecule with cysteine at the tail end is Thiol-click reaction, and the reaction mechanism is shown in figure 9. Since thiol-click reactions have been widely used, the specific reaction process of each reaction is not described in detail.
Samples were prepared as follows by the following procedure, examples 1-5:
the experimental methods in the examples are all conventional methods unless otherwise specified.
The raw materials and reagent materials used in the examples are all commercial products unless otherwise specified.
Wherein, the purchase conditions of partial reagents and raw materials are as follows:
polypeptide is prepared by the peptide biotechnology Co.Ltd.
C16-NLVPMVATVKKQYIKANSKFIGITELC (abbreviated as G1, N end is modified by hexadecanoic acid), SEQ NO 01, theoretical molecular weight 3248.03G/mol, found mass spectrum molecular weight 3247.95G/mol.
NLVPMVATVKKQYIKANSKFIGITELC (abbreviated as G2), SEQ NO 01, theoretical molecular weight 3009.63G/mol, mass spectrum measured molecular weight 3009.45G/mol.
Pam 2 Cys-Cys (abbreviated Pam) mass spectra measured molecular weight 410.5g/mol.
C16-NLVPMVATVKKQYIKANSKFIGITELKKK (abbreviated as P1), SEQ NO 01.
Triethylamine (TEA): a national drug reagent;
dimethyl sulfoxide (DMSO): ara Ding Shiji
8-Arm PEG-mal (20K): the mass spectrum of Guangzhou city carbohydrate science, inc., found a molecular weight of 14.4K.
An embodiment I relates to a preparation method of a high molecular polypeptide coupled nanometer vaccine. The molar ratio of the macromolecular framework to the grafted molecule is 1:8.1. the preparation method comprises the following steps:
1) Preparing mother solution: dissolving 8ARM-PEG-mal in DMSO solution to obtain PEG solution with concentration of 5mg/ml, and marking as S (PEG) solution; dissolving Pam adjuvant in DMSO solution to obtain Pam solution with concentration of 4mg/ml, and marking as S (Pam) solution;
2) In a glass bottle of a specification of 2mg of G1 polypeptide, 220. Mu.l of S (PEG) solution, 750. Mu.l of DMSO solution and 7. Mu.l to 1.5ml of TEA, magnetic stirring was performed at a speed of 700rpm, the reaction was conducted at room temperature in the absence of light for 6 hours, and then 100mM cysteine was added to quench the reaction.
3) The above mixed solution was dialyzed with a dialysis bag having a molecular weight cut-off of 10000Da to remove unreacted substances. 200ml of water was changed every 4 hours and dialyzed for a total of 16 hours, after which the resulting sample solution was collected, sample No. 8ARM-PEG-G1 8 Pam 0 。
Embodiment II, a method for preparing a high molecular polypeptide coupled nanometer vaccine.
The molar ratio of the macromolecular framework to the grafted molecule is 1:8.1.
the preparation method comprises the following steps:
1) Preparing mother solution: dissolving 8ARM-PEG-mal in DMSO solution to obtain PEG solution with concentration of 5mg/ml, and marking as S (PEG) solution; dissolving Pam in DMSO solution to obtain Pam solution with concentration of 4mg/ml, and marking as S (Pam) solution;
2) In a glass bottle of a specification of 2mg of G1 polypeptide, 63. Mu.l of S (Pam) solution, 438. Mu.l of S (PEG) solution, 499. Mu.l of DMSO solution and 7. Mu.l to 1.5ml of TEA, magnetic stirring was performed at a speed of 700rpm, the reaction was conducted at room temperature for 6 hours in the absence of light, and then 100mM cysteine was added to quench the reaction.
3) The above mixed solution was dialyzed with a dialysis bag having a molecular weight cut-off of 10000Da to remove unreacted substances. 200ml of water was changed every 4 hours and dialyzed for a total of 16 hours, after which the resulting sample solution was collected, sample No. 8ARM-PEG-G1 4 Pam 4 。
Embodiment three, a method for preparing a high molecular polypeptide coupled nanometer vaccine. High molecular weight 1:8.1. the preparation method comprises the following steps:
1) Preparing mother solution: dissolving 8ARM-PEG-mal in DMSO solution to obtain PEG solution with concentration of 5mg/ml, and marking as S (PEG) solution; dissolving Pam in DMSO solution to obtain Pam solution with concentration of 4mg/ml, and marking as S (Pam) solution;
2) 2mg of G2 polypeptide, 204.5. Mu.l of S (Pam) solution, 957. Mu.l of S (PEG) solution and 8. Mu.l to 1.5ml of TEA were taken out in glass bottles of a standard, magnetically stirred at 700rpm, reacted at room temperature for 6 hours in the absence of light, and then quenched by adding 100mM cysteine.
3) The above mixed solution was dialyzed with a dialysis bag having a molecular weight cut-off of 10000Da to remove unreacted substances. 200ml of water was changed every 4 hours and dialyzed for a total of 16 hours, after which the resulting sample solution was collected, sample No. 8ARM-PEG-G2 2 Pam 6 。
The fourth embodiment is a method for preparing a high molecular polypeptide coupled nanometer vaccine. High molecular weight 1:8.1. the preparation method comprises the following steps:
1) Preparing mother solution: dissolving 8ARM-PEG-mal in DMSO solution to obtain PEG solution with concentration of 5mg/ml, and marking as S (PEG) solution; dissolving Pam in DMSO solution to obtain Pam solution with concentration of 4mg/ml, and marking as S (Pam) solution;
2) 2mg of G2 polypeptide, 68.25. Mu.l of S (Pam) solution, 478. Mu.l of S (PEG) solution, 453.7. Mu.l of DMSO and 7. Mu.l of TEA to 1.5ml of a glass bottle were magnetically stirred at 700rpm, reacted at room temperature for 6 hours in the absence of light, and then quenched by adding 100mM cysteine.
3) The above mixed solution was dialyzed with a dialysis bag having a molecular weight cut-off of 10000Da to remove unreacted substances. 200ml of water was changed every 4 hours and dialyzed for a total of 16 hours, after which the resulting sample solution was collected, sample No. 8ARM-PEG-G2 4 Pam 4 。
Fifth embodiment is a method for preparing a polymer polypeptide coupled nanometer vaccine. High molecular weight 1:8.1. the preparation method comprises the following steps:
1) Preparing mother solution: dissolving 8ARM-PEG-mal in DMSO solution to obtain PEG solution with concentration of 5mg/ml, and marking as S (PEG) solution; dissolving Pam in DMSO solution to obtain Pam solution with concentration of 4mg/ml, and marking as S (Pam) solution;
2) 2mg of G2 polypeptide, 22.75. Mu.l of S (Pam) solution, 320. Mu.l of S (PEG) solution, 657. Mu.l of DMSO, and 7. Mu.l of TEA to 1.5ml of a glass bottle were magnetically stirred at 700rpm, reacted at room temperature for 6 hours in the absence of light, and then quenched by adding 100mM cysteine.
3) The above mixed solution was dialyzed with a dialysis bag having a molecular weight cut-off of 10000Da to remove unreacted substances. 200ml of water was changed every 4 hours and dialyzed for a total of 16 hours, after which the resulting sample solution was collected, sample No. 8ARM-PEG-G2 6 Pam 2 。
Experimental verification of the technical effects of the present invention was carried out using the samples of the above five examples:
experiment one:
experiment name: regulating and controlling polymer polypeptide coupling experiments with different stoichiometric ratios of grafted molecules;
experimental facilities: frozen samples were prepared by a FEI Vitrobot frozen sample preparation instrument and frozen electron micrographs were taken by Talos F200C200kV frozen electron microscopy.
Experimental materials: examples samples: 8ARM-PEG-G1 8 Pam 0 The method comprises the steps of carrying out a first treatment on the surface of the Example two samples: 8ARM-PEG-G1 4 Pam 4 The method comprises the steps of carrying out a first treatment on the surface of the Example three samples: 8ARM-PEG-G2 2 Pam 6 The method comprises the steps of carrying out a first treatment on the surface of the Example four samples: 8ARM-PEG-G2 4 Pam 4 The method comprises the steps of carrying out a first treatment on the surface of the Example five samples: 8ARM-PEG-G2 6 Pam 2 。
The experimental process comprises the following steps: mu.l of the sample solution (concentration 100. Mu.g/ml) was added dropwise to the TEM copper mesh, and the frozen sample preparation instrument was used 2 times, each time for 5 seconds, followed by vitrification in an ethane medium at a temperature of-180 ℃. Samples after vitrification were stored in liquid nitrogen (77K), after which the morphology of the samples was photographed using a Talos F200C200kV cryo-electron microscope.
Experimental results:
the multicenter polymer skeletons used in the examples of this patent were all 8-Arm PEG-Mal, i.e., 8-Arm PEG modified with maleimide functional groups. Under mild conditions, maleimide functionality can achieve selective modification with thiol functionality at the end of the grafted molecule.
As shown in fig. 2, the experimental result of the sample obtained in example one shows that the feeding molar ratio of G1 to Pam is 8:0, 8ARM-PEG-G1 formed 8 Pam 0 Has a uniform particle size, and the size is about 15nm.
As shown in fig. 3, the experimental result of the sample obtained in example two shows that the feeding molar ratio of G1 to Pam is 4:4, 8ARM-PEG-G1 formed 4 Pam 4 Has a uniform particle size, and the size is about 12nm.
As shown in fig. 4, the experimental result of the sample obtained in example three shows that the feeding molar ratio of G2 to Pam is 2:6, 8ARM-PEG-G2 formed 2 Pam 6 Has a uniform particle size, and the size is about 7nm.
As shown in fig. 5, the experimental result of the sample obtained in example four shows that the feeding molar ratio of G1 to Pam is 4:4, 8ARM-PEG-G1 formed 4 Pam 4 Has a uniform particle size, and the size is about 7nm.
As shown in fig. 6, the experimental result of the sample obtained in example five shows that the feeding molar ratio of G1 to Pam is 6:2, 8ARM-PEG-G1 formed 6 Pam 2 Having a size of about 8nm.
The experimental results show that the first, second, third, fourth and fifth samples of the invention can form uniform nano-sized particles, which verifies the feasibility of the strategy for coupling the macromolecular polypeptide provided by the patent, and simultaneously shows that the chemical composition of the nano-macromolecular polypeptide conjugate can be regulated and controlled by precisely regulating and controlling the stoichiometric ratio of the grafting molecules (polypeptide and Pam adjuvant molecules); namely the feeding mole ratio of the antigen polypeptide and the adjuvant molecule, and influences the particle size of the macromolecule coupled polypeptide nanometer vaccine.
Wherein, the antigen polypeptide grafted in the third, fourth and fifth embodiments is hexadecane (C16) modified G2 polypeptide, and the modified fragment has stronger hydrophobicity because C16 is long-chain saturated alkane. The size of the polymer polypeptide conjugate obtained in the first and second embodiments is about 12-15 nm, and the size of the polymer polypeptide conjugate obtained in the third, fourth and fifth embodiments is about 7-8 nm.
The difference of the sizes of the significant macromolecular polypeptide conjugates shows that the polypeptide grafted with the C16 hydrophobic fragment modification can self-assemble to form the larger-size nano conjugate under the drive of hydrophobic acting force. The specific mechanism by which the hydrophobic effect drives the formation of self-assembled particles is: because the high molecular polypeptide conjugate has both a hydrophilic region and a hydrophobic region, in an aqueous solution environment, the hydrophobic region is hydrophobic, so that the compound molecule spontaneously undergoes conformational collapse, the hydrophobic region is in the self-assembled inner core, and the hydrophilic region is on the surface of the self-assembled shell layer. Because the first and second embodiments have hydrophobic cores with larger molecular weight due to C16 modification, the polypeptide modified by the C16 hydrophobic segment is grafted to form a nano-conjugate with larger size; that is, the particle size of the polymer conjugate polypeptide nano vaccine obtained by using the antigen polypeptide modified by the C16 hydrophobic fragment is larger than that of the polymer conjugate polypeptide nano vaccine obtained by using the antigen polypeptide without the C16 hydrophobic fragment.
Experiment II:
experiment name: compared with the amphiphilic polypeptide self-assembled sample, the high molecular polypeptide conjugate has stable structure.
Experimental facilities: the model of the fluorescence spectrophotometer is Shimadzu RF-5301PC
Experimental materials: examples samples: 8ARM-PEG-G18Pam0, and P1 polypeptide self-assembled samples.
The experimental process comprises the following steps: 10ml of an aqueous pyrene-containing solution was prepared at a concentration of 1.3X10-5M. 1ml of the above aqueous solution was taken to dissolve polypeptides of different masses to give P1 solutions with concentrations of 0.976, 1.95313, 3.90625, 7.8125, 15.625, 31.25, 62.5, 125, 250 and 500. Mu.g/ml. The P1 solutions of different concentrations were shaken for 4 hours in a dark room temperature environment. The corresponding P1 solution was then tested for light intensity using a fluorescence spectrophotometer. Wherein the setting parameter of the fluorescence spectrophotometer is excitation wavelength 335nm, emission wavelength is 360-400nm, and the emission bandwidth and excitation bandwidth are both 2.5nm. And comparing the fluorescence intensity of the P1 sample with different concentrations at the wavelength of 383nm to obtain the critical micelle concentration (CMCPoint) of the P1 sample. Mu.l of the sample solution of example 8ARM-PEG-G18Pam0 (concentration 10. Mu.g/ml) was added dropwise to a TEM copper grid, and the mixture was subjected to a frozen sample preparation instrument (Blots) 2 times, each for 5 seconds, and then vitrified in an ethane medium at a temperature of-180 ℃. Samples after vitrification were stored in liquid nitrogen (77K), after which the morphology of the samples was photographed using a Talos F200C200kV cryo-electron microscope.
Experimental results:
compared with a polypeptide sample P1 which is not coupled (C16-NLVPMVATVKKQYIKANSKFIGITELC cannot be dissolved in water, so that 3K are additionally modified at the C end to increase water solubility), the P1 has an amphiphilic structure, and can self-assemble in an aqueous solution to form regular morphology micelles, but if the concentration is lower than the critical micelle concentration, the micelles can be disintegrated and particles cannot be formed. In order to determine the critical micelle concentration of P1, the characteristic that the fluorescent probe molecule pyrene is sensitive to microenvironment is utilized to determine the light intensity of P1 with different concentrations by utilizing a fluorescence spectrophotometer, and as shown in FIG. 7, the critical micelle concentration of P1 is 100 mug/ml. Below this data, P1 cannot form a micelle structure, but is present in the form of a free polypeptide in aqueous solution. Whereas the high molecular polypeptide conjugate 8Arm-PEG G18Pam0 formed stable nanoparticle structures even at 10. Mu.g/ml (FIG. 8). This is because the G1 polypeptide is coupled to the polymer backbone via a covalent bond, which has a bond energy much higher than that of non-covalent bonds such as hydrogen bonds, and thus the polymer polypeptide conjugate can remain stable in chemical structure in aqueous solution even at very low sample concentrations without the application of additional energy input (e.g., high temperature, high power ultrasound, intense radiation).
In summary, the method of the present invention can not only control the particle size of the nano vaccine by controlling the stoichiometric ratio of the grafted molecule (polypeptide and Pam adjuvant molecule), but also the polymer polypeptide conjugate formed by the method of the present invention has a stable structure and is hardly affected by the concentration compared with the conventional self-assembled micelle particles.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be appreciated by persons skilled in the art that the above embodiments are not intended to limit the invention in any way, and that all technical solutions obtained by means of equivalent substitutions or equivalent transformations fall within the scope of the invention.

Claims (10)

1. The high molecular coupled polypeptide nanometer vaccine is characterized by comprising the following components in mole ratio: 1 a polymeric backbone having multiple reaction centers: 8.1 grafting molecules: 49167-213900 organic solvent: 170-657 organic base;
the grafting molecule is an organic molecule covalently coupled to a polymeric backbone, comprising: an antigenic polypeptide comprising a cysteine at the end and an adjuvant molecule comprising a cysteine at the end; the adjuvant molecule is an auxiliary substance which can enhance the immune response capability of an organism to the antigen or change the type of the immune response;
the polymer skeleton with multiple reaction centers comprises: 8-Arm PEG-Acrylate (Acrylate modification), 8-Arm PEG-Mal (Maleimide modification), 8-Arm PEG-OPSS (Orthopyridyl Disulfide modification), 8-Arm PEG-VS (vinyl sulfate modification), 8-Arm PEG-epoxy (epoxy modification), 8-Arm PEG-Alkyne (Alkyne modification).
2. The polymer conjugate polypeptide nanovaccine of claim 1, wherein the antigenic polypeptide comprises:
C16-NLVPMVATVKKQYIKANSKFIGITELC，SEQ NO 01；
NLVPMVATVKKQYIKANSKFIGITELC，SEQ NO 02；
C16-NLVPMVATVKKQYIKANSKFIGITELKKK，SEQ NO 03。
3. the polymer conjugate polypeptide nanovaccine of claim 1, wherein the adjuvant molecule is Pam 2 CysSerLysLysLysLys-Cys、Pam 2 CysSer-Cys、Pam 3 CysSer-Cys、Pam 3 One of CysSerLysLysLysLys-Cys.
4. The polymer conjugate polypeptide nanovaccine of claim 1, wherein the organic solvent comprises: one or more of dimethyl sulfoxide, N-methylpyrrolidone, N-dimethylformamide or N, N-dimethylacetamide.
5. The polymer conjugate polypeptide nanovaccine of claim 1, wherein the organic base comprises: one or more of ethanolamine, methylamine, urea, dimethylamine, ethylenediamine, trimethylamine, triethylamine, propylamine, isopropylamine, 1, 3-propylenediamine, 1, 2-propylenediamine, tripropylamine, butylamine, isobutylamine, tert-butylamine.
6. The polymer conjugate polypeptide nanovaccine of claim 1, wherein the molar ratio of the antigen polypeptide to the adjuvant molecule affects the particle size of the polymer conjugate polypeptide nanovaccine.
7. The polymer conjugate polypeptide nanovaccine of claim 1, wherein the particle size of the polymer conjugate polypeptide nanovaccine obtained using the antigen polypeptide modified with the C16 hydrophobic fragment is greater than the particle size of the polymer conjugate polypeptide nanovaccine obtained using the antigen polypeptide without the C16 hydrophobic fragment modification.
8. The preparation method of the macromolecule coupled polypeptide nanometer vaccine is characterized by comprising the following steps:
step one, according to the mole ratio: 1 a polymeric backbone having multiple reaction centers: 8.1 grafting molecules: 49167-213900 organic solvent: 170-657 of organic base preparation material; dissolving a polymer skeleton with multiple reaction centers and grafted molecules in an organic solvent;
the grafting molecule is an organic molecule covalently coupled to a polymeric backbone, comprising: an antigenic polypeptide comprising a cysteine at the end and an adjuvant molecule comprising a cysteine at the end; the adjuvant molecule is an auxiliary substance which can enhance the immune response capability of an organism to the antigen or change the type of the immune response;
the polymer skeleton with multiple reaction centers comprises: 8-Arm PEG-Acrylate (Acrylate modification), 8-Arm PEG-Mal (Maleimide modification), 8-Arm PEG-OPSS (Orthopyridyl Disulfide modification), 8-Arm PEG-VS (vinyl sulfate modification), 8-Arm PEG-epoxy (epoxy modification), 8-Arm PEG-Alkyne (Alkyne modification);
adding organic alkali, stirring, and performing light-shielding reaction at room temperature;
step three, adding cysteine with the concentration of 100mM to quench the reaction;
and step four, dialyzing and purifying to obtain a high molecular polypeptide coupled nano vaccine sample.
9. The method for preparing a polymer conjugate polypeptide nano vaccine according to claim 8, wherein the reaction time in the second step is 4-8h.
10. The method for preparing the polymer conjugate polypeptide nano vaccine according to claim 8, wherein the specific steps of dialysis and purification in the fourth step are as follows: and (3) dialyzing the reaction mixture obtained in the step (III) by using a dialysis bag with the molecular weight cut-off of 10000Da, removing unreacted substances, changing 200ml of water every 4 hours, and dialyzing for 16 hours.
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CN111249453A (en) * 2020-02-26 2020-06-09 浙江大学 Nano vaccine and preparation method thereof
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