CN115010918A - Polyamino acid, preparation method thereof and nano vaccine - Google Patents

Polyamino acid, preparation method thereof and nano vaccine Download PDF

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CN115010918A
CN115010918A CN202210860411.2A CN202210860411A CN115010918A CN 115010918 A CN115010918 A CN 115010918A CN 202210860411 A CN202210860411 A CN 202210860411A CN 115010918 A CN115010918 A CN 115010918A
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陈学思
宿元祯
许维国
丁建勋
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Changchun Institute of Applied Chemistry of CAS
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Abstract

The invention relates to the technical field of biology, and particularly discloses a polyamino acid, a preparation method thereof and application thereof in preparation of a vaccine adjuvant. One end of the polyamino acid is hydrophobic amino acid comprising tryptophan, phenylalanine, valine, leucine, isoleucine, alanine and methionine, the other end of the polyamino acid is hydrophilic poly-D-lysine, poly-D-histidine or poly-D-arginine, and the amphiphilic polyamino acid with a hydrophilic chain segment and a hydrophobic chain segment can be self-assembled in water to form the nano micelle. The result shows that the nano-particles prepared by self-assembly of the polyamino acid in water or aqueous solvent can cause dendritic cell activation and enhanced antigen cross-presentation capability, and enhance adaptive immune response reaction, thereby achieving the effects of immune enhancement and treatment.

Description

Polyamino acid, preparation method thereof and nano vaccine
Technical Field
The invention relates to the technical field of biology, in particular to polyamino acid, a preparation method thereof and a nano vaccine.
Background
Vaccination remains a key tool for the protection and eradication of disease. Vaccination plays a great role in the prevention of infectious diseases, and has potential applications in other fields such as cancer and allergy. In addition to ensuring that the vaccine contains the best antigenic components to stimulate the immune response, the addition of vaccine adjuvants that stimulate the immune response and promote antigen presentation is contemplated. By the simultaneous administration of vaccine adjuvants, the host immune response to a given vaccine antigen can be greatly enhanced. Therefore, it is important to develop an effective vaccine adjuvant to stimulate the body to produce an enhanced immune response.
Disclosure of Invention
In view of the above, the invention provides a polyamino acid, a preparation method thereof and application thereof in preparing an immunologic adjuvant. The polyamino acid can cause dendritic cell activation and enhanced antigen cross-presentation capacity and adaptive immune response reaction, thereby achieving the effects of immune enhancement and treatment.
In order to achieve the above object, the present invention provides the following technical solutions:
a polyamino acid having a structure represented by formula (I):
Figure BDA0003758182050000011
wherein R is selected from- (CH) 2 ) 4 -NH 2 ，-(CH 2 ) 3 -NH-C(-NH)-NH 2 Or
Figure BDA0003758182050000012
The Q is selected from any one of the following structures:
Figure BDA0003758182050000013
3≤x≤100，2≤y≤200，2≤z≤500。
in some embodiments, 5 ≦ y ≦ 60 and 10 ≦ z ≦ 60.
The invention also provides a preparation method of the polyamino acid, which comprises the following steps of 1) and 2):
1) reacting primary amine with a structure shown in a formula (II) with L-amino acid-N-internal carboxylic anhydride shown in a formula (III) to obtain polyamino acid with a structure shown in a formula (IV);
2) reacting the polyamino acid with the structure of formula (IV) with a compound with any structure of formulae (V) to (VII), and deprotecting to obtain the polyamino acid with the structure of formula (I);
Figure BDA0003758182050000021
wherein, in the formula (I), R is- (CH) 2 ) 4 -NH 2 ，-(CH 2 ) 3 -NH-C(-NH)-NH 2 And
Figure BDA0003758182050000022
x is more than or equal to 3 and less than or equal to 100, y is more than or equal to 2 and less than or equal to 200, and z is more than or equal to 2 and less than or equal to 500; in the formula (II), x is more than or equal to 3 and less than or equal to 100; in the formula (IV), y is more than or equal to 2 and less than or equal to 200. Q is selected from any one of the following structures:
Figure BDA0003758182050000023
in some embodiments, the molar ratio of primary amine of formula (II) to L-amino acid-N-carboxyanhydride of formula (III) is 1: 2 to 200.
In some embodiments, the molar ratio of the primary amine of formula (IV) to the compound of formula (V), (VI), or (VII) is 1: 2 to 500.
In some embodiments, in steps 1) to 3), the solvent for the reaction is at least one of N, N-dimethylformamide, dichloromethane, and chloroform; the reaction temperature is 10-65 ℃ and the reaction time is 24-120 h.
The invention also provides application of the polyamino acid in preparation of an immunologic adjuvant or a vaccine.
The invention also provides a nano particle prepared by self-assembling the polyamino acid in water or an aqueous solvent.
In some embodiments, the nanoparticles are made by:
and mixing the polyamino acid with water or an aqueous solvent, and performing ultrasonic treatment to obtain the nanoparticles.
In some embodiments, the temperature of the ultrasound is 5-30 ℃, the time is 20-80 min, and the power is 0.3-0.5 KW.
In some embodiments, the aqueous solvent comprises at least one of PBS buffer, PB buffer, pure water.
The invention also provides application of the nano-particle as an immunologic adjuvant in preparation of nano-vaccines.
The invention also provides a nano vaccine which comprises the nano particles, the antigen and acceptable auxiliary materials in the vaccine.
In some embodiments, the antigen comprises a tumor antigen and a viral antigen.
The invention provides a polyamino acid, which has a structure shown in a formula (I), and nanoparticles prepared by self-assembly of the polyamino acid in water or an aqueous solvent can cause dendritic cell activation and enhanced antigen cross-presentation capacity and adaptive immune response reaction, so that the effects of immune enhancement and treatment are achieved.
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FIG. 1 shows a nuclear magnetic resonance hydrogen spectrum of a polyamino acid according to example 1 of the present invention;
FIG. 2 shows the topographical features of the nano-vaccine of example 2 of the present invention;
FIG. 3 shows the flow analysis results of the cells after the nano-vaccine treatment of example 2 of the present invention and the control group;
FIG. 4 shows the cell supernatant factor concentration after the nano-vaccine treatment of example 2 of the present invention and a control group;
FIG. 5 shows the activation of DC cells in lymph nodes by the nano-vaccine of example 2 and the control group;
FIG. 6 shows the effect of the nano-vaccine of example 2 and the control group of the present invention in preventing and treating tumor;
FIG. 7 shows a NMR spectrum of a polyamino acid of example 7 of the present invention;
FIG. 8 shows a NMR spectrum of a polyamino acid of example 8 of the present invention.
Detailed Description
The invention provides a polyamino acid, a preparation method thereof and application thereof in preparation of vaccine adjuvants. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The polyamino acid provided by the invention has a structure shown in a formula (I):
Figure BDA0003758182050000041
wherein R is selected from- (CH) 2 ) 4 -NH 2 ，-(CH 2 ) 3 -NH-C(-NH)-NH 2 Or
Figure BDA0003758182050000042
Q is selected from any one of the following structures:
Figure BDA0003758182050000043
3≤x≤100，2≤y≤200，2≤z≤500。
in the examples of the present invention, in the formula (I), y and z are preferably: y is more than or equal to 5 and less than or equal to 60, and z is more than or equal to 10 and less than or equal to 60; more preferably: y is more than or equal to 5 and less than or equal to 30; more preferably, 30. ltoreq. y.ltoreq.50 or 50. ltoreq. y.ltoreq.80.
Preferably, 2. ltoreq. z.ltoreq.100; more preferably, z is 100. ltoreq. z.ltoreq.200; specifically, y is 12. ltoreq. y.ltoreq.18, y 5, y 15, y 25, and y 30.
In the embodiment of the invention, the number average molecular weight of the polyamino acid with the structure shown in the formula (I) is 1000-100000; in some embodiments, the polyamino acid having a structure represented by formula (I) has a number average molecular weight of 3500 to 90000; in some embodiments, the polyamino acid having the structure of formula (I) has a number average molecular weight of 4000 to 80000.
The invention also provides a preparation method of the polyamino acid, which comprises the following steps:
1) reacting primary amine with a structure shown in a formula (II) with L-amino acid-N-internal carboxylic anhydride shown in a formula (III) to obtain polyamino acid with a structure shown in a formula (IV);
2) reacting the polyamino acid with the structure of formula (IV) with a compound with any structure of formulae (V) to (VII), and deprotecting to obtain the polyamino acid with the structure of formula (I);
Figure BDA0003758182050000051
wherein, in the formula (I), R is- (CH) 2 ) 4 -NH 2 ，-(CH 2 ) 3 -NH-C(-NH)-NH 2 And
Figure BDA0003758182050000052
x is more than or equal to 3 and less than or equal to 100, y is more than or equal to 2 and less than or equal to 200, and z is more than or equal to 2 and less than or equal to 500; in the formula (II), x is more than or equal to 3 and less than or equal to 100; in the formula (IV), y is more than or equal to 2 and less than or equal to 200. Q is selected from any one of the following structures:
Figure BDA0003758182050000053
in the invention, the polyamino acid with the structure of the formula (I) is prepared according to the following method:
a first reaction stage: reacting primary amine with a structure shown in a formula (II) with L-amino acid-N-inner carboxylic anhydride (III) in N, N-dimethylformamide serving as a solvent to obtain polyamino acid with a structure shown in a formula (IV);
and (3) a second reaction stage: reacting the polyamino acid with the structure of formula (IV) with a compound shown as the structures of formula (V), (VI) or (VII), and deprotecting to obtain the polyamino acid with the structure of formula (I).
In some embodiments, the molar ratio of the primary amine having the structure of formula (II) to the L-amino acid-N-carboxyanhydride having the structure of formula (III) is 1: 2-200; preferably, the molar ratio of the primary amine with the structure of formula (II) to the L-amino acid-N-internal carboxylic anhydride with the structure of formula (III) is 1: 5 to 60.
In some embodiments, the molar ratio of the primary amine having the structure of formula (IV) to the compound having the structure of formula (V), (VI), or (VII) is 1: 2-200; preferably, the molar ratio of the primary amine with the structure of formula (IV) to the compound with the structure of formula (V), (VI) or (VII) is 1: 10 to 60.
In some embodiments, in steps 1) to 2), the solvent for the reaction is at least one of N, N-dimethylformamide, dichloromethane, and chloroform; the reaction temperature is 10-65 ℃, preferably 18-35 ℃, and the reaction time is 24-120 h, preferably 72-85 h.
In the examples of the present invention, the L-amino acid-N-carboxyanhydride has a structure represented by the formula (III). The present invention is not particularly limited in its source, and may be generally commercially available.
Figure BDA0003758182050000061
Q is selected from any one of the following structures:
Figure BDA0003758182050000062
in the examples of the present invention, the compound of formula (V) is prepared according to methods well known to those skilled in the art by:
reacting N-benzyloxycarbonyl-D-lysine with triphosgene in a tetrahydrofuran solvent to obtain a compound having a structure represented by formula (V).
In the embodiment of the invention, the reaction temperature of the N-benzyloxycarbonyl-D-lysine and the triphosgene is 40-70 ℃; preferably, the reaction temperature is 50-65 ℃. The reaction time is 0.5-6 h, and preferably, the reaction temperature is 3-5 h. In the present example, N-benzyloxycarbonyl-D-lysine and triphosgene were reacted under nitrogen protection.
In the embodiment of the invention, the mass ratio of the N-benzyloxycarbonyl-D-lysine to the triphosgene is 1: 0.5 to 2; preferably, the mass ratio of the N-benzyloxycarbonyl-D-lysine to the triphosgene is 1: 0.5 to 1.
In the embodiment of the invention, after the reaction of the N-benzyloxycarbonyl-D-lysine and triphosgene is completed, the product is settled by using ice petroleum ether, and a crude product is obtained by means of suction filtration. Further, the crude product was dissolved with low temperature ethyl acetate and transferred to a separatory funnel. Then, washing off insoluble impurities and byproducts by using a low-temperature saturated sodium chloride aqueous solution to obtain a compound solution with the structure of the formula (V). And then, adding anhydrous magnesium sulfate to dry the compound solution for 6-24 hours. Finally, removing anhydrous magnesium sulfate by a filtration mode, and drying the solution to obtain the pure compound with the structure of the formula (V).
In the examples of the present invention, the compound of formula (vi) is prepared according to methods well known to those skilled in the art by:
a250 ml dry flask with a magnet was charged with tris-N-benzyloxycarbonyl-D-arginine (2.0g, 3.5mmol) which had been dried in vacuo for 1 hour. Dry dichloromethane (100mL) was added to the flask through a cannula under nitrogen. After the amino acid had dissolved, α' -dichloromethyl ether (0.5mL of 5.5mmol) was added via cannula under nitrogen. The flask was then equipped with a reflux condenser under nitrogen and heated at 48 ℃ for 36 hours. The volatiles were then removed under vacuum and the reaction flask was transferred to a glove box under nitrogen. The residue was crystallized five times in succession from a mixture of tetrahydrofuran and n-hexane (1:3) in a drying oven to give the compound of the formula (vi) (0.85 g, 73%) as a white solid.
In the examples of the present invention, the compounds having the structure of formula (VII) are prepared according to methods well known to those skilled in the art. The preparation method comprises the following steps:
under stirring in an ice bath, 30mL of anhydrous tetrahydrofuran was quickly added to a single-neck flask containing 6g of dry Boc-D-His (trt) -OH powder, and 10mL of 1.5mL of thionyl chloride diluted in anhydrous tetrahydrofuran was slowly added dropwise to the above solution. The reaction was carried out for 3h under ice-bath conditions, and then precipitated with 300mL of anhydrous ether and filtered. The crude product obtained by filtration was dissolved with low temperature ethyl acetate and transferred to a separatory funnel. Then, washing off insoluble impurities and byproducts by using a low-temperature saturated sodium chloride aqueous solution to obtain a compound solution with the structure of the formula (VII) through purification. And then, adding anhydrous magnesium sulfate to dry the compound solution for 6-24 hours. Finally, removing anhydrous magnesium sulfate by a filtration mode, and drying the solution to obtain the pure compound with the structure of the formula (VII).
The invention also provides application of the polyamino acid in preparation of an immunologic adjuvant or a vaccine.
The invention also provides a nano particle prepared by self-assembling the polyamino acid in water or an aqueous solvent.
In the present invention, the nanoparticles may be prepared in a manner well known to those skilled in the art. In the embodiment of the invention, the preparation method specifically adopts an ultrasonic method, and comprises the following steps:
and adding polyamino acid into water or a water-containing solvent, and carrying out ultrasonic treatment to obtain the polyamino acid nano vaccine adjuvant.
In the embodiment of the invention, the temperature of the ultrasonic wave is 5-30 ℃, the time is 20-80 min, and the power is 0.3-0.5 KW.
In an embodiment of the present invention, the aqueous solvent includes at least one of PBS buffer, PB buffer, and pure water.
The invention also provides application of the nano-particle as an immunologic adjuvant in preparation of nano-vaccines.
The invention also provides a nano vaccine, which comprises the nano particles, the antigen and acceptable auxiliary materials in the vaccine.
In embodiments of the invention, the antigens include tumor antigens and viral antigens.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
In the following examples, the starting materials used are either commercially available or prepared according to conventional methods, the yields of the respective products being the actual product mass/theoretical product mass x 100%.
The invention is further illustrated by the following examples: in some embodiments, the polyamino acids provided herein have structures represented by formulas (I-1), (II-1), and (III-1):
Figure BDA0003758182050000081
poly (L-phenylalanine-block-D-lysine) formula (I-1)
Figure BDA0003758182050000091
Poly (L-phenylalanine-block-D-histidine) of formula (II-1)
Figure BDA0003758182050000092
Poly (L-phenylalanine-block-D-arginine) formula (III-1)
EXAMPLE 1 preparation of polyamino acid having the structure of formula (I-1)
In the present invention, the polyamino acid having a structure of formula (I-1) is prepared according to the following method:
L-phenylalanine-N-carboxyanhydride (1.434g, 7.5mmol) was weighed out dry in a dry ampoule and dissolved in 30mL of anhydrous N, N-dimethylformamide with stirring. N-hexylamine (0.051g, 0.5mmol) was then added to the glove box filled with argon and stirred at room temperature for three days. After three days, N-benzyloxycarbonyl-D-lysine-N-carboxyanhydride (1.532g, 5.0mmol) was weighed out and added to the ampoule for the above reaction, and the reaction was continued at room temperature for three days. At the end of the reaction, it was precipitated with ice dry ether and dried by centrifugation to give poly (L-phenylalanine-block-N.epsilon. -benzyloxycarbonyl-D-lysine) (1.98g, 80.8%). The above-mentioned non-deprotected polyamino acid (1.00g, 0.2mmol) was weighed into a clean flask, 10mL of trifluoroacetic acid was added and dissolved with stirring, and after complete dissolution, 3mL of a 30 wt% hydrogen bromide glacial acetic acid 50:50(v/v) mixture was added and deprotected with stirring for 1 hour. After the reaction was completed, the reaction mixture was dried by settling with ice-dry ether, and the dried product was dissolved in 20ml N, N-dimethylformamide, and the solution was dialyzed for 72 hours or more using 3500MWCO membrane. Dialysis was complete and then lyophilized to obtain the final product poly (L-phenylalanine-block-D-lysine) (0.635g, 87.6%). The nuclear magnetic results are shown in FIG. 1.
Example 2 preparation of nanoparticles (immunoadjuvants) and Nanoprovices
Preparing nano particles: weigh 4mg of the polyamino acid of example 1 into a clean glass vial, add 4mL of PBS, and make up a 1mg/mL PBS solution. And then carrying out ultrasonic treatment on the small bottle (the ultrasonic temperature is 25 ℃, the ultrasonic time is 60min, and the ultrasonic power is 0.45KW) to obtain the amino acid nano particles.
Preparing a nano vaccine: the nanoparticles are compounded with 1mg/mL and antigen (OVA) PBS solution of 0.2mg/mL at 4 ℃ and stirred for 15 min.
Example 3 morphological characterization of NanoVans
The nano-vaccine of example 2 was diluted to 0.05mg/mL PBS solution. A10. mu.L sample was dropped onto a carbon-supported membrane and allowed to stand overnight in a fume hood. And photographed using a JEOL JEM-1400 projection electron microscope at an accelerating voltage of 120kV, and the result is shown in fig. 2.
Comparative example 1 preparation of poly (L-phenylalanine-Block-L-lysine) and Nanoprotein
The poly (L-phenylalanine-block-L-lysine) has the following structural formula:
Figure BDA0003758182050000101
the preparation method comprises the following steps:
L-phenylalanine-N-carboxyanhydride (1.434g, 7.5mmol) was weighed out dry in a dry ampoule and dissolved by adding 30mL of anhydrous solvent N, N-dimethylformamide with stirring. N-hexylamine (0.051g, 0.5mmol) was then added to the argon-filled glove box and stirred at room temperature for three days. After three days, N-benzyloxycarbonyl-L-lysine-N-carboxyanhydride (1.532g, 5.0mmol) was weighed out and added to the ampoule for the above reaction, and the reaction was continued at room temperature for three days. After the reaction was completed, the reaction mixture was precipitated with ice-dry ether and centrifugally dried to obtain poly (L-phenylalanine-block-N.epsilon. -benzyloxycarbonyl-L-lysine) (2.05g, 83.7%). The above-mentioned non-deprotected polyamino acid (1.00g, 0.2mmol) was weighed into a clean flask, 10mL of trifluoroacetic acid was added and dissolved with stirring, and after complete dissolution, 3mL of a 30 wt% hydrogen bromide glacial acetic acid 50:50(v/v) mixture was added and deprotected with stirring for 1 hour. After the reaction was completed, the reaction mixture was dried by settling with ice-dry ether, and the dried product was dissolved in 20ml N, N-dimethylformamide, and the solution was dialyzed for 72 hours or more using 3500MWCO membrane. Dialysis was complete and then lyophilized to obtain the final product poly (L-phenylalanine-block-L-lysine) (0.625g, 86.2%).
Vaccine adjuvants and nano-vaccines were prepared according to the method of example 2.
Example 4 bone marrow-derived dendritic cell (BMDC) activation assay
Preparation of BMDC: dendritic cells were isolated from bone marrow cells of C57BL/6N mice. Bone marrow was collected from femur and tibia, single cell suspensions were cultured in RPMI 1640, 10% heat-inactivated fetal bovine serum, 1% penicillin streptomycin, 20ng/mL GM-CSF, and 10ng/mL IL-4 were added to RPMI 1640. Non-adherent and loosely adherent cells from day 7-10 of differentiation were collected for study.
Test procedure
1. BMDC was added to 25 million/well in 24-well plates per well.
2. A blank control, a free OVA solution group and a poly (L-phenylalanine-block-L-lysine) control (comparative example 1) were set.
3. The formulated nano-vaccine of example 2 was added to each well to give a final nanoparticle concentration of 8. mu.g/mL.
4. The 24-well plate was placed in a 37 ℃ incubator for 12 hours.
5. After each treatment, BMDCs were harvested, resuspended in flow cytometry (FACS) buffer (1% fetal bovine serum in PBS), and then flow analyzed after 30min staining with fluorescently labeled antibodies to CD11c, CD86, CD40, MHC-II and MHC-I, the results of which are shown in FIG. 3. And cell supernatants were collected and the levels of cytokines TNF-. alpha., IFN-. gamma., IL-6 and IL-12p70 were measured using enzyme-linked immunosorbent assay (ELISA) analysis, and the results are shown in FIG. 4.
As shown by the results of FIGS. 3 and 4, the poly (L-phenylalanine-block-D-lysine) represented by formula I-1 of the present invention and the control group poly (L-phenylalanine-block-L-lysine), poly (L-phenylalanine-block-D-lysine) significantly promoted CD86, CD40, MHC-II and MHC-I of BMDC, causing dendritic cell activation and antigen cross-presentation. And obviously improve the secretion of IL-6, IL-12p70, IFN-gamma and TNF-alpha, enhance the immune response reaction and improve the body protection capability.
Example 5 in vivo Lymph Node (LN) assay
The mice were injected subcutaneously with the nano-vaccines of examples 3 and 4 on day 0, and the inguinal lymph node of the mice was dissected on day 3 and treated by mechanical disruption at 37 ℃, and then the samples were passed through a 200-mesh nylon mesh filter to obtain single cell suspensions, and cell counting was performed by a cell counting plate. DC maturity was then analyzed by incubation with anti-CD 11c, anti-CD 80, anti-CD 40, and anti-MHC-II for 30 minutes at 4 ℃. Samples were flow data acquired on a flow cytometer (BD FACSCelesta) and analyzed by FlowJo software. The results are shown in FIG. 5.
In vivo experiments were performed to evaluate the immune effect of the nano-vaccine of example 2, and the results of fig. 4 show that the injection of the vaccine (poly-L-phenylalanine-block-D-lysine) nano-vaccine of example 2 causes an increase in the number of lymphocytes in lymph nodes compared to the poly (L-phenylalanine-block-L-lysine) nano-vaccine of comparative example 1. Also, the DC cell activation markers CD80, CD40 and MHC-II high in lymph nodes indicate DC cell activation, which is consistent with in vitro experimental results.
Example 6 in vivo tumor prevention experiments
In a prophylactic study, each group of female C57BL/6N mice was injected subcutaneously with the nano-vaccine of example 2 1 time every 1 week and three consecutive inoculations. 7 days after the last inoculation, the inoculated mice were injected subcutaneously with 2 x 10 5 B16-OVA cells. The tumor volume was measured every other day with a caliper according to the formula V length width 2mm 3 ). When the tumor volume reaches 2000mm 3 At that time, the mice were euthanized.
In vivo tumor prevention experiments were performed to evaluate the immune effect of the nano-vaccine of example 2, and the results are shown in fig. 6. The results in fig. 6 show that the poly (L-phenylalanine-block-D-lysine) nano vaccine injected in example 2 can effectively inhibit the growth of tumor and prolong the survival time of mice, and compared with the vaccine prepared by the commercial aluminum adjuvant and the poly (L-phenylalanine-block-L-lysine) nano vaccine in comparative example 1, the vaccine has statistical difference and obvious action advantage.
Example 7 preparation of polyamino acid having the Structure of formula (II-1)
In the present invention, the polyamino acid having a structure of formula (II-1) is prepared according to the following method:
L-phenylalanine-N-carboxyanhydride (0.613g, 3.2mmol) was weighed out dry in a dry ampoule and dissolved in 30mL of anhydrous N, N-dimethylformamide with stirring. N-hexylamine (0.020g, 0.2mmol) was then added to the argon-filled glove box and stirred at room temperature for three days. After three days, N (IM) -trityl-D-histidine-N-carboxyanhydride (0.512g, 1.2mmol) was weighed into the ampoule for the above reaction and allowed to react at room temperature for three additional days. At the end of the reaction, it was precipitated with ice dry ether and dried by centrifugation to give poly (L-phenylalanine-block-trityl-D-histidine) (0.74g, 78.8%). The above-mentioned non-deprotected polyamino acid (0.50g, 0.1mmol) was weighed into a clean flask, and dissolved by adding 4mL of dichloromethane and 4mL of trifluoroacetic acid with stirring for 1 hour to deprotect. After the reaction was completed, triethylsilane was added in an equimolar amount to the imidazole group, and then precipitated and dried with ice anhydrous ether, and the dried product was dissolved in 20ml N, N-dimethylformamide and the solution was dialyzed with a 2000MWCO membrane for 72 hours or more. Dialysis was complete and then lyophilized to obtain the final product poly (L-phenylalanine-block-D-histidine) (0.346g, 90.5%). The nuclear magnetic results are shown in FIG. 7.
Example 8 preparation of a polyamino acid having a Structure of formula (III-1)
In the present invention, the polyamino acid having a structure of formula (III-1) is prepared according to the following method:
L-phenylalanine-N-carboxyanhydride (0.613g, 3.2mmol) was weighed out dry in a dry ampoule and dissolved in 30mL of anhydrous N, N-dimethylformamide with stirring. N-hexylamine (0.020g, 0.2mmol) was then added to the argon-filled glove box and stirred at room temperature for three days. After three days, di-N-benzyloxycarbonyl-D-arginine-N-carboxyanhydride (0.523g, 1.2mmol) was weighed out and added to the ampoule for the above reaction, and the reaction was continued at room temperature for three days. At the end of the reaction, it was precipitated with ice dry ether and dried by centrifugation to give poly (L-phenylalanine-block-dibenzyloxycarbonyl-D-arginine) (0.82g, 82.6%). The above-mentioned non-deprotected polyamino acid (0.50g, 0.1mmol) was weighed into a clean flask, 20mL of trifluoroacetic acid was added and dissolved with stirring, and after complete dissolution 6mL of a 30 wt% hydrogen bromide glacial acetic acid 50:50(v/v) mixture was added and deprotected with stirring for 1 hour. After the reaction was completed, the reaction mixture was dried by settling with ice-dry ether, and the dried product was dissolved in 20ml of N, N-dimethylformamide, and the solution was dialyzed for 72 hours or more using a 2000MWCO membrane. Dialysis was complete and then lyophilized to obtain the final product poly (L-phenylalanine-block-D-arginine) (0.377g, 88.8%). The nuclear magnetic results are shown in FIG. 8.
Example 9 average particle size of polyamino acid nanoparticles
Preparing nano particles: the polyamino acids shown in Table 1 were weighed out in an amount of 4mg and added to a clean glass vial, and 4mL of PBS was added to prepare a 1mg/mL PBS solution. Then, the vial is subjected to ultrasonic treatment (the ultrasonic temperature is 25 ℃, the ultrasonic time is 60min, and the ultrasonic power is 0.45KW), so that amino acid nanoparticles can be obtained, wherein the average particle size of the nanoparticles is shown in Table 1.
TABLE 1 average particle diameter of polyamino acid nanoparticles
Figure BDA0003758182050000131
Figure BDA0003758182050000141
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Claims (12)

1. A polyamino acid having a structure represented by formula (I):
Figure FDA0003758182040000011
wherein R is selected from- (CH) 2 ) 4 -NH 2 ，-(CH 2 ) 3 -NH-C(-NH)-NH 2 Or
Figure FDA0003758182040000012
Q is selected from any one of the following structures:
Figure FDA0003758182040000013
3≤x≤100，2≤y≤200，2≤z≤500。
2. the polyamino acid of claim 1, wherein y is 5. ltoreq. y.ltoreq.60 and z is 10. ltoreq. z.ltoreq.60.
3. The method for producing a polyamino acid according to claim 1 or 2, which comprises:
1) reacting primary amine with a structure shown in a formula (II) with L-amino acid-N-internal carboxylic anhydride shown in a formula (III) to obtain polyamino acid with a structure shown in a formula (IV);
2) reacting the polyamino acid with the structure of formula (IV) with a compound with any structure of formulae (V) to (VII), and deprotecting to obtain the polyamino acid with the structure of formula (I);
Figure FDA0003758182040000014
wherein, in the formula (I), R is- (CH) 2 ) 4 -NH 2 ，-(CH 2 ) 3 -NH-C(-NH)-NH 2 And
Figure FDA0003758182040000021
x is more than or equal to 3 and less than or equal to 100, y is more than or equal to 2 and less than or equal to 200, and z is more than or equal to 2 and less than or equal to 500; in the formula (II), x is more than or equal to 3 and less than or equal to 100; in the formula (IV), y is more than or equal to 2 and less than or equal to 200; q is selected from any one of the following structures:
Figure FDA0003758182040000022
4. the process according to claim 3, wherein the molar ratio of the primary amine of formula (II) to the L-amino acid-N-carboxyanhydride of formula III is 1: 2 to 200.
5. The method according to claim 3, wherein the molar ratio of the primary amine having the structure of formula (IV) to the compound having the structure of formula (V), (VI) or (VII) is 1: 2 to 500.
6. The method according to claim 3, wherein in steps 1) to 3), the solvent for the reaction is at least one of N, N-dimethylformamide, dichloromethane and chloroform; the reaction temperature is 10-65 ℃ and the reaction time is 24-120 h.
7. Use of the polyamino acid according to claim 1 or the polyamino acid prepared by the preparation method according to any one of claims 3 to 6 for the preparation of an immunoadjuvant or a vaccine.
8. Nanoparticles, which are prepared by self-assembly of the polyamino acid according to claim 1 or the polyamino acid prepared by the preparation method according to any one of claims 3 to 6 in water or an aqueous solvent.
9. A nanoparticle according to claim 8, wherein the aqueous solvent comprises at least one of PBS buffer, PB buffer, pure water.
10. Use of a nanoparticle according to claim 8 or 9 as an immunoadjuvant in the preparation of a nano-vaccine.
11. A nano-vaccine comprising the nanoparticle of claim 8 or 9, an antigen and an adjuvant acceptable in a vaccine.
12. The nano-vaccine of claim 11, wherein the antigens comprise tumor antigens and viral antigens.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0503301A2 (en) * 1991-02-14 1992-09-16 Fuji Photo Film Co., Ltd. Peptide derivatives and application thereof
CN1691889A (en) * 2002-08-23 2005-11-02 沃尔特及伊莱萨霍尔医学研究院 Curing and preventing method

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0503301A2 (en) * 1991-02-14 1992-09-16 Fuji Photo Film Co., Ltd. Peptide derivatives and application thereof
CN1691889A (en) * 2002-08-23 2005-11-02 沃尔特及伊莱萨霍尔医学研究院 Curing and preventing method

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