CN114716679B - Poly (beta-amino ester) polymer with reactive oxygen species and preparation method and application thereof - Google Patents

Poly (beta-amino ester) polymer with reactive oxygen species and preparation method and application thereof Download PDF

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CN114716679B
CN114716679B CN202210369190.9A CN202210369190A CN114716679B CN 114716679 B CN114716679 B CN 114716679B CN 202210369190 A CN202210369190 A CN 202210369190A CN 114716679 B CN114716679 B CN 114716679B
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CN114716679A (en
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谭松巍
丁家辉
袁彩燕
张菡丹
张冲
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Huazhong University of Science and Technology
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G75/00—Macromolecular compounds obtained by reactions forming a linkage containing sulfur with or without nitrogen, oxygen, or carbon in the main chain of the macromolecule
    • C08G75/02—Polythioethers
    • C08G75/04—Polythioethers from mercapto compounds or metallic derivatives thereof
    • C08G75/045—Polythioethers from mercapto compounds or metallic derivatives thereof from mercapto compounds and unsaturated compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0091—Purification or manufacturing processes for gene therapy compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
    • C08G73/02—Polyamines

Abstract

The invention discloses a poly (beta-amino ester) polymer with reactive oxygen species, a preparation method and application thereof, wherein the poly (beta-amino ester) polymer contains an A block, a B block and a capping agent residue, and the structural formula of the A block is as follows:
Figure DDA0003587241420000011
the structural formula of the B block is as follows:

Description

Poly (beta-amino ester) polymer with reactive oxygen species and preparation method and application thereof
Technical Field
The invention relates to a poly (beta-amino ester) polymer with active oxygen responsiveness, a preparation method and application thereof.
Background
The gene therapy is a novel anti-tumor therapy and has good application prospect. Exogenous genes are delivered into cells as a special drug, so that the knocking-in, knocking-out, activating or silencing of the specific genes can be realized, and the occurrence and development processes of diseases are changed.
However, in order to function in the nucleus, the therapeutic gene needs to overcome the tissue, cell, etc. weight barrier, and the plasmid is easily degraded by nuclease under physiological environment, and the vector is the key to solve the problem. The vectors mainly comprise two types, namely a viral vector and a non-viral vector, and the viral vector limits clinical application due to safety problems. Poly (beta-amino ester) (PBAE) in a non-viral vector is a biodegradable cationic polymer, has good biocompatibility and pH responsiveness, and has been applied to gene vectors.
Chinese patent CN106554499B discloses a disulfide bond-containing poly (beta-amino ester) polymer gene carrier, a synthetic method and application thereof, wherein the polymer is prepared from a disulfide bond-containing diacrylate monomer with the following structural formula
Figure BDA0003587241400000011
And the polymer is synthesized by carrying out addition polymerization reaction with 5-amino-1-amyl alcohol and then adopting small molecular amine end capping, but the transfection efficiency of the polymer is still lower.
Disclosure of Invention
In view of the shortcomings and drawbacks of the prior art, the present invention provides an improved poly (β -amino ester) based polymer with reactive oxygen species that can increase the transfection efficiency of cells.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a poly (β -amino ester) based polymer having reactive oxygen species, characterized by: the poly (beta-amino ester) polymer comprises an A block, a B block and a capping agent residue, wherein the A block has the following structural formula:
Figure BDA0003587241400000012
wherein R is 1 Is C2-C26 alkylene, C2-C26 alkylene interrupted by 1-8 oxygen atoms, R 2 Is C2-C10 alkylene, C2-C10 alkylene interrupted by 1-2 oxygen atoms or +.>
Figure BDA0003587241400000013
The structural formula of the B block is as follows: />
Figure BDA0003587241400000021
Wherein R is 1 Is C2-C26 alkylene, C2-C26 alkylene interrupted by 1-8 oxygen atoms, R 3 Is C3-C5 alkyl, - (CH) 2 ) a OH、-(CH 2 ) a N(CH 3 ) 2 、-(CH 2 ) a O(CH 2 ) a OH or
Figure BDA0003587241400000022
Wherein a is an integer of 2 to 5.
In some embodiments of the invention, R 1 Is C2-C10 alkylene, C5-C26 alkylene interrupted by 1-8 oxygen atoms, R 2 Is C2-C10 alkylene, C8 alkylene interrupted by 2 oxygen atoms, or
Figure BDA0003587241400000023
R 3 Is propyl, amyl, - (CH) 2 ) a OH、-(CH 2 ) a N(CH 3 ) 2 、-(CH 2 ) a O(CH 2 ) a OH or
Figure BDA0003587241400000024
Wherein a is 2,3 or 5.
In some embodiments of the invention, the a block is obtained by addition polymerization of a diacrylate and a dithiol and the B block is obtained by addition polymerization of a diacrylate and an amino group containing monomer.
In some embodiments of the invention, the capping agent has the structure of
Figure BDA0003587241400000025
Figure BDA0003587241400000026
Wherein a is an integer of 2 to 5, R 4 Is a C3-C6 alkylene group, a C8-C12 alkylene group interrupted by 1 to 3 oxygen atoms.
In some embodiments of the invention, the diacrylates are independently selected from the following formulas:
Figure BDA0003587241400000031
preferably, the diacrylate is selected from the following structural formulas:
Figure BDA0003587241400000032
in some embodiments of the invention, the dithiol is selected from the following structural formulas:
Figure BDA0003587241400000041
preferably, the dithiol is selected from the following structural formulas:
Figure BDA0003587241400000042
in some embodiments of the invention, the amino group-containing monomer is selected from the following structural formulas:
Figure BDA0003587241400000051
preferably, the amino group-containing monomer is selected from the following structural formulas:
Figure BDA0003587241400000052
in some embodiments of the invention, the capping agent is selected from the following structural formulas:
Figure BDA0003587241400000053
preferably, the capping agent is selected from the following structural formulas:
Figure BDA0003587241400000061
according to the research, the inventor finds that dithiol monomer units which are added and polymerized with diacrylate are introduced into a traditional PBAE main chain, the obtained PBAE polymer main chain is provided with thioether building, the chemical bond can respond to high-concentration Reactive Oxygen Species (ROS) in cancer cells, when the polymer is used as a gene therapy carrier, the targeted release of gene therapy can be realized, meanwhile, the intracellular ROS can be reduced, the oxidative stress is reduced, the transfection efficiency is improved, and the targets of high efficiency and low toxicity are realized.
In some embodiments of the invention, the poly (β -amino ester) based polymer has a number average molecular weight of 1000 to 100000 Da.
In some embodiments of the invention, the poly (β -amino ester) based polymer has a mass percent of thioether bonds of 0.1 to 25%.
The invention also provides a preparation method of the poly (beta-amino ester) polymer with active oxygen responsiveness, which comprises the following steps: the diacrylates and dithiols are subjected to addition polymerization in an organic solvent, and then amino-containing monomers are added for addition polymerization, and the method further comprises the step of adding a blocking agent for blocking.
In some embodiments of the invention, the organic solvent is selected from the group consisting of one or more of dichloromethane, N-dimethylformamide, dimethyl sulfoxide, methanol, and ethanol.
In some embodiments of the invention, the reaction temperature is 15 to 30 ℃ and the reaction time is 2 to 8 hours in the addition polymerization of the diacrylate and the dithiol.
In some embodiments of the invention, the addition polymerization is carried out with the addition of the amino-containing monomer at a reaction temperature of 50 to 120℃for a reaction time of 24 to 72 hours.
In some embodiments of the invention, the capping step is carried out at a reaction temperature of 15 to 30℃for a reaction time of 16 to 30 hours.
In some embodiments of the invention, the method specifically comprises the steps of:
1) Performing addition polymerization on the diacrylate and the dithiol in an organic solvent;
2) Removing the organic solvent, and adding an amino-containing monomer for addition polymerization to obtain an unfilled polymer;
3) And dissolving the non-terminated polymer in a solvent, and adding the capping agent to carry out capping reaction to obtain the poly (beta-amino ester) polymer with the active oxygen responsiveness.
The molar feed ratio of the diacrylate to the dithiol to the amino-containing monomer is 100:1:99-100:99:1.
By adjusting the types and molar feed ratios of the diacrylates, dithiols, amino-containing monomers, and capping agents, a range of different PBAE-type polymers can be obtained.
In some embodiments of the invention, after the addition polymerization in step 2), the purification is performed by precipitation with methanol and diethyl ether.
In some embodiments of the invention, the solvent is selected from the group consisting of one or more of dimethyl sulfoxide, methylene chloride, N-dimethylformamide, methanol, and ethanol.
In some embodiments of the present invention, after the capping reaction, ether precipitation, centrifuge precipitation, and vacuum drying are employed to obtain the poly (β -amino ester) based polymer with reactive oxygen species.
In some embodiments of the invention, the method specifically comprises the steps of:
(1) Weighing diacrylate (B series) and dithiol (S series) in a specific molar ratio by using an analytical balance, adding a proper amount of dichloromethane, reacting for 2-8 hours at room temperature, removing the dichloromethane by using a rotary evaporator, adding a proper amount of amino-containing monomer (N series), heating to 50-120 ℃ and reacting for 24-72 hours;
(2) Stopping the reaction, dissolving the reaction solution with a proper amount of methanol, dripping the reaction solution into diethyl ether with the volume of 4-10 times for precipitation, discarding the supernatant diethyl ether, washing twice with diethyl ether, and drying in a vacuum drying oven to obtain an end-free polymer;
(3) Dissolving the non-terminated polymer by using a proper amount of dimethyl sulfoxide (DMSO), adding a 2-10 times molar quantity of a blocking agent (E series) relative to the non-terminated polymer, and reacting for 24 hours at room temperature;
(4) Stopping the reaction, dripping the reaction solution into diethyl ether with the volume of 6-10 times for precipitation, washing twice again, centrifuging by using a centrifugal machine to obtain precipitation, drying in a vacuum drying oven, and obtaining the poly (beta-amino ester) polymer with the active oxygen responsiveness after the diethyl ether is completely pumped away.
The invention also provides the application of the poly (beta-amino ester) polymer with active oxygen responsiveness in gene vectors.
The invention also provides a gene vector which is prepared from the poly (beta-amino ester) polymer with active oxygen responsiveness.
The invention also provides a gene therapy drug, which comprises nano particles, wherein the nano particles are formed by compounding the poly (beta-amino ester) polymer with active oxygen responsiveness and genes.
Preferably, the mass ratio of the poly (beta-amino ester) polymer to the gene is 30-100:1.
In some embodiments of the invention, the nanoparticle is prepared by dissolving the aforementioned poly (β -amino ester) based polymer having active oxygen responsiveness in an acidic buffer, and then mixing with the gene.
Preferably, the poly (beta-amino ester) polymer with active oxygen responsiveness is dissolved in an acidic buffer solution to obtain a polymer solution with the concentration of 15-60mg/mL, the polymer solution and the gene are uniformly mixed, and incubated for 0.4-2 hours at 15-30 ℃ to obtain the nanoparticle.
Further, the acidic buffer is selected from one or more of an acetate buffer, a citrate buffer, and a phosphate buffer.
Compared with the prior art, the invention has the following advantages:
according to the invention, dithiol monomer units are introduced into a traditional PBAE main chain through addition polymerization, and when the obtained PBAE polymer is used as a gene therapy carrier, the targeted release of gene therapy can be realized, meanwhile, intracellular ROS (reactive oxygen species) can be reduced, the oxidative stress is reduced, and meanwhile, the transfection efficiency can be improved.
According to the invention, a series of different PBAE polymers can be obtained by adjusting the types and the molar feed ratios of the diacrylate, the dithiol, the amino-containing monomer and the end capping agent, and the various polymers can be used for efficiently transfecting cells.
Drawings
FIG. 1 is a nuclear magnetic resonance diagram of a polymer B1S5N4E 3;
FIG. 2 is a nuclear magnetic resonance diagram of polymer B3S2N5E 1;
FIG. 3 is an infrared spectrum of polymer B3S2N5E 1;
FIG. 4 is a DLS particle size plot of nanoparticles after complexing each polymer with GFP, respectively;
FIG. 5 is a transmission electron microscope image of nanoparticles after the polymer B12S8N2E6 is compounded with GFP;
FIG. 6 shows the results of flow transfection efficiency of Polymer B4S5N6E1 at 293T cells transfected at three different monomer ratios;
FIG. 7 is a flow-based quantitative statistical result of transfection of each polymer nanoparticle in 293T cells;
FIG. 8 is a fluorescence microscope image of the polymer B5S3N5E4, B3S5N9E4, B6S8N4E6 composite nanoparticle transfected at 293T;
FIGS. 9 a-9 f are the results of transfection flow of polymer B5S3N5E4, B3S5N9E4, B6S8N4E6 composite nanoparticles at 293T;
FIG. 10 is a fluorescence microscope image of the transfection of polymer B4S5N6E1 composite nanoparticles in Siha, hela, me180, C666-1 cells;
FIGS. 11 a-11 f are transfection flow type results of polymer B4S5N6E1 composite nanoparticles in Siha, hela, me180, C666-1 cells;
FIG. 12 is ROS responsiveness on Siha cells after complexing polymer B5S3N5E4 and PBAE with RFP plasmid;
FIG. 13 shows the results of in vivo transfection of polymers B7S4N4E6 and B10S1N7E2 and GFP complexes in SD rat vagina.
Detailed Description
The invention is further described below with reference to examples. The present invention is not limited to the following examples. The implementation conditions adopted in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions which are not noted are conventional conditions in the industry. The technical features of the various embodiments of the present invention may be combined with each other as long as they do not form a conflict with each other.
The corresponding species are indicated in the following examples by the numbers of diacrylates, dithiols, amino-containing monomers, and end-capping agents in the formulae of the specification.
Example 1
This example provides a poly (β -amino ester) based polymer with reactive oxygen species, abbreviated as B1S5N4E3.
Weighing specific mass of B1, S5 and N4 (according to the molar ratio of B1 to S5 to N4=5 to 1 to 4) by using an analytical balance, adding a proper amount of dichloromethane into the B1 and the S5, reacting for 4 hours at room temperature, removing the dichloromethane by using a rotary evaporator, adding the N4, and heating to 90 ℃ for reacting for 24 hours; stopping the reaction, dissolving the reaction solution with a proper amount of methanol, dripping the reaction solution into diethyl ether with the volume of 6 times for precipitation, discarding the supernatant diethyl ether, washing twice with diethyl ether, and drying in a vacuum drying oven to obtain an uncapped polymer; dissolving the non-terminated polymer by using a proper amount of DMSO, adding E3 with a molar ratio of 4 times that of the non-terminated polymer, and reacting for 24 hours at room temperature; stopping the reaction, dripping the reaction solution into diethyl ether with the volume of 6 times for precipitation, washing twice again, centrifuging by using a centrifugal machine to obtain precipitation, drying in a vacuum drying oven, and obtaining a final product B1S5N4E3 after the diethyl ether is completely pumped away.
10mg of B1S5N4E3 was weighed, dissolved by 200. Mu.l of deuterated chloroform and put into a nuclear magnetic resonance tube with a diameter of 5mm, and hydrogen spectrum measurement was performed by a nuclear magnetic resonance spectrometer, and the result is shown in FIG. 1. Wherein, sigma 4.065 (g) is-OCH on the diacrylate chain 2 CH 2 The ratio of the peak areas of the hydrogen signal peak of O-and the peak areas of Sigma 1.530 (e) and 1.411 (d) is 1:1, and the peak area ratio is methylene-CH on the 4-amino-1-butanol chain 2 -CH 2 -a hydrogen signal peak, sigma 3.580 (f) is a methylene hydrogen signal peak on the o-hydroxy group on 4-amino-1-butanol, and a peak on 1, 5-dimercapto-pentanol is shown in the graph h, i, j, indicating successful synthesis of B1S5N4E3.
The number average molecular weight of B1S5N4E3 is 18500 calculated by nuclear magnetic resonance hydrogen spectrum.
Example 2
This example provides a poly (beta-amino ester) based polymer with reactive oxygen species abbreviated as B3S2N5E1.
Weighing specific mass of B3, S2 and N5 (according to the molar ratio of B3 to S2 to N5=5 to 1:4) by using an analytical balance, adding a proper amount of dichloromethane into the S2 in the B3, reacting for 6 hours at room temperature, removing the dichloromethane by using a rotary evaporator, adding the N5, and heating and reacting for 36 hours at 70 ℃; stopping the reaction, dissolving the reaction solution with a proper amount of methanol, dripping the reaction solution into diethyl ether with the volume of 5 times for precipitation, discarding the supernatant diethyl ether, washing twice with diethyl ether, and drying in a vacuum drying oven to obtain an uncapped polymer; dissolving the polymer with a proper amount of DMSO, adding E1 with a molar ratio of 5 times that of the non-terminated polymer, and reacting for 24 hours at room temperature; stopping the reaction, dripping the reaction solution into diethyl ether with the volume of 6 times for precipitation, washing twice again, centrifuging by using a centrifugal machine to obtain precipitation, drying in a vacuum drying oven, and obtaining a final product B3S2N5E1 after the diethyl ether is completely pumped away.
10mg of B3S2N5E1 was weighed, dissolved in 200. Mu.l of deuterated dimethyl sulfoxide (d-DMSO), and placed in a nuclear magnetic tube having a diameter of 5mm, and hydrogen was measured by a nuclear magnetic resonance spectrometer, and the results are shown in FIG. 2. Wherein, sigma 4.062 (f) is-COOCH on the 1, 4-butanediol diacrylate chain 2 The ratio of the areas of the five peaks of the hydrogen signal peak, sigma 1.421 (p), 1.322 (q) and 1.118 (r) is 1:1:1, and the peak area is methylene-CH on the 5-amino-1-amyl alcohol chain 2 -CH 2 -CH 2 -a hydrogen signal peak, sigma 4.750 (a) is the signal peak on the hydroxyl group on 5-amino-1-pentanol, sigma 4.301 (u) is the signal peak on the hydroxyl group on dithiothreitol, sigma 3.571 (B) is the signal peak on the carbon attached to the hydroxyl group on dithiothreitol, these two groups of peaks indicate successful introduction of dithiothreitol in the polymer chain, i.e. the thioether bond, sigma 1.113 is the hydrogen atom peak at 1- (3-aminopropyl) -4-methylpiperazine l, m, N, and almost no peak is seen between sigma 5.5-6.5, indicating that the double bond end was blocked successfully, the above results indicate successful synthesis of B3S2N5E1.
Mixing B3S2N5E1 and KBr powder, tabletting, loading into Fourier infrared spectrometer, and measuring at 4000-450cm -1 Scan 32 times in range. The spectrum is shown in FIG. 3, at 3500cm -1 The broad peak appearing nearby is 5-ammonia1735cm due to stretching movement of hydroxyl groups on the base-1-pentanol and dithiothreitol -1 The nearby spike is a telescopic vibration peak of the carbonyl group in the ester in the 1, 4-butanediol diacrylate chain, indicating successful synthesis of B3S2N5E1.
Examples 3 to 18
The procedure for the synthesis of each polymer was essentially the same as in example 1, except that the molar ratio of B, S, N was varied, the reaction time at room temperature after addition of dichloromethane, the reaction temperature and time after removal of methylene chloride, and the molar ratio of E to the non-terminated polymer were varied, as shown in Table 1 below.
Respectively dissolving each polymer in sodium acetate-acetic acid buffer solution with pH of 5.0, ultrasonically dissolving to obtain carrier solutions, uniformly mixing each carrier solution with GFP plasmid according to the mass ratio of 30:1, 50:1, 75:1 and 100:1 respectively, mixing by vortex oscillation, and incubating for 30min at room temperature.
TABLE 1 conditions for synthesizing the respective polymers
Figure BDA0003587241400000101
After dilution of each of the incubated complex solutions, particle size was measured by a dynamic light scattering particle sizer (DLS). As shown in FIG. 4, the particle size of most nanoparticles is between 150-400 nm. For the nanoparticles formed after the B1S4N5E3 or B5S3N5E4 is compounded with GFP plasmid, the particle size gradually increases with the increase of the molar ratio (material ratio) of the polymer to GFP plasmid of 30, 50, 75, 100; in contrast, the particle size of the nanoparticles after the B9S22N5E2 or B6S8N4E6 was compounded with the GFP plasmid gradually decreased as the molar ratio of the polymer to the GFP plasmid increased. The B2S6N1E5 or the B7S7N3E2 has the minimum particle size under the specific material ratio. Most polymers are able to successfully control nanoparticle size below 250nm, exhibiting effective electrostatic compaction and encapsulation of DNA by the polymer.
The morphology of the nanoparticles with the material ratio of 75:1 was observed by using a Transmission Electron Microscope (TEM), and as shown in fig. 5, it can be seen that the nanoparticles formed after electrostatic compression of GFP plasmid by B12S8N2E6 were spherical in shape, and the particle size of the nanoparticles was slightly reduced as measured by DLS, possibly caused by shrinkage due to water loss during infrared baking of the nanoparticles on a copper mesh.
Example 19
B4S5N6E1 polymer synthesis: the synthesis was performed by the method of example 4 according to the molar ratios of B4 to S5 to N6 of 10:1 to 9, 10:2 to 8 and 10:4 to 6, respectively, to obtain the final product B4S5N6E1 at different molar ratios.
The following procedure was used to transfect the polymer B4S5N6E1 in 293T cells
(1) Cell culture: cells were cultured in Corning dishes with d=9 cm, 293T cells were prepared with Gibco DMEM, fetal bovine serum FBS and double antibody (penicillin-streptomycin solution) to a cell complete medium containing 100IU/mL penicillin (penicillin) and 100 μg/mL streptomycin (streptomyin), 10% FBS, and cultured in a cell incubator with 5% co2 at 37 ℃ and saturated humidity, and after the cells had adhered to the wall and grown to a density of 80-90%, the cells were digested with trypsin and passaged in a three dish ratio;
(2) Seed plate: after counting the cells with a hemocytometer, the cells were counted at about 2X 10 cells per well 5 Inoculating the cells into a six-hole plate, and placing the cells into a cell culture box for adherent culture for 24 hours;
(3) Pharmaceutical preparation: filtering and sterilizing a polymer solution dissolved by a sodium acetate-acetic acid buffer solution with the pH of 5.0 by using a 0.22 mu m water-based filter head for later use, compositing the polymer solution with GFP according to different material ratios of 30:1, 50:1, 75:1, 100:1 to obtain nanoparticle solution, standing at room temperature for 30min, preparing a composite of cationic liposome hp and GFP, and standing at room temperature for 15min to serve as a positive control group for later use;
(4) Adding the medicine: when the cell density of the attached wall reaches 70% -80%, the medicine can be administered, the original complete culture medium of each hole is sucked, each hole is washed twice by 1mL of sterile PBS, then 2mL of serum-free culture medium DMEM is added, the compounded nanoparticle solution is added, a culture dish is gently shaken and uniformly mixed, and the culture is carried out in an incubator, after 4 hours of culture, the serum-free culture medium DMEM is sucked by a dropper, and 2mL of complete culture medium is directly added to each hole for continuous culture for 36 hours;
(5) And (3) detection: after 36h of transfection, the transfection condition and the morphology of cells in a six-hole plate are respectively observed by using blue light and white light of an inverted fluorescence microscope, then the cells are digested, cell sediment is collected by centrifugation at 2000rpm multiplied by 5min, 300 mu LPBS is added, the cells are fully dispersed by vortex, and finally the transfection efficiency is detected by a flow cytometer.
293T derived from human embryonic kidney cells is a very common cell line for expression studies of foreign genes. As shown in FIG. 6, the transfection efficiency of the polymer B4S5N6E1 at a molar ratio of 10/1/9 reached a maximum of about 68% at a material ratio of 100. At a 10/2/8 molar ratio, the transfection efficiency can reach 89% at a material ratio of 75, and then when the molar ratio of S5 to B4 is increased to 4/10, the maximum transfection efficiency reaches 41% at an optimal material ratio of 100.
Example 20
Each polymer was transfected into 293T cells using the same method as in example 19
The results of flow quantification of the transfection efficiency of each polymer on 293T cells using non-dosed cells as blank (blank) and 25kDa PEI transfection group (N/P=7.5) as positive control are shown in FIG. 7, and three of these representative polymers B5S3N5E4, B3S5N9E4, B6S8N4E6 were selected for transfection fluorescence and flow testing, and the results are shown in FIGS. 8 and 9 a-9 f, respectively. The microscope results show that under the condition of white light, the cell forms are normal under various material ratios, and under the condition of fluorescence, the transfection efficiency of B5S3N5E4 and B6S8N4E6 is higher than that of PEI, the transfection efficiency of B3S3N5E4 on 293T cells is not very strong, and the transfection efficiency is gradually increased along with the increase of the material ratio, so that the material ratio is equivalent to that of PEI at 50.
From the flow results, FIGS. 9 a-9 f, it can be seen that B5S3N5E4 achieved 95% higher transfection efficiency than PEI (56.6%) at a texture ratio of 30, and the increase in texture ratio had little effect on transfection efficiency. The transfection efficiency of B3S5N9E4 increased from 12.0% of the material ratio 30 to 61.4% of the material ratio 100 with the increase of the material ratio, and the transfection efficiency was equivalent to that of PEI from 50. The transfection efficiency of B6S8N4E6 is over 90% after the material ratio exceeds 50, and the material is an excellent transfection material.
Example 21
In this example, the polymer B4S5N6E1 and GFP plasmid composite nanoparticles were used to transfect tumor cells
Siha and Hela are HPV16 positive cervical cancer cells and HPV18 positive cervical cancer cells respectively, me180 cells are HPV18 and HPV68 double-positive cervical cancer cells, and C666-1 cells are EBV positive nasopharyngeal carcinoma cells, wherein the Siha, hela and Me180 cells are similar to 293T cells in culture mode and same in culture condition; the culture conditions of C666-1 cells were identical to those of 293T cells except that 1640 was used in place of DMEM. Cell plating and drug administration and detection were as in example 19.
From the microscope results in FIG. 10, it is seen that the transfection ability of the polymer B4S5N6E1 on three cervical cancer cells of Siha, hela and Me180 is significantly higher than that on the nasopharyngeal carcinoma cell C666-1, and the fluorescence intensity of the polymer B4S5N6E1 on Hela in the cervical cancer cells is significantly higher than that on Siha and Me 180.
The flow results FIGS. 11 a-11 f show that the transfection efficiency of the polymer B4S5N6E1 on Siha cells was between 40% -60% higher than PEI at 100, and on HeLa cells, the transfection efficiency was reduced from 85.2% of 30 to 74.4% of 100 with increasing material ratio, both of which were much higher than PEI (51.4%) and reached 87.3% at 50. The transfection efficiency increased with increasing material ratio at Me180, 25.2% at 30, 43.0% higher than PEI when 50 (51.4%) was reached and 67.6% maximum at 100. The overall transfection efficiency on C666-1 cells is not high, the positive control PEI has only 6.5% of transfection efficiency on C666-1, the transfection efficiency of the material B4S5N6E1 on C666-1 is gradually increased along with the increase of the material ratio, and the transfection efficiency under the condition of the optimal material ratio of 100 is 23.2%.
Example 22
ROS responsiveness test with Polymer B5S3N5E4 and plasmid composite nanoparticles
(1) Preparation of cells: cell culture and plating operations were the same as in example 19;
(2) Pharmaceutical preparation: preparation of a sufficient amount of a composite solution with a material ratio of 75 using a thioether bond-free PBAE (B5N 5E 4) and B5S3N5E4 was performed in the same manner as in example 19 except that the plasmid was a model non-fluorescent plasmid, avoiding coincidence of the fluorescent light of the green fluorescent protein and DCF;
(3) Administration: the administration was performed in reverse order, at 8 hours, followed by 4 hours, 2 hours, 1 hour, and 0.5 hour. Two multiple wells of six well plates were set at each time point, while a non-dosing group was set as a blank control group. Before each administration, the complete culture medium in the corresponding hole is firstly sucked, each hole is washed twice by 1ml of sterile PBS, then 2ml of pure Gibco DMEM is added, the prepared medicine is added, and the mixture is gently shaken to be uniform and placed into an incubator for continuous culture;
(4) And (3) detection: cells in six well plates were digested, cell pellet was collected by centrifugation at 2000rpm x 5min, washed once with 300 μlpbs, resuspended in 200 μl of DCFH-DA solution (volume ratio DCFH-DA: dmem=1:1000), incubated at 37 ℃ for 20-30min, vortexed once every 3-5min, washed three times with 200 μl of pure DMEM, resuspended in 300 μlpbs, and fluorescence of intracellular DCF was detected by flow cytometry.
As shown in FIG. 12, the intracellular ROS content of the PBAE group increased continuously from 0.5h to 2h over time, reached a peak after 2h, decreased slightly at 4h, decreased slightly at 8h, but still was ten or more times higher than the ROS level at 0h, and the cells were put in oxidative stress. In contrast, the total level of ROS level induced by the polymer B5S3N5E4 and the plasmid composite nanoparticle in Siha cells is lower by an order of magnitude than that of PBAE, after reaching a peak value after 1h, the ROS level is continuously reduced at a later time point, and when the ROS level is reduced to be close to 0h at 8h, the ROS level is reduced to be close to 0h, so that thioether bonds in the material can respond to the intracellular elevated ROS, and excessive ROS are consumed, so that the oxidative stress in the cells is reduced, the normal physiological process of the cells is restored, the cytotoxicity is reduced and the transfection efficiency is improved.
Example 23
Transfection in SD rat vagina with Polymer B7S4N4E6 or B10S1N7E2 and GFP plasmid composite nanoparticles
About 3 female SD rats (250 g) were taken, and polymers B7S4N4E6 and B10S1N7E2 were compounded with GFP at a material ratio of 75 at a dose of 50. Mu.g GFP per SD rat, respectively, to form nanoparticles. After the SD rat is anesthetized, the rat is placed in a posture with the head slightly downward and the tail upward, nanoparticle solution is added from the vagina of the rat, after the anesthesia is maintained for 3 hours, the SD rat is leveled, after the SD rat is cultured for two days, the vagina of the rat is flushed through PBS, and vaginal epithelial cells are taken. The vaginal epithelial cells of SD rats were not dosed as BLANK, and then stained with DAPI, and after smear preparation, they were observed under a fluorescence microscope.
As shown in FIG. 13, the group without BLANK was not administered to express GFP in the blue light channel, but the group with partial vaginal epithelial cells was administered to express GFP, and the transfection efficiency of B7S4N4E6-GFP in rats was approximately 20% and the transfection efficiency of B10S1N7E2-GFP in rats was approximately 30% as estimated by DAPI transfection. It can be seen that the B7S4N4E6 and the B10S1N7E2 are better in-vitro cell transfection, have a certain transfection capacity in vivo, and have wide application prospects.
The above embodiments are provided to illustrate the technical concept and features of the present invention and are intended to enable those skilled in the art to understand the content of the present invention and implement the same, and are not intended to limit the scope of the present invention. All equivalent changes or modifications made in accordance with the spirit of the present invention should be construed to be included in the scope of the present invention.
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.

Claims (8)

1. A poly (β -amino ester) based polymer having reactive oxygen species, characterized by: the poly (beta-amino ester) polymer consists of an A block, a B block and a capping agent residue, wherein the A block has the structural formulaThe following are provided:
Figure 763882DEST_PATH_IMAGE001
wherein R is 1 Is C2-C26 alkylene, C2-C26 alkylene interrupted by 1-8 oxygen atoms, R 2 Is C2-C10 alkylene, C2-C10 alkylene interrupted by 1-2 oxygen atoms or +.>
Figure 149864DEST_PATH_IMAGE002
The method comprises the steps of carrying out a first treatment on the surface of the The structural formula of the B block is as follows:
Figure 664022DEST_PATH_IMAGE003
wherein R is 1 Is C2-C26 alkylene, C2-C26 alkylene interrupted by 1-8 oxygen atoms, R 3 Is C3-C5 alkyl, - (CH) 2 ) a OH、-(CH 2 ) a N(CH 3 ) 2 、-(CH 2 ) a O(CH 2 ) a OH or
Figure 918286DEST_PATH_IMAGE004
Wherein a is an integer of 2 to 5;
the A block is obtained by addition polymerization of diacrylate and dithiol, and the B block is obtained by addition polymerization of diacrylate and an amino-containing monomer;
the diacrylates are independently selected from the following structural formulas:
Figure 467079DEST_PATH_IMAGE006
the dithiol is selected from the following structural formulas:
Figure 23962DEST_PATH_IMAGE007
；
the amino group-containing monomer is selected from the following structural formulas:
Figure 884471DEST_PATH_IMAGE008
；
the structure of the end capping agent is that
Figure 817791DEST_PATH_IMAGE009
、/>
Figure 955512DEST_PATH_IMAGE010
、
Figure 807930DEST_PATH_IMAGE011
Or->
Figure 296680DEST_PATH_IMAGE012
Wherein a is an integer of 2 to 5, R 4 Is a C3-C6 alkylene group or a C8-C12 alkylene group interrupted by 1 to 3 oxygen atoms.
2. The active oxygen responsive poly (β -amino ester) based polymer of claim 1, wherein:
the end capping agent is selected from the following structural formulas:
Figure 768113DEST_PATH_IMAGE013
。
3. the active oxygen responsive poly (β -amino ester) based polymer of claim 1, wherein: the number average molecular weight of the poly (beta-amino ester) polymer is 1000-100000 Da; and/or the mass percentage of thioether bonds in the poly (beta-amino ester) polymer is 0.1-25%.
4. A method for preparing the poly (β -amino ester) based polymer having active oxygen responsiveness according to any one of claims 1 to 3, characterized in that: the method comprises the steps of carrying out addition polymerization on the diacrylate and the dithiol in an organic solvent, adding an amino-containing monomer for carrying out addition polymerization, and adding a blocking agent for blocking.
5. The method according to claim 4, wherein: the method specifically comprises the following steps:
1) Performing addition polymerization on the diacrylate and the dithiol in an organic solvent;
2) Removing the organic solvent, and adding an amino-containing monomer for addition polymerization to obtain an unfilled polymer;
3) And dissolving the non-terminated polymer in a solvent, and adding the capping agent to carry out capping reaction to obtain the poly (beta-amino ester) polymer with active oxygen responsiveness.
6. Use of a poly (β -amino ester) based polymer having reactive oxygen species according to any one of claims 1 to 3, characterized in that: the poly (beta-amino ester) polymer with active oxygen responsiveness is used for preparing a gene vector.
7. A gene therapy agent characterized in that: the gene therapy drug comprises nanoparticles, wherein the nanoparticles are formed by compounding the poly (beta-amino ester) polymer with the reactive oxygen species and the genes, wherein the poly (beta-amino ester) polymer has the reactive oxygen species of any one of claims 1 to 3.
8. The gene therapy drug according to claim 7, characterized in that: the nanoparticle is prepared by dissolving the poly (beta-amino ester) polymer having active oxygen responsiveness according to any one of claims 1 to 3 in an acidic buffer, and mixing with the gene.
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