CN110538319B - Deformable nano vaccine and preparation method and application thereof - Google Patents
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Abstract
The invention provides a deformable nano vaccine and a preparation method and application thereof, wherein the nano vaccine comprises a polymer-polypeptide assembly and tumor antigen polypeptide, has monodispersity, is spherical particles under the condition of physiological pH, has the particle diameter of 100-200nm, and is changed into a two-dimensional lamellar structure of 5-8 mu m from the spherical particles under the acidic environment of endocytosis; the invention combines nanotechnology and biological immunity, prepares the pH response and self-assembly nano vaccine through complex experiments, and has the advantages of simple preparation process, ingenious principle, obvious effect, wide application prospect and market value.
Description
Technical Field
The invention relates to the field of nano vaccines, in particular to a deformable nano vaccine and a preparation method and application thereof.
Background
Tumor immunotherapy is considered to have great promise. Among them, immune checkpoint therapy, chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T) therapy, tumor vaccine, and the like have attracted increasing attention. The tumor vaccine adopts tumor specific antigen to lead antigen presenting cells to recognize and selectively kill target antigen, and has larger application potential. However, effective antitumor immunity is achievedThe premise is to achieve cross-presentation of antigens, since antigen-presenting cells often do not deliver well to the cytoplasm after uptake of the vaccine, enzymes in their endosomes or lysosomes degrade these antigenic polypeptides and present them to CD4 via MHC class II molecules + T cells, but CD4 + T cells have limited tumor killing ability, so efficient intracellular delivery is achieved, leading to efficient cross-presentation of antigens, which is a critical scientific problem facing the field of tumor vaccines.
The rise of nanotechnology has brought hope to solve the above problems, and in the prior art, the adopted strategy comprises using a polymer material with a 'proton sponge effect', which absorbs a large amount of protons in endocytosis bodies, so that the endocytosis bodies are damaged by osmotic pressure, and the endocytosis bodies are damaged; adopting polypeptide with membrane perforation effect such as melittin, which is selectively released at endocytosis site, perforating on endocytosis membrane, and releasing carried gene or protein to cytoplasm; the method adopts a charge reversal method, the nano particles are negatively charged under physiological conditions, if the nano particles are endocytosed into an endosome, the surfaces of the nano particles can be rapidly positively charged, the nano particles with positive charges interact with the endosome membrane to cause the endosome membrane to be damaged, and thus, the carried genes or proteins are released to a cytoplasmic site. CN105214097A relates to a technology for implementing and detecting pH-responsive PMMMA membrane shells for vaccine oral immunization. The PLGA/antigen and other micro-nano vaccine systems are taken as the basis, the poly (methyl methacrylate-methyl acrylate-methacrylic acid) (PMMMA) membrane shell layer reinforced oral vaccine which is stable and resistant to enzymolysis under the acidic condition is obtained by a surface free radical polymerization method, and a subject (such as tilapia mossambica) can obtain high-level immunity with better persistence in a short period by means of orally taking the vaccine to implement immunity. However, the prior art has the defects of complex preparation process, high cost and uneven treatment effect.
Recent developments in the field of nano-self-assembly have reached the level of living organisms, where nano-self-assembly at the cellular or living animal level can have very unique biological properties, which are not possible with traditional biological or chemical methods. Therefore, a controllable living polypeptide self-assembly method is developed and prepared into the nano vaccine, the carried antigen polypeptide is directly delivered into cytoplasm, the high-efficiency tumor immunotherapy effect is realized, and the nano vaccine has wide application prospect and great market value.
Disclosure of Invention
Aiming at the defects and actual needs of the prior art, the invention provides a deformable nano vaccine and a preparation method and application thereof, wherein the nano vaccine comprises a macromolecule-polypeptide assembly and tumor antigen polypeptides,
in order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a deformable nano-vaccine, comprising a polymer-polypeptide assembly and a tumor antigen polypeptide;
wherein, the nano vaccine has a structure shown as the following formula I:
wherein M is independently selected from integers from 2 to 6, N is independently selected from integers from 20 to 24, K is independently selected from integers from 28 to 32, and P is independently selected from 4 or 5.
The M may be, for example, 2, 3, 4, 5 or 6,N may be, for example, 20, 21, 22, 23 or 24, K may be, for example, 28, 29, 30, 31 or 32.
The polymer-polypeptide assembly is a nanometer deformer, and has a particle size of 100-150nm, such as 100nm, 110nm, 120nm, 130nm, 140nm or 150nm.
Preferably, the nano-vaccine is monodisperse, is spherical particles at physiological pH, and has a particle size of 100-200nm, which may be, for example, 100nm, 120nm, 140nm, 160nm, 180nm, or 200nm.
Preferably, the nano vaccine is changed into a two-dimensional lamellar structure of 5-8 μm from a spherical particle under an acidic environment.
Preferably, the acidic environment has a pH of 5.6.
Preferably, the acidic environment is that of an endocytosis.
In the invention, the inventor deeply researches the advantages and disadvantages of the vaccine field, combines nanotechnology and biological immunity technology, prepares a macromolecule-polypeptide assembly through long-term complex scientific research exploration, and the assembly has an amphiphilic structure and can be self-assembled to form a nanostructure (a nano deformer) and carry hydrophilic antigen polypeptide through an emulsification-solvent evaporation method.
The nano vaccine provided by the invention has the characteristic of sensitive shape change in an acidic environment, maintains spherical shape and nano-scale particle size under the condition of physiological pH, can change the shape under the acidic environment, changes spherical nanoparticles (100 nm) into a 5-8 mu m lamellar structure, can cause the endosome of a cell to be damaged by mechanical force, and causes the carried tumor antigen to be released into cytoplasm, so that the antigen can be efficiently presented to the surface of Dendritic Cells (DCs) through a Major Histocompatibility Complex I (Major Histocompatibility Complex I, MHC-I), and the DCs interact with T cell receptors of the T cells through an antigen polypeptide-MHC-I Complex thereof, thereby efficiently transferring antigen information to CD8 + T lymphocytes, thereby inducing strong antigen-specific CD8+ T cell proliferation and antigen-specific tumor cell killing.
In a second aspect, the present invention provides a method for preparing the nano-vaccine according to the first aspect, comprising the steps of:
(1) Through RAFT polymerization, DMAEMA and OEGMA are used as monomers, azobisisobutyronitrile is used as an initiator, and polymer p (OEGMA) is synthesized through reaction M -DMAEMA N )；
(2) P (OEGMA) obtained in step (1) M -DMAEMA N ) Is a macromolecular chain transfer agent, takes MA as a monomer,polymerization reaction is carried out under initiation of AIBN, and p (OEGMA) is synthesized by constant temperature stirring M -DMAEMA N )-p(MA) K ；
(3) P (OEGMA) obtained in step (2) M -DMAEMA N )-p(MA) K By reacting carboxyl on MA with VE to obtain p (OEGMA) M -DMAEMA N )-p(MAVE) K ；
(4) Linking PDP to p (OEGMA) by acid-sensitive covalent bond M -DMAEMA N )-p(MAVE) K Reaction to synthesize p (OEGMA) M -DMAEMA N )-p((MAVE) K1 -(MAVE-PDP) K2 )；
(5) Encapsulating the antigen polypeptide in the p (OEGMA) obtained in the step (4) by an emulsification-solvent evaporation method M -DMAEMA N )-p((MAVE) K1 -(MAVE-PDP) K2 ) And (c) forming the nano vaccine of the first aspect.
Wherein M is independently selected from integers from 2 to 6, N is independently selected from integers from 20 to 24, K is independently selected from integers from 28 to 32, P is independently selected from 4 or 5, the sum of K1 and K2 is K, and the values of K1 and K2 can be adjusted by varying the ratio of PDP to polymer, for example, K1 is 26, 22 or 18, and K2 is 4, 8 or 12.
Specifically, the deformable nano vaccine is synthesized by a four-step method, firstly, dimethyl aminoethyl methacrylate (2- (Dimethylamino) ethyl methacrylate, DMAEMA), oligo (ethylene glycol) methyl methacrylate, OEGMA, are used as monomers, azobisisobutyronitrile (2,2' -Azobis (2-methyl propionitril), AIBN) is used as an initiator, and p (OEGMA) is synthesized M -DMAEMA N )；
Then with p (OEGMA) M -DMAEMA N ) For macromolecular chain transfer agent, using Methacrylic Acid (MA) as monomer, under the initiation of azobisisobutyronitrile (2,2' -Azobis (2-methylpiperonitrile), AIBN) synthesizing p (OEGMA) M -DMAEMA N )-p(MA) K ；
With p (OEGMA) M -DMAEMA N )-p(MA) K The raw material is prepared by reacting carboxyl on Methacrylic Acid (MA) with 2-chloroethyl vinyl ether (2-chloroethyl)Reaction of vinyl ether, VE) to give p (OEGMA) M -DMAEMA N )-p(MAVE) K Taking a certain amount of p (OEGMA) M -DMAEMA N )-p(MA) K Dissolving in anhydrous DMF, adding 2-chloroethyl Vinyl Ether (VE) in excess under the catalysis of potassium hydroxide (KOH), reacting for 24 hr, and dialyzing to obtain p (OEGMA) M -DMAEMA N )-b-p(MAVE) K ；
Linking Pyrene-linked D-type polypeptide (PDP) to p (OEGMA) by acid sensitive covalent bond M -DMAEMA N )-p(MAVE) K Synthesis of p (OEGMA) M -DMAEMA N )-p((MAVE) K1 -(MAVE-PDP) K2 ) The method comprises the following specific steps: adding a certain amount of p (OEGMA) M -DMAEMA N )-b-p(MAVE) K Dissolving in dry DMF, adding a certain amount of PDP polypeptide and p-toluenesulphonic acid (p-TSA), and dissolving in N 2 Under protective conditions, in
Reacting for 72h in the presence of a molecular sieve to obtain p (OEGMA) M -DMAEMA N )-p-(MAVE) K1 -(MAVE-PDP) K2 ；
And then antigen polypeptide is encapsulated in the nanometer deformer by an emulsification-solvent volatilization method to form the deformable nanometer vaccine.
Preferably, the molar weight ratio of AIBN, OEGMA and DMAEMA in step (1) is 1: (70-280) for example, a 1.
Preferably, the number of monomers on the synthetic polymer chain in step (1) is 6 to 50, for example, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 34, 38, 40, 46, 48 or 50, preferably 10 to 30.
Preferably, the reaction time in step (1) is 1-8h, and may be, for example, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h, 6h, 6.5h, 7h, 7.5h or 8h.
Preferably, the reaction temperature in step (1) is 55-76 ℃, for example 55 ℃, 58 ℃, 60 ℃, 62 ℃, 64 ℃, 66 ℃, 68 ℃, 70 ℃, 72 ℃, 74 ℃ or 76 ℃.
Preferably, the addition of p (OEGMA) in step (2) M -DMAEMA N ) The molar mass ratio to MA is 1 (10-400), and may be, for example, 1.
Preferably, the ratio of the molar amount of AIBN added in step (2) to the molar amount of MA is 1 (100-900), and may be, for example, 1.
Preferably, the methacrylic acid of step (2) is dissolved in N, N-Dimethylformamide (DMF) or 1,4-dioxane (1,4-dioxane, DOA).
Preferably, the polymerization reaction in the step (2) is carried out under the constant temperature condition of an oil bath pan.
Preferably, the rotation speed of the stirring in the step (2) is 1000-10000rpm, such as 1000rpm, 2000rpm, 2500rpm, 3000rpm, 3800rpm, 4000rpm, 5000rpm, 6000rpm, 7500rpm, 8000rpm or 10000rpm, preferably 1000-5000rpm.
Preferably, the polymerization reaction time in step (2) is 2-8h, and may be, for example, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h, 6h, 6.5h, 7h, 7.5h or 8h.
Preferably, the addition of p (OEGMA) in step (3) M -DMAEMA N )-p(MA) O The molar mass ratio to VE is 1 (30-300), and may be, for example, 1.
Preferably, the reaction time of step (3) is 12-48h, for example 12h, 18h, 24h, 32h or 48h, preferably 24h.
Preferably, p (OEGMA) is added in step (4) M -DMAEMA N )-p(MAVE) K The ratio of the molar amount to the molar amount of the PDP is 1 (30-400), and may be, for example, 1.
Preferably, the reaction temperature in step (4) is from 15 to 37 deg.C, such as 15 deg.C, 20 deg.C, 25 deg.C, 30 deg.C or 37 deg.C, preferably 25 deg.C.
Preferably, the reaction time in step (4) is 20-48h, for example, 20h, 25h, 28h, 32h, 36h, 40h, 44h or 48h, preferably 24h.
Preferably, the solvent for the reaction of step (4) comprises N, N-Dimethylformamide (DMF) or dimethyl sulfoxide (DMSO).
Preferably, step (5) is performed using p (OEGMA) M -DMAEMA N )-p((MAVE) K1 -(MAVE-PDP) K2 ) And the ratio of the antigen polypeptide is 1 (5-100), and can be, for example, 1:5, 1.
Preferably, step (1) is performed using p (OEGMA) M -DMAEMA N ) The purification method of (2) is a dialysis method with a molecular weight cut-off of 1000-2000Da, which may be, for example, 1000Da, 1500Da, 1800Da or 2000Da, preferably 1000Da.
Preferably, the p (OEGMA) in the step (2) M -DMAEMA N )-p(MA) K The purification method of (4) is a dialysis method having a molecular weight cut-off of 10 to 30kDa, and may be, for example, 10kDa, 12kDa, 14kDa, 16kDa, 18kDa, 20kDa, 23kDa, 25kDa, 28kDa or 30kDa.
Preferably, the p (OEGMA) in the step (3) M -DMAEMA N )-p(MAVE) K The purification method of (4) is a dialysis method having a molecular weight cut-off of 10 to 30kDa, and may be, for example, 10kDa, 12kDa, 14kDa, 16kDa, 18kDa, 20kDa, 23kDa, 25kDa, 28kDa or 30kDa.
Preferably, the method for encapsulating the antigen polypeptide in the step (5) comprises the following steps:
(1') adding p (OEGMA) M -DMAEMA N )-p-(MAVE) K1 -(MAVE-PDP) K2 Dissolving the polypeptide and the antigen in chloroform;
(2 ') adding PBS buffer solution in the step (1'), performing ultrasonic treatment for 5min by using an ultrasonic instrument to obtain an emulsion white liquid, and removing chloroform in the system.
Preferably, the volume of chloroform in step (1') is 100-1000. Mu.L, for example, 100. Mu.L, 200. Mu.L, 300. Mu.L, 500. Mu.L, 800. Mu.L or 1000. Mu.L, preferably 200. Mu.L.
Preferably, the pH of PBS in step (2') is from 7.2 to 7.4, and may be, for example, 7.2, 7.3, or 7.4.
Preferably, the volume of PBS in step (2') is 0.5-10mL, and can be, for example, 0.5mL, 1mL, 3mL, 4mL, 5mL, or 10mL, preferably 2mL.
Preferably, the power of the ultrasound in step (2') is 30-150W, such as 30W, 50W, 60W, 100W or 150W, preferably 50W.
Preferably, the time of ultrasound in step (2') is 1-20min, for example, 1min, 2min, 3min, 4min, 6min, 7min, 8min, 9min, 10min, 15min or 20min, preferably 5min.
Preferably, the chloroform is removed in step (2') by rotary evaporation.
Preferably, the purification method of the nano vaccine in the step (5) is centrifugation.
Preferably, the rotation speed of the centrifugation is 3000-5000rpm, which may be, for example, 3000rpm, 3500rpm, 4000rpm, 4500rpm or 5000rpm.
Preferably, the purification further comprises redispersion and washing with phosphate buffer at pH 7.2-7.4.
In a specific embodiment, a variable nano vaccine is exemplified, wherein M is 4,N selected from 22, K is 30, P is 4-5, and the specific preparation method is as follows:
specifically, the deformable nano vaccine is synthesized by a four-step method, firstly, dimethyl aminoethyl methacrylate (2- (Dimethylamino) ethyl methacrylate, DMAEMA), oligo (ethylene glycol) methyl methacrylate, OEGMA, are used as monomers, azobisisobutyronitrile (2,2' -Azobis (2-methyl propionitril), AIBN) is used as an initiator, and p (OEGMA) is synthesized 4 -DMAEMA 22 )；
Then with p (OEGMA) 4 -DMAEMA 22 ) For macromolecular chain transfer agent, using Methacrylic Acid (MA) as monomer, under the initiation of azobisisobutyronitrile (2,2' -Azobis (2-methylpiperonitrile), AIBN) synthesizing p (OEGMA) 4 -DMAEMA 22 )-p(MA) 30 ；
With p (OEGMA) 4 -DMAEMA 22 )-p(MA) 30 Reacting carboxyl on Methacrylic Acid (MA) with 2-chloroethyl Vinyl Ether (VE) to obtain p (OEGMA) as raw material 4 -DMAEMA 22 )-p(MAVE) 30 Specifically, a certain amount of p (OEGMA) is taken 4 -DMAEMA 22 )-p(MA) 30 Dissolving in anhydrous DMF, adding 2-chloroethyl Vinyl Ether (VE) in excess under the catalysis of potassium hydroxide (KOH), reacting for 24 hr, and dialyzing to obtain p (OEGMA) 4 -DMAEMA 22 )-b-p(MAVE) 30 ；
Linking Pyrene-linked D-type polypeptide (PDP) to p (OEGMA) by acid sensitive covalent bond 4 -DMAEMA 22 )-p(MAVE) 30 Synthesis of p (OEGMA) 4 -DMAEMA 22 )-p((MAVE) 18 -(MAVE-PDP) 12 ) The method comprises the following specific steps: adding a certain amount of p (OEGMA) 4 -DMAEMA 22 )-b-p(MAVE) 30 Dissolving in dry DMF, adding a certain amount of PDP polypeptide and p-toluenesulphonic acid (p-TSA), and reacting in N 2 Under protective conditions, in
Reacting for 72h in the presence of a molecular sieve to obtain p (OEGMA) 4 -DMAEMA 22 )-p-(MAVE) 18 -(MAVE-PDP) 12 ；
And then antigen polypeptide is encapsulated in the nanometer deformer by an emulsification-solvent volatilization method to form the deformable nanometer vaccine.
In a third aspect, the invention relates to a deformable nano-vaccine according to the first aspect for use in the preparation of a medicament for the treatment of tumors.
Compared with the prior art, the invention has the following beneficial effects:
the method for preparing the nano vaccine has the characteristics of simple process, high yield and the like; the deformable nanometer vaccine provided by the invention has the advantages of 100-200nm particle size, uniform particle size and good dispersibility, the nanometer deformer can release polypeptide to be reassembled into a two-dimensional sheet material with the particle size of 5-8 mu m under the acid environment of endocytosis, the shape change of the nanometer deformer can lead the endocytosis of cells to be broken, and further the carried tumor antigen polypeptide is directly delivered into cytoplasm, and the tumor antigen is presented to cytotoxic CD8 through MHC I (major histocompatibility complex) path + T lymphocytes, then CD8 + The T cells are selectively identified and killed; the research finds that the deformable nano vaccine has better tumor cell killing power and can be used for preparing the medicine for tumor immunotherapy.
Drawings
FIG. 1 is a schematic diagram of a method for preparing a deformable nano vaccine according to the present invention;
FIG. 2 is a schematic of the synthesis route of a nano-deformer made according to the present invention;
FIG. 3 shows a nano-deformer prepared according to the present invention 1 H-NMR characterization results;
FIG. 4 is a transmission electron micrograph of a deformable nano-vaccine prepared according to the present invention, wherein FIG. 4 (A) and FIG. 4 (B) are TEM images of the deformable nano-vaccine under physiological pH and simulated endocytosis pH conditions;
FIG. 5 is a graph showing the results of dynamic light scattering characterization of the nano-vaccine prepared according to the present invention, wherein FIGS. 5 (A) and 5 (B) are particle size distribution diagrams under physiological pH and simulated endocytosis pH conditions;
FIG. 6 is a intracellular distribution diagram of OVA antigen carried by the deformable nano-vaccine prepared by the invention;
FIG. 7 is a methylene orange staining experimental graph of the nano-vaccine prepared by the present invention, which reflects the effect of the nano-vaccine on the integrity of endocytosis;
FIG. 8 is a diagram of the results of in vivo tumor suppression experiments of the deformable nano-vaccine prepared by the present invention.
Detailed Description
To further illustrate the technical means and effects of the present invention, the following further describes the technical solutions of the present invention by way of specific embodiments with reference to the drawings, but the present invention is not limited to the scope of the embodiments.
Example 1
In this example, the deformable nano-vaccine was prepared by the following method, specifically including the following steps (the preparation method is schematically shown in fig. 1):
(1) P (OEGMA) was synthesized by RAFT polymerization using DMAEMA (17.2 mmol) and OEGMA (3.44 mmol) as starting materials and AIBN (0.058 mmol) as initiator at 60 ℃ for 12 hours 4 -DMAEMA 22 )；
(2) P (OEGMA) obtained in step (1) 4 -DMAEMA 22 ) P (OEGMA) was synthesized as a macromolecular chain transfer agent (0.01 mmol) using MA (2 mmol) as a monomer and AIBN (0.005 mmol) as an initiator at 60 ℃ for 2 hours 4 -DMAEMA 22 )-p(MA) 30 ；
(3) P (OEGMA) obtained in step (2) 4 -DMAEMA 22 )-p(MA) 30 Based on the reaction of the side-chain carboxyl group of methacrylic acid with VE to obtain p (OEGMA) 4 -DMAEMA 22 )-p-(MAVE) 30 ；
(4) P (OEGMA) obtained in step (2) 4 -DMAEMA 22 )-p-(MAVE) 30 Based on the fact that different numbers of PDPs, e.g. 4, 8, 12, are attached to their MAVE side chains to give p (OEGMA) 4 -DMAEMA 22 )-p-(MAVE) 26 -(MAVE-PDP) 4 ，p(OEGMA 4 -DMAEMA 22 )-p-(MAVE) 22 -(MAVE-PDP) 8 Or p (OEGMA) 4 -DMAEMA 22 )-p-(MAVE) 18 -(MAVE-PDP) 12 Namely a nano deformer, and the synthetic route is shown in figure 2; p (OEGMA) 4 -DMAEMA 22 )-p-(MAVE) 18 -(MAVE-PDP) 12 Is/are as follows 1 The results of H-NMR characterization are shown in FIG. 3, and the average graft number of PDP can be calculated by the ratio of the number of aromatic hydrogens on the benzene ring of PDP to the integral of the methyl hydrogens of DMAEMA on the backbone of the polymer chain.
(5) P (OEGMA) obtained in the step (4) 4 -DMAEMA 22 )-p-(MAVE) 18 -(MAVE-PDP) 12 Using OVA model polypeptide as antigen (2 mg) as carrier (100 mg), preparing deformable nanometer vaccine carrying antigen polypeptide by emulsification-solvent evaporation method, and the transmission electron microscope picture is shown in figure 4;
as can be seen from FIG. 4, the prepared nano vaccine has uniform particle size and monodispersity.
The dynamic light scattering characterization result diagram of the deformable nano vaccine is shown in fig. 5, and as can be seen from fig. 5, the deformable nano vaccine has a spherical shape under the physiological pH condition, the particle size is 100nm, and when the nano vaccine is taken by antigen presenting cells, the acidic environment of endocytosis bodies of the nano vaccine can cause the nano vaccine to deform to 5-8 μm;
example 2
In this example, the deformable nano-vaccine was prepared by the following method, specifically including the following steps (the preparation method is schematically shown in fig. 1):
(1) P (OEGMA) was synthesized by RAFT polymerization using DMAEMA (17.2 mmol) and OEGMA (3.44 mmol) as starting materials and AIBN (0.088 mmol) as initiator at 60 ℃ for 8 hours 4 -DMAEMA 22 )；
(2) P (OEGMA) obtained in step (1) 4 -DMAEMA 22 ) P (OEGMA) was synthesized as a macromolecular chain transfer agent (0.01 mmol) using MA (3 mmol) as a monomer and AIBN (0.004 mmol) as an initiator at 65 ℃ for 1 hour 4 -DMAEMA 22 )-p(MA) 30 ；
(3) P (OEGMA) obtained in step (2) 4 -DMAEMA 22 )-p(MA) 30 Based on the reaction of the side chain carboxyl group of methacrylic acid with VE to obtain p (OEGMA) 4 -DMAEMA 22 )-p-(MAVE) 30 ；
(4) P (OEGMA) obtained in step (2) 4 -DMAEMA 22 )-p-(MAVE) 30 Based on the attachment of different numbers of PDPs to their MAVE side chains, p (OEGMA) is obtained 4 -DMAEMA 22 )-p-(MAVE) 18 -(MAVE-PDP) 12 ；
(5) P (OEGMA) obtained in the step (4) 4 -DMAEMA 22 )-p-(MAVE) 18 -(MAVE-PDP) 12 Using OVA model polypeptide as antigen (1 mg) as carrier (100 mg), and emulsifying and solvent evaporating to obtain deformable nanometer vaccine carrying antigen polypeptide.
Example 3
In this example, the deformable nano-vaccine was prepared by the following method, specifically including the following steps (the preparation method is schematically shown in fig. 1):
(1) P (OEGMA) was synthesized by RAFT polymerization using DMAEMA (17.2 mmol) and OEGMA (3.44 mmol) as starting materials and AIBN (0.035 mmol) as initiator at 70 deg.C for 6 hours 4 -DMAEMA 22 )；
(2) P (OEGMA) obtained in step (1) 4 -DMAEMA 22 ) As a macromolecular chain transfer agent (0.01 mmol), using MA (4 mmol) as a monomer and AIBN (0.002 mmol) as an initiator, p (OEGMA) was synthesized at 60 ℃ for 1.5 hours 4 -DMAEMA 22 )-p(MA) 30 ；
(3) P (OEGMA) obtained in step (2) 4 -DMAEMA 22 )-p(MA) 30 Based on the reaction of the side-chain carboxyl group of methacrylic acid with VE to obtain p (OEGMA) 4 -DMAEMA 22 )-p-(MAVE) 30 ；
(4) P (OEGMA) obtained in step (2) 4 -DMAEMA 22 )-p-(MAVE) 30 Based on the different number of PDPs attached to their MAVE side chains, p (OEGMA) is obtained 4 -DMAEMA 22 )-p-(MAVE) 18 -(MAVE-PDP) 12 ；
(5) P (OEGMA) obtained in the step (4) 4 -DMAEMA 22 )-p-(MAVE) 18 -(MAVE-PDP) 12 Is used as a carrier (100 mg), and an OVA model polypeptide is used as an antigen (0.8 mg), and the deformable nano-vaccine carrying the antigen polypeptide is prepared by an emulsification-solvent evaporation method.
Example 4
In this example, the deformable nano-vaccine was prepared by the following method, specifically including the following steps (the preparation method is schematically shown in fig. 1):
(1) By RAFT polymerization using DMAEMA (17.2 mmol) and OEGMA (1.72 mmol) as raw materials and AIBN (0.03 mmol) as a guideHair agent, reacting at 60 deg.C for 24 hr to synthesize p (OEGMA) 4 -DMAEMA 22 )；
(2) P (OEGMA) obtained in step (1) 4 -DMAEMA 22 ) As a macromolecular chain transfer agent (0.01 mmol), p (OEGMA) was synthesized at 65 ℃ for 1.5 hours using MA (2 mmol) as a monomer and AIBN (0.006 mmol) as an initiator 4 -DMAEMA 22 )-p(MA) 30 ；
(3) P (OEGMA) obtained in step (2) 4 -DMAEMA 22 )-p(MA) 30 Based on the reaction of the side-chain carboxyl group of methacrylic acid with VE to obtain p (OEGMA) 4 -DMAEMA 22 )-p-(MAVE) 30 ；
(4) P (OEGMA) obtained in step (2) 4 -DMAEMA 22 )-p-(MAVE) 30 Based on the attachment of different numbers of PDPs to their MAVE side chains, p (OEGMA) is obtained 4 -DMAEMA 22 )-p-(MAVE) 18 -(MAVE-PDP) 12 ；
(5 p (OEGMA) obtained in the step (4) 4 -DMAEMA 22 )-p-(MAVE) 18 -(MAVE-PDP) 12 Using OVA model polypeptide as antigen (2.5 mg) as carrier (100 mg), and preparing deformable nano vaccine carrying antigen polypeptide by emulsification-solvent evaporation method.
Example 5
The effect of the deformer on cytoplasmic delivery of antigen in dendritic cells was examined in this example as follows:
DC2.4 cells are inoculated in a laser confocal dish at the density of 50000 cells per hole, after the cells are attached to the wall, the nano vaccine prepared in example 1 is treated for 24 hours at the speed of 60 mu g/mL, and the distribution of the polypeptide with the fluorescent label in the cells is detected, as shown in figure 6; meanwhile, the Lyso-Tracker is adopted to mark endocytosis bodies/lysosomes of the cells, whether the nano vaccine has the capacity of helping the antigen to be delivered to cytoplasm is researched, and meanwhile, the lysosome/endocytosis body integrity is researched by adopting a methylene orange staining method for the cells treated by the nano deformable vaccine, and the result is shown in FIG. 7;
from fig. 6 and fig. 7, it can be concluded that the nano deformable vaccine has the ability to break down endocytic structures, resulting in the carried antigen entering cytoplasm.
Example 6
The inhibition of tumor growth by the deformable nano-vaccine was examined in this example as follows:
B16-OVA cells were revived, expanded, and C57BL/6 mice were injected with 1X 10 cells on the right back on day zero 6 B16-OVA cells, after 5 days, the tumors of the mice reached 50mm 3 (ii) a The mice were given subcutaneous injections (125. Mu.g/mouse) once every 7 days; and the tumor growth condition is monitored in real time by adopting a vernier caliper to measure the tumor once every other day. Tumor volume was calculated by the formula (volume =1/2 (length × width) 2 ) Mice were weighed every other day and recorded, the results are shown in fig. 8;
as can be seen from FIG. 8, in the saline control group, tumors grew to 1500mm on day 25 3 The tumor volumes of the mice of the nano-distorter group and the free antigen polypeptide group are all more than 1500mm 3 However, the volume of the tumor was reduced to about 55mm in the mice of the nano deformable vaccine administration group 3 The prepared nano vaccine has good tumor inhibition effect and certain potential in the aspect of being used as a vaccine carrier.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Claims (17)
1. A deformable nano vaccine, characterized in that the nano vaccine comprises a macromolecule-polypeptide assembly p (OEGMA) M -DMAEMA N )-p((MAVE) K1 -(MAVE-PDP) K2 ) And a tumor antigen polypeptide;
wherein the polymer-polypeptide assembly has the following formula
The structure is as follows:
formula (II)
Wherein M is independently selected from an integer from 2 to 6, N is independently selected from an integer from 20 to 24, K is independently selected from an integer from 28 to 32, P is independently selected from 4 or 5, K1 is 26, 22 or 18, K2 is 4, 8 or 12;
the pyrene-linked D-type polypeptide (PDP) has the following structure:
2. the nano-vaccine of claim 1, wherein the nano-vaccine is monodisperse, spherical particles under physiological pH conditions, and has a particle size of 100-200nm;
the nano vaccine is changed into a two-dimensional lamellar structure with the thickness of 5-8 mu m from spherical particles in an acidic environment.
3. The nano-vaccine of claim 2, wherein the acidic environment has a pH of 5.6.
4. The nano-vaccine of claim 3, wherein the acidic environment is that of an endocytosis.
5. A method for preparing a nano-vaccine according to any one of claims 1 to 4, comprising the steps of:
(1) By using a RAFT polymerization method, DMAEMA and OEGMA are taken as monomers,azodiisobutyronitrile (AIBN) is used as an initiator to react and synthesize polymer p (OEGMA) M -DMAEMA N )；
(2) P (OEGMA) obtained in step (1) M -DMAEMA N ) Is a macromolecular chain transfer agent, methacrylic Acid (MA) is used as a monomer, polymerization reaction is carried out under the initiation of AIBN, and p (OEGMA) is synthesized by stirring at constant temperature M -DMAEMA N )-p(MA) K ；
(3) P (OEGMA) obtained in step (2) M -DMAEMA N )-p(MA) K The p (OEGMA) is obtained by reacting carboxyl on MA with 2-chloroethyl Vinyl Ether (VE) as a raw material M -DMAEMA N )-p(MAVE) K ；
(4) Linking PDP to p (OEGMA) by acid-sensitive covalent bond M -DMAEMA N )-p(MAVE) K Reaction to synthesize p (OEGMA) M -DMAEMA N )-p((MAVE) K1 -(MAVE-PDP) K2 )；
(5) Encapsulating the antigen polypeptide in the p (OEGMA) obtained in the step (4) by an emulsification-solvent evaporation method M -DMAEMA N )-p((MAVE) K1 -(MAVE-PDP) K2 ) To obtain the nano vaccine;
wherein M is independently selected from an integer of 2 to 6, N is independently selected from an integer of 20 to 24, K is independently selected from an integer of 28 to 32, P is independently selected from 4 or 5, the sum of K1 and K2 is K, K1 is 26, 22 or 18, and K2 is 4, 8 or 12.
6. The method of claim 5, wherein the molar ratio of AIBN, OEGMA and DMAEMA in step (1) is 1 (70-280): (80-290);
the number of monomers on the synthetic polymer chain in the step (1) is 6-50;
the reaction time in the step (1) is 1-8 h;
the reaction temperature in the step (1) is 55-76 ℃.
7. The process of claim 6, wherein the molar ratio of AIBN, OEGMA and DMAEMA in step (1) is 1;
the number of monomers on the synthetic polymer chain in the step (1) is 10-30.
8. The process of claim 5 wherein step (2) adds p (OEGMA) M -DMAEMA N ) The molar weight ratio of the monomer to MA is 1 (10-400);
the ratio of the molar weight of the AIBN added in the step (2) to the molar weight of the MA is 1 (100-900);
the stirring speed of the step (2) is 1000-10000rpm;
the polymerization reaction time in the step (2) is 2-8 h.
9. The process of claim 8 wherein step (2) adds p (OEGMA) M -DMAEMA N ) The molar weight ratio of the monomer to MA is 1;
the ratio of the molar weight of AIBN added in the step (2) to the molar weight of MA is 1;
the stirring speed of the step (2) is 1000-5000rpm.
10. The process of claim 5 wherein step (3) adds p (OEGMA) M -DMAEMA N )-p(MA) K The molar weight ratio of VE to VE is 1 (30-300);
the reaction time of the step (3) is 12-48h;
p (OEGMA) added in step (4) M -DMAEMA N )-p(MAVE) K The ratio of the molar amount of (b) to the molar amount of PDP is 1 (30-400);
the reaction temperature in the step (4) is 15-37 ℃;
the reaction time in the step (4) is 20-48h;
step (5) the p (OEGMA) M -DMAEMA N )-p((MAVE) K1 -(MAVE-PDP) K2 ) The ratio of the antigen polypeptide to the antigen polypeptide is 1 (5-100).
11. The method of claim 10The method is characterized in that p (OEGMA) is added in the step (3) M -DMAEMA N )-p(MA) K The molar weight ratio to VE is 1;
the reaction time of the step (3) is 24 hours;
p (OEGMA) added in step (4) M -DMAEMA N )-p(MAVE) K The ratio of the molar amount of (b) to the molar amount of the PDP is 1;
the reaction temperature in the step (4) is 25 ℃;
the reaction time in the step (4) is 24 hours;
the solvent for the reaction in the step (4) is DMF;
step (5) the p (OEGMA) M -DMAEMA N )-p((MAVE) K1 -(MAVE-PDP) K2 ) The ratio to antigenic polypeptide is 1.
12. The method of claim 5, wherein step (1) said p (OEGMA) M -DMAEMA N ) The purification method of (1) is a dialysis method, the molecular weight cut-off of the dialysis is 1000-2000Da;
step (2) the p (OEGMA) M -DMAEMA N )-p(MA) K The purification method of (2) is a dialysis method, and the dialysis has a molecular weight cut-off of 10-30 kDa;
step (3) the p (OEGMA) M -DMAEMA N )-p(MAVE) K The purification method of (1) is a dialysis method, and the molecular weight cut-off of the dialysis is 10-30 kDa.
13. The method of claim 12, wherein the dialysis of step (1) has a molecular weight cut-off of 1000Da.
14. The method according to claim 5, wherein the method for entrapping the antigenic polypeptide in step (5) comprises the steps of:
(1') reacting p (OEGMA) M -DMAEMA N )-p-(MAVE) K1 -(MAVE-PDP) K2 Dissolving the polypeptide and the antigen in chloroform;
(2 ') adding a PBS buffer solution into the step (1'), performing ultrasonic treatment for 5min by using an ultrasonic instrument to obtain an emulsion white liquid, and removing chloroform in the system;
the volume of the chloroform in the step (1') is 100-1000 mu L;
the pH value of the PBS in the step (2') is 7.2-7.4;
the volume of PBS in the step (2') is 0.5-10 mL;
the power of the ultrasound in the step (2') is 30-150W;
the ultrasonic treatment time in the step (2') is 1-20min.
15. The method according to claim 14, wherein the chloroform of step (1') has a volume of 200 μ L;
the volume of PBS in step (2') was 2 mL;
the power of the ultrasound in the step (2') is 50W;
the time for ultrasound in step (2') was 5min.
16. The method according to claim 5, wherein the purification method of the nano vaccine in the step (5) is centrifugation;
the rotating speed of the centrifugation is 3000-5000rpm;
the purification also includes redispersion and washing with phosphate buffer at pH 7.2-7.4.
17. Use of a deformable nano-vaccine according to any of claims 1-4 for the preparation of a medicament for the treatment of tumors.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103172806A (en) * | 2013-03-16 | 2013-06-26 | 太原理工大学 | Core-crosslinked multi-responsiveness miktoarm star-like polymer and preparation method thereof |
| CN103421195A (en) * | 2013-08-19 | 2013-12-04 | 苏州大学 | Acid-sensitive cationic block copolymer, and preparation method and application for same |
| CN105497891A (en) * | 2015-12-23 | 2016-04-20 | 南开大学 | Application of polypeptide hydrogel serving as protein vaccine adjuvant and protein vaccine |
| CN105833272A (en) * | 2016-04-20 | 2016-08-10 | 国家纳米科学中心 | Multifunctional nano-medicinal composition, as well as preparation method and application thereof |
| CN105833287A (en) * | 2016-04-20 | 2016-08-10 | 国家纳米科学中心 | Slow-release nano drug carrier as well as preparation method and application thereof |
-
2018
- 2018-05-28 CN CN201810523012.0A patent/CN110538319B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103172806A (en) * | 2013-03-16 | 2013-06-26 | 太原理工大学 | Core-crosslinked multi-responsiveness miktoarm star-like polymer and preparation method thereof |
| CN103421195A (en) * | 2013-08-19 | 2013-12-04 | 苏州大学 | Acid-sensitive cationic block copolymer, and preparation method and application for same |
| CN105497891A (en) * | 2015-12-23 | 2016-04-20 | 南开大学 | Application of polypeptide hydrogel serving as protein vaccine adjuvant and protein vaccine |
| CN105833272A (en) * | 2016-04-20 | 2016-08-10 | 国家纳米科学中心 | Multifunctional nano-medicinal composition, as well as preparation method and application thereof |
| CN105833287A (en) * | 2016-04-20 | 2016-08-10 | 国家纳米科学中心 | Slow-release nano drug carrier as well as preparation method and application thereof |
Non-Patent Citations (5)
| Title |
|---|
| Development of switchable polymers to address the dilemma of stability and cargo release in polycationic nucleic acid carriers.;Yilong Cheng et al.;《Biomaterials.》;20170301;第127卷;89-96 * |
| Enzyme-Instructed Self-Assembly of Small D-Peptides as a Multiple-Step Process for Selectively Killing Cancer Cells.;Jie Zhou et al.;《J Am Chem Soc.》;20160311;第38卷(第11期);3813-3823 * |
| Hyaluronic acid-shelled acid-activatable paclitaxel prodrug micelles effectively target and treat CD44-overexpressing human breast tumor xenografts in vivo.;Yinan Zhong et al.;《Biomaterials.》;20160123;第84卷;250-261 * |
| pH-responsive stealth micelles composed of cholesterol-modified PLA as a nano-carrier for controlled drug release.;Massoumeh Bagheri et al.;《Prog Biomater.》;20140403;第3卷(第1期);文章号：22 * |
| Proton-driven transformable nanovaccine for cancer immunotherapy.;Ningqiang Gong et al.;《Nat Nanotechnol.》;20201026;第15卷(第12期);1053-1064 * |
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