CN117586524A - Composition of PEI polymer material for in vivo delivery of nucleic acid substances and preparation method thereof - Google Patents

Composition of PEI polymer material for in vivo delivery of nucleic acid substances and preparation method thereof Download PDF

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CN117586524A
CN117586524A CN202311645598.5A CN202311645598A CN117586524A CN 117586524 A CN117586524 A CN 117586524A CN 202311645598 A CN202311645598 A CN 202311645598A CN 117586524 A CN117586524 A CN 117586524A
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parts
nucleic acid
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polymer material
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王栋
黄菁
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Suzhou Xinjirui Biotechnology Co ltd
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Suzhou Xinjirui Biotechnology Co ltd
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Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to a composition of PEI polymer materials for in-vivo transfer of nucleic acid substances and a preparation method thereof, wherein the composition comprises the following components in parts by weight and the dosage: polyethylene imine, polyether polyol, modifier and amide solvent, wherein the PEI is 30-45 parts, and/or the polyether polyol is 40-60 parts, and/or the modifier is 25-40 parts, and/or the amide solvent is 20-25 parts. The composition is efficient, low in toxicity and degradable into small molecules in an initial state, can be used for transferring and conveying nucleic acid substances, and PEI is used as a cationic hydrophilic area to bind nucleic acid so as to increase cellular uptake; the preparation method is simple, low in cost and convenient to operate.

Description

Composition of PEI polymer material for in vivo delivery of nucleic acid substances and preparation method thereof
Technical Field
The invention belongs to the technical field of biomedical materials, and particularly relates to a composition of PEI polymer materials for in-vivo transfer of nucleic acid substances and a preparation method thereof.
Background
In the study of gene therapy, a key issue is how to efficiently and safely deliver a gene vector into a target cell, the gene delivery vector being an important factor affecting the effect of gene therapy. Various gene delivery systems have been developed, including viral vectors and non-viral vectors. The transfection efficiency of viral vectors is relatively high, and most of them are now viral vectors. However, there is a possibility that some replication competent viruses are brought in when transformed into pathogenic viruses or purified, and there is a possibility that an immune response may be caused due to immunogenicity, and there is a lack of targeting costs. The non-viral vector is mainly a synthesized positive cationic gene vector, and has the advantages of safety, high adaptability and easy preparation. Non-viral systems mainly include polypeptides, liposomes and cationic polymers. Synthetic polymers have attracted increasing attention in terms of nucleic acid delivery due to their wide range of uses, good safety and ease of production. Among the numerous cationic polymer carriers, polyethylenimine PEI has been of great interest due to its proton sponge effect.
Polyethylenimine (PEI), also known as polyazacyclopropane, has the formula H (NHCH) 2 CH 2 )nNH 2 The molecular structure isIs a positively charged polymeric amine with a linear or branched structure which is chelated with a variety of metal ions, is highly soluble in water and is used as an effective base in aqueous solutions, and PEI has primary, secondary, tertiary amine and other bonds in its molecular structure. Has higher charge density and proton sponge effect. Due to its excellent physicochemical properties, it is widely used in biomedical, water purification, biological imaging, gene transfer and self-healing materials.
The molecular weight of a typical PEI carrier is about 2.5 x 10 4 On the right, there is a good transfer effect, the transfer mechanism is shown in fig. 1. However, PEI has the technology that PEI is not degradable in vivo and has high toxicity as a basic building block of a carrier of nucleic acid substancesProblems. And PEI can be directly used in vivo as a gene transfection carrier to cause aggregation of erythrocytes, can be combined with non-specificity such as albumin in blood to cause formation of polymers, and has low gene transfection efficiency in vivo.
Disclosure of Invention
In order to solve the technical problems, the invention provides a composition of PEI polymer material for transferring nucleic acid substances in vivo and a preparation method thereof, wherein the composition is efficient, low in toxicity and capable of degrading into small molecules in an initial state, and can be used for transferring and conveying nucleic acid substances, and PEI is used as a cationic hydrophilic area to combine nucleic acid so as to increase cellular uptake; the preparation method is simple, low in cost and convenient to operate.
The composition and the preparation method for the PEI polymer material for in vivo transfer of nucleic acid substances solve the technical problems, and comprise the following components in parts by weight: polyethylene imine (PEI), polyether polyol, modifier and amide solvent, wherein the PEI is 30-45 parts, and/or the polyether polyol is 40-60 parts, and/or the modifier is 25-40 parts, and/or the amide solvent is 15-25 parts.
Preferably, the composition and the preparation method comprise the following components in parts by weight: polyethylene imine (PEI), polyether polyol, modifier and amide solvent, wherein the PEI is 35-40 parts, and/or polyether polyol is 45-50 parts, and/or modifier is 30-35 parts, and/or amide solvent is 18-22 parts.
The PEI is low molecular weight polyethylenimine PEI, and the weight average molecular weight Mw of the low molecular weight PEI is any low molecular weight PEI below 5000 Da. The weight average molecular weight is measured by the LS method and the number average molecular weight is measured by GPC, for example.
Preferably, the low molecular weight PEI has a molecular weight of 2000Da, 1800Da, 800Da or 600Da.
The polyether polyol is selected from one or more of 2, 4 functionality polyether polyols, preferably selected from one or more of polytetrahydrofuran ether glycol, polyethylene glycol and polypropylene glycol, preferably the polyether polyol has a weight average molecular weight of 800-4000 and a hydroxyl number of 10300mgKOH/g.
In the optimized scheme, the polyether polyol is polyethylene glycol.
The PEGylation improves the stability of the carrier, reduces the non-specific binding of the carrier and the in vivo anionic substance, prolongs the in vivo circulation time, can keep the advantages of PEG, and can also increase the binding capacity of ligand-receptor.
The modifier is one or more of cholesterol, cholesterol formyl chloride, deoxycholic acid and lipoic acid. PEI is modified with cholesterol, deoxycholic acid or lipoic acid to increase the ability of the polymer to protect and deliver genes to cells, forming more lipophilic PEI derivatives that promote interactions with cells.
In the optimized scheme, the modifier is cholesterol and/or cholesterol formyl chloride, and the cholesterol formyl chloride is synthesized by cholesterol and di (trichloromethyl) carbonic ester.
The amide solvent is N, N-Dimethylacetamide (DMA) or maleimide; preferably, the amide solvent is maleimide.
PEG-MAL-Chol compound composed of Cholesterol (Cholesterol), polyethylene glycol (Polyethylene Glycol, PEG) and Maleimide (MAL).
In a further optimization scheme, amino functional groups in PEI can be subjected to chemical reaction with the surface of UCNPs, surface modification and functionalization, PEI is anchored to the surface of UCNPs, and the surface of UCNPs is provided with the molecular structure of PEI, so that the possibility is provided for subsequent functionalization or biological markers. Upconverting nanoparticles (UCNPs) are nanoscale particles that have the special property of converting lower energy light, such as Near Infrared (NIR) or Infrared (IR) light, to higher energy visible or ultraviolet light, by upconverting luminescence.
The preparation method of the PEI polymer material composition for in vivo delivery of nucleic acid substances comprises the following steps:
(1) Mixing an amide solvent, polyether polyol and a modifier;
(2) Mixing Polyethylenimine (PEI) and polyether polyol;
(3) Mixing Polyethylenimine (PEI) with a modifier;
(4) Mixing the above steps (1) - (3), spin-drying the solvent by rotary evaporation, dialyzing in ethanol solution and deionized water respectively with dialysis bag, and freeze-drying.
The reaction temperature in the steps (1) - (4) is 30-40 ℃.
In the invention, PEI with small molecular weight is comprehensively treated to obtain the PEI derivative composition with high transfection efficiency of the PEI level and low toxicity.
The compositions of the present invention have repeating structural units and flexible polymer chains, which can be modified or functionalized to obtain specific properties such as sensitivity, specificity and biocompatibility, high water solubility due to the large number of amino groups present on PEI.
The composition reduces the toxicity of PEI, improves the stability of PEI and DNA complex, improves targeting, reduces non-specific binding with albumin and prolongs in vivo time.
Drawings
FIG. 1 is a diagram of a cationic liposome mediated delivery mechanism.
Detailed Description
The invention is further illustrated by the following description of specific embodiments:
PEI (molecular weight 1800 Da) was purchased from Aldrich, and other starting materials were also commercially available.
Example 1
30 parts of PEI, 40 parts of polyethylene glycol, 10 parts of cholesterol, 15 parts of cholesterol formyl chloride and 20 parts of maleimide. The PEG is bonded to PEI through a degradable amide bond, so that the biocompatibility is good, and the degradable PEI-g-PEG graft copolymer is provided with a plurality of active group sites (amino groups), so that the PEI-g-PEG graft copolymer has potential application prospects in drug modification.
The preparation method comprises the following steps:
(1) Mixing maleimide, polyethylene glycol (the dosage is 1/3 of the total polyethylene glycol) and cholesterol, and stirring at 30-40deg.C to obtain PEG-MAL-Chol compound; cholesterol is a hydrophobic molecule with very low water solubility that activates cholesterol PEG with carboxylic acid function to react with amine or hydroxyl groups. The targeted delivery of the drug, the improvement of the stability and the bioavailability of the drug and the improvement of the intracellular uptake of the drug through cholesterol targeting effect can be realized.
(2) Mixing Polyethylenimine (PEI) (the dosage is 1/2 of the total PEI) and the rest polyethylene glycol, stirring and dissolving at 30-40 ℃ to form polyethylenimine-graft-polyethylene glycol (PEI-g-PEG) graft copolymer; the PEI-g-PEG graft copolymer has a plurality of active group sites (amino groups).
Characterization was performed by IR,1H-NMR, DSC, GPC, as conventional methods, indicating that the small molecule drug was bound to PEG; the product can be dissolved in most organic solvents such as dichloromethane, ethanol, tetrahydrofuran (THF), benzene, dimethylformamide (DMF), dimethyl sulfoxide (DMSO) and the like, so that the solubility of the micromolecular drug in water is improved.
(3) Mixing the rest polyethylene imine (PEI) with cholesterol formyl chloride, and stirring uniformly at 30-40 ℃ to obtain Chol-PEI; cholesterol (Chol) modifies Polyethyleneimine (PEI), and cholesterol formyl chloride (Chol-Cl) links the amino groups of polyethyleneimine to form a PEI-Chol lipid copolymer. The synthesis method of cholesterol formyl chloride by di (trichloromethyl) carbonic ester and cholesterol is conventional.
Lipid-derived PEI also self-assembles to form NPs, providing more controlled interactions with nucleic acids, anchoring PEI molecules to Nanoparticle (NPs) surfaces of specific size, and allowing more efficient and controlled interactions with nucleic acids. The nano complex can protect nucleic acid substances from degradation, prolong retention time and enhance organ targeting.
The PEI-Chol lipid copolymer was formed by linking the amino groups of the polyethyleneimine with cholesterol formyl chloride (Chol-Cl), and the polymer was characterized by IR,1H-NMR, gel chromatography, DSC, respectively, and the carbonyl characteristic peak shifted after PEI-Chol formation, indicating successful attachment of polyethyleneimine to cholesterol.
(4) Mixing in the steps (1) - (3), and uniformly stirring at 30-40 ℃; or adding other needed substances for further polymerization; the obtained mixed solution is spin-dried by adopting a rotary evaporation method, and is dialyzed for 5-6 times in 50% ethanol solution by using a dialysis bag with the molecular weight cut-off of 1000, wherein the dialysis time is 6-9 hours each time, and then is dialyzed for 20-24 hours in deionized water, and the carrier compound is prepared after freeze drying for 48-52 hours. Rotary evaporation, dialysis and freeze-drying are conventional techniques.
The polyethylene glycol is adopted to crosslink the non-cytotoxic small molecular weight PEI to form the degradable PEI with the imine bond connection, the crosslinked small molecular weight PEI has greatly enhanced accounting delivery efficiency compared with the parent PEI, and the crosslinked chemical bond is hydrolyzed or hydrolyzed under a certain condition, so that the crosslinked small molecular weight PEI can be degraded into the non-cytotoxic small molecular weight PEI, and the safety is high. The carrier generated in the invention combines a plurality of composite structures, has diversity and improves the transfer efficiency under the combined action.
Example 2
Other matters are as in example 1, 45 parts of PEI, 60 parts of polyethylene glycol, 15 parts of cholesterol, 25 parts of cholesterol formyl chloride and 25 parts of maleimide.
Example 4
The other contents are as in example 1, 35 parts of PEI, 45 parts of polyethylene glycol, 10 parts of cholesterol, 20 parts of cholesterol formyl chloride and 20 parts of maleimide.
Example 5
Other contents are as in example 1, PEI 40 parts, polyethylene glycol 50 parts, cholesterol 10 parts, cholesterol formyl chloride 25 parts, maleimide 22 parts.
Example 6
Other contents are as in example 1, 38 parts PEI, 48 parts polyethylene glycol, 10 parts cholesterol, 22 parts cholesterol formyl chloride and 22 parts maleimide.
The amino functional group in PEI can chemically react with the surface of UCNPs, surface modification and functionalization are carried out, PEI is anchored to the surface of UCNPs, and the surface of UCNPs is provided with the molecular structure of PEI, so that the possibility is provided for subsequent functionalization or biological marking. Upconverting nanoparticles (UCNPs) are nanoscale particles that have the special property of converting lower energy light, such as Near Infrared (NIR) or Infrared (IR) light, to higher energy visible or ultraviolet light, by upconverting luminescence.
The term "nucleic acid" as used herein refers to a polymer of nucleotides linked by 3'-5' -phosphodiester bonds, wherein the nucleotides include ribonucleotides and deoxyribonucleotides. The nucleic acid includes ribonucleic acid (RNA) composed of ribonucleotides (adenine ribonucleotide (A), guanine ribonucleotide (G), cytosine ribonucleotide (C), uracil ribonucleotide (U)) and deoxyribonucleic acid (DNA) composed of deoxyribonucleotides (deoxyadenine ribonucleotide (A), deoxyguanine ribonucleotide (G), deoxycytosine ribonucleotide (C), deoxythymine ribonucleotide (T)) in a single-stranded or double-stranded form, linear or circular form. The nucleic acid may include any desired modification, such as methylation, thio, glycosylation, and the like.
Herein, the nucleic acid to be delivered refers to a nucleic acid that needs to be transferred into a target cell, for example, a construct comprising a transgene such as an expression vector or a transfer vector for gene recombination, and the like. "delivery" refers to the process of passing the desired nucleic acid through the cell membrane into the cytoplasm or into the nucleus using PEI as a delivery vehicle. Conditions, steps and conventional reagents required for delivering nucleic acids to cells using PEI as a delivery vehicle are known in the art. The ratio of nucleic acid to PEI to be delivered can be adjusted according to conventional technical knowledge and the aqueous solution can be a medium.
Test-cytotoxicity and delivery evaluation test
Mouse melanoma B16 cells are inoculated into a 96-well plate at the density of 4X 103/well, after the culture is carried out for 32 hours, old culture medium is sucked and removed, 100 mu l of serum-free RPMI1640 culture medium with the concentration of 4 mu g/ml of the composition carrier in the invention is added, after the continuous culture is carried out for 4 hours, old culture medium is sucked and removed, each well is respectively added with the culture medium containing 0.5mg/ml of MTT for continuous culture for 4 hours, solution is sucked and removed, 150 mu l of DMSO is added to dissolve formazan crystals, then an enzyme-labeled instrument is used for measuring the OD value of each well at 570nm, and the OD value of each well without the composition solution is used as a control, so that the survival rate of the cells under the action condition of the composition is calculated. The results show that the inventive composition carrier is substantially non-toxic or less toxic to B16 cells at the experimental concentration of 4 μg/ml compared to untreated PEI precursor (PEI 1800), indicating that the inventive composition carrier has a certain safety profile.
It was also shown that the vector of the composition of the present invention successfully delivered pDNA plasmid to the liver and lung of mice, and the composition was able to enter liver and lung cells and further release plasmid DNA and gene expression. The composition provided by the invention has the advantages of low toxicity, high transfection efficiency, released gene medicine and the like when PEI is used as one of the gene delivery carrier units.
The nucleic acid delivery vehicle of the present invention comprises modified PEI cationic liposomes and cationic materials, and a polymer covalently bonded to polyethylene glycol phospholipids. The drug delivery carrier can be used for preparing a drug preparation for co-delivering multiple therapeutic agents, the particle size of the drug preparation is 40-260 nm, the stability is good, and multiple therapeutic agents can be simultaneously delivered to similar target sites, so that the multiple therapeutic agents can exert synergistic therapeutic effects, or multiple research directions are provided.
The above examples/experiments are only examples for clarity of illustration and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.

Claims (10)

1. A composition of PEI polymer material for in vivo delivery of nucleic acid materials, characterized by: the formula comprises the following components in parts by weight: polyethylene imine, polyether polyol, modifier and amide solvent, wherein the PEI is 30-45 parts, and/or the polyether polyol is 40-60 parts, and/or the modifier is 25-40 parts, and/or the amide solvent is 20-25 parts.
2. A composition of PEI polymer material for in vivo delivery of nucleic acid substances according to claim 1, wherein: the formula comprises the following components in parts by weight: polyethylene imine (PEI), polyether polyol, modifier and amide solvent, wherein the PEI is 35-40 parts, and/or the polyether polyol is 45-50 parts, and/or the modifier is 30-35 parts, and/or the amide solvent is 20-22 parts.
3. A composition of PEI polymer material for in vivo delivery of nucleic acid substances according to claim 1, wherein: the PEI is low molecular weight polyethylenimine PEI, and the weight average molecular weight Mw of the low molecular weight PEI is any low molecular weight PEI below 5000 Da.
4. A composition of matter for in vivo delivery of PEI polymer material for nucleic acid according to claim 3, wherein: the molecular weight of the low molecular weight PEI is 2000Da, 1800Da, 800Da or 600Da.
5. A composition of PEI polymer material for in vivo delivery of nucleic acid substances according to claim 1, wherein: the polyether polyol is selected from one or more of 2, 4-functionality polyether polyols, preferably selected from one or more of polytetrahydrofuran ether glycol, polyethylene glycol and polypropylene glycol, preferably the polyether polyol has a weight average molecular weight of 800-4000 and a hydroxyl value of 10300mgKOH/g; further preferably, the polyether polyol is polyethylene glycol.
6. A composition of PEI polymer material for in vivo delivery of nucleic acid substances according to claim 1, wherein: the modifier is one or more than two of cholesterol, cholesterol formyl chloride, deoxycholic acid and lipoic acid; preferably the modifier is cholesterol and/or cholesterol formyl chloride.
7. A composition of PEI polymer material for in vivo delivery of nucleic acid substances according to claim 1, wherein: the amide solvent is N, N-Dimethylacetamide (DMA) or maleimide; preferably, the amide solvent is maleimide.
8. A composition of PEI polymer material for in vivo delivery of nucleic acid substances according to claim 1, wherein: the amino functional group in PEI can chemically react with the surface of UCNPs, surface modification and functionalization are carried out, PEI is anchored to the surface of UCNPs, and the surface of UCNPs is provided with the molecular structure of PEI.
9. The method of preparing a composition of PEI polymer material for in vivo delivery of nucleic acid substances according to claim 1, wherein: the method comprises the following steps:
(1) Mixing the amide solvent, the polyether polyol and the modifier, and uniformly stirring;
(2) Mixing polyethylene imine (PEI) and polyether polyol, and uniformly stirring;
(3) Mixing polyethylene imine (PEI) with a modifier, and uniformly stirring;
(4) Mixing in the steps (1) - (3), and uniformly stirring; the obtained mixed solution is spin-dried by adopting a rotary evaporation method, dialyzed in ethanol solution and deionized water respectively by using a dialysis bag, and then freeze-dried to obtain the product.
10. The method of preparing a composition of PEI polymer material for in vivo delivery of nucleic acid substances according to claim 1, wherein: the reaction temperature in the steps (1) - (4) is 30-40 ℃.
CN202311645598.5A 2023-12-04 2023-12-04 Composition of PEI polymer material for in vivo delivery of nucleic acid substances and preparation method thereof Pending CN117586524A (en)

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