WO2012068870A1 - Complexe ternaire, liquide contenant un complexe ternaire, procédé de préparation et utilisation correspondante - Google Patents

Complexe ternaire, liquide contenant un complexe ternaire, procédé de préparation et utilisation correspondante Download PDF

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Publication number
WO2012068870A1
WO2012068870A1 PCT/CN2011/075735 CN2011075735W WO2012068870A1 WO 2012068870 A1 WO2012068870 A1 WO 2012068870A1 CN 2011075735 W CN2011075735 W CN 2011075735W WO 2012068870 A1 WO2012068870 A1 WO 2012068870A1
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group
copolymer
ternary complex
substance
component
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PCT/CN2011/075735
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English (en)
Chinese (zh)
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董岸杰
郭术涛
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苏州瑞博生物技术有限公司
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Publication of WO2012068870A1 publication Critical patent/WO2012068870A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • 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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the present invention relates to a ternary composite, a liquid containing a ternary composite, a method for preparing a liquid containing a ternary composite, and the use of a ternary composite and a liquid containing a ternary composite.
  • Synthetic cationic polymers have attracted extensive attention as non-viral vectors for genes such as polyethyleneimine (PEI), chitosan (CS), polylysine (PLL), and poly- ⁇ -amino esters (Poly(P). -ami n0 esters) , PBAEs ), cationic polymers such as polyamide-amine dendrimer (PAMAM), polydimethylamino acrylate (PDMAEMA) or copolymers containing these cationic polymer chains, through static electricity The interaction is combined with negatively charged DNA or RA to form nanoparticles.
  • nano gold particles having a positive charge on the surface, nano silica gel particles, and the like can also be used as a carrier for DNA or RNA.
  • the nanoparticle formed by the combination of the above cationic polymer or nanoparticle with DNA or RA can promote the transfection effect of the gene, but at the same time, the surface of the composite is too strong, and the toxicity is large, and the running time in the body is short.
  • Studies have shown that the introduction of polyethylene glycol segments on the above cationic polymers or nanoparticles can greatly improve biocompatibility, reduce the toxicity of nanocomposites, prolong the circulation time of nanoparticles in the body, etc., but also lead to nanocomposite systems. The efficiency of cell transfection is greatly reduced. Summary of the invention
  • the object of the present invention is to overcome the above drawbacks and to provide a ternary composite having both low toxicity and high transfection efficiency, a liquid thereof, a preparation method and application thereof.
  • the present invention provides a ternary composite characterized in that the ternary composite contains an A component, a B component and a C component, the A component is a positively charged carrier, and the B component is provided with A negatively charged nucleic acid, the C component is a negatively charged copolymer.
  • the present invention also provides a liquid containing a ternary composite, characterized in that the liquid containing the ternary composite contains water and the above ternary composite.
  • the present invention provides a method for preparing a liquid containing a ternary composite, which comprises mixing an aqueous solution of substance Y with an aqueous solution of substance D, and mixing the resulting mixture with an aqueous solution of substance M.
  • the Y substance is a carrier capable of forming a positively charged group in an aqueous solution
  • the D substance is a nucleic acid
  • the M substance is a copolymer capable of forming a negatively charged group in an aqueous solution.
  • a liquid containing the ternary complex prepared by the above method is provided.
  • the present invention also provides the use of the above ternary complex or liquid containing a ternary complex in cell transfection, disease prevention, diagnosis and gene therapy. And the use of the above ternary complex or a liquid containing a ternary complex in the preparation of a cell transfection reagent for the preparation of a medicament for the prevention, diagnosis or gene therapy of diseases.
  • the toxicity of the complex is much lower than that of the binary complex; HeLa cell transfection experiments were carried out on the ternary complex provided by the present invention and the liquid containing the ternary complex, and the cell transformation of the ternary complex compared to the binary complex was confirmed. The dyeing efficiency has also been greatly improved.
  • the RA interference experiment is performed on the liquid containing the ternary complex prepared by the preferred embodiment of the present invention, and it is confirmed that the ternary complex of the present invention and/or the liquid containing the ternary complex can be efficiently used as a nucleic acid delivery system. High specificity allows nucleic acids to enter target cells and is capable of ensuring the interfering activity of the delivered nucleic acids.
  • the ternary complex provided by the present invention and the liquid containing the ternary complex can be widely used in cell transfection, disease prevention, diagnosis and gene therapy, and can be widely used for preparing cell transfection reagents. , in the preparation of drugs for the prevention, diagnosis or gene therapy of diseases.
  • Figure 1 is a gel electrophoresis pattern of a liquid containing PCL-g-PDMAEMAl/D/M-1 ternary complex and six control groups in four embodiments of the present invention.
  • FIG. 2 is a transmission electron micrograph of a PCL-g-PDMAEMAl/D/M-1 ternary composite in one embodiment of the present invention.
  • Fig. 3 is a bar graph showing the transfection efficiency of HeLa cells containing liquid of PCL-g-PDMAEMAl/D/M-1 ternary complex and four groups of control groups in two embodiments of the present invention.
  • Figure 4 is a bar graph showing the toxicity of HeLa cells containing a PCL-g-PDMAEMAl/D/M-1 ternary complex and six groups of control groups in four embodiments of the present invention.
  • Fig. 5 is a bar graph showing the efficiency of inhibiting the expression of luciferase protein in HeLa-Luc cells by the liquid containing the PCL-g-PDMAEMAl/siR Al/M-2 ternary complex and the control group in the two embodiments of the present invention.
  • the present invention provides a ternary composite characterized in that the ternary composite contains an A component, a B component and a C component, the A component is a positively charged carrier, and the B component is provided with A negatively charged nucleic acid, the C component is a negatively charged copolymer.
  • the copolymer may be various negatively charged copolymers in the art, as long as it can be combined with the A component by electrostatic interaction to form the ternary composite, in order to form the three
  • the meta-complex has better biological activity.
  • the C component is a copolymer of polyglutamic acid and polyethylene glycol and/or polyglutamic acid and a polyethyl group having a targeting group at the end. a copolymer formed from a diol.
  • the positively charged support i.e. the A component
  • the positively charged support is preferably a positively charged nanoparticle and/or a positively charged cationic polymer, further preferably said positively charged nanoparticle
  • Both the particles and the positively charged cationic polymer contain a group Z capable of binding hydrogen ions and forming a positively charged group, and at least a portion of the group Z is combined with hydrogen ions to form a positively charged group, the positive charge carried by the A component is provided by the positively charged group; the negative charge of the B component is lost by at least a portion of the hydrogen ion by the phosphodiester bond in the nucleic acid.
  • the resulting group provides, wherein the phosphodiester bond is a well-known concept in the art, namely: a chemical group, which refers to two ester bonds formed by esterification of one molecule of phosphoric acid with two molecules of alcohol (hydroxyl), in an aqueous solution.
  • a chemical group which refers to two ester bonds formed by esterification of one molecule of phosphoric acid with two molecules of alcohol (hydroxyl), in an aqueous solution.
  • at least The hydrogen atom of the hydroxyl group of the partial phosphoric acid is dissociated, thereby causing the nucleic acid to have a negative charge; preferably, the negative charge carried by the C component is provided by a group obtained by losing a carboxyl group at least a part of the hydrogen ion in the copolymer.
  • the carboxyl group is a carboxyl group which is not used for polymerization on the polyglutamic acid, and in the aqueous solution, at least a part of the carboxyl group can be ionized, thereby causing the C component to have a negative charge.
  • the number of moles of the carboxyl group in the copolymer in the present invention means the sum of the number of moles of the carboxyl group in the ionized state and the number of moles of the carboxyl group not in the ionized state, that is, corresponding to the glutamic acid structural unit in the copolymer. The number of moles.
  • the ratio of the number of moles of the group Z (denoted as N), the number of moles of nucleotides in the nucleic acid (denoted as P) to the number of moles of carboxyl groups in the copolymer (denoted as C), Referred to as N/P/C value, it can be changed within a large range.
  • the N/P/C value is preferably 1-50:1:1-50, further preferably, the N/P/C value is 1-30:1:1-30, and, under preferred conditions, the A component carries positive The charge, the negative charge carried by the B component, and the negative charge of the C component make the zeta potential of the ternary complex -20 mV to 30 mV, and more preferably -15 mV to 20 mV.
  • the group Z is any group capable of binding a hydrogen ion and having a positive charge, such as an amino group having a more electronegative nitrogen atom, specifically, the group Z described in the present invention It is preferably one or more of a primary amino group, a secondary amino group and a tertiary amino group, and more preferably a tertiary amino group.
  • the cationic polymer in the present invention further includes various cationic polymer type transfection reagents which are currently commercially available, such as cationic liposome and Tnmsfectam reagent, etc., and it should be particularly noted that, in the present invention, commercially available The process of preparing a ternary complex by a cationic liposome and a Tnmsfectam reagent, both according to the procedures in the commercially available kit, depending on the number of N atoms or positive charges carried. A ternary composite that satisfies a certain N/P/C value is obtained.
  • the positively charged nanoparticles are used as a carrier for nucleic acids, preferably having a particle diameter of 10 to 400 nm; and the positively charged nanoparticles can be formed in a solution in the field with positive Nanoparticles of charge groups, such as positively charged gold nanoparticles and/or positively charged silica nanoparticles formed by various means, when the positively charged nanoparticles are gold nanoparticles and / Or a positively charged silica gel nanoparticle, preferably having a particle diameter of 10 to 200 nm, and most preferably 10 to 100 nm; the positively charged nanoparticles in the present invention are recombined on the surface of the carboxylated gold nanoparticle.
  • a cationic polymer such as polyethyleneimine, is formed.
  • the number average molecular weight of the cationic polymer may vary within a wide range.
  • the cationic polymer has a number average molecular weight of from 1 KDa to 150 KDa, and more preferably, the cationic polymer The number average molecular weight is from 5 KDa to 120 KDa, and most preferably, the cationic polymer has a number average molecular weight of from 10 KDa to 100 KDa.
  • cationic polymers are conventionally defined in the art and refer to polymers having cationic groups on the chain, generally referred to as water soluble polymers.
  • Commonly used cationic polymers include polydiallyldimethylammonium chloride, polyfluorene, polyvinylamine, cationic polyacrylamide, and the like.
  • the cationic polymer is used as a carrier for nucleic acid transfected cells, and the cationic polymer may be any cationic polymer capable of being used as a carrier for cell transfection.
  • the cationic polymer is selected from the group consisting of Polyethyleneimine, cationic liposome, poly- ⁇ -amino ester, chitosan, chitosan quaternary ammonium salt, containing formula (1)
  • R & lt 2 is a C r C 4 alkylene group, preferably, R 2 is an alkylene group having 2 to dC;
  • R 3 and R4 is each independently alkyl of dC 4 , preferably, R 3 and each are independently methyl or ethyl, and further preferably, R 3 and R 4 are both methyl or ethyl.
  • the amphiphilic comb-shaped graft copolymer may be a plurality of two types having a hydrophobic main chain and a hydrophilic side chain, provided that a positively chargeable group can be formed in an aqueous solution.
  • the affinity comb copolymer, the preparation method of the amphiphilic comb copolymer in the present invention is not particularly limited, preferably, the amphiphilic comb copolymer is under atom transfer radical polymerization conditions,
  • the bromine-functional polyester is contacted with at least one material having an ethylenic terminal at the end, and the molar ratio of the bromine-functional polyester to the at least one terminal having an ethylenic bond may be a conventional ratio in the art, preferably The molar ratio of the bromine-functional polyester to the at least one substance having an ethylenic bond at the end is from 1 to 100, more preferably from 1:30 to 70.
  • the at least one terminal is an ethylenic bond, the monomer having the structure represented by the formula (2), the polymer containing the structural unit represented by the formula (1), and one or more of polyethyleneimine. That is, the bromine-functional polyester may react with the monomer or with the polymer.
  • R 2 is an alkyl group of H or dC 4 , preferably 11 or a methyl group
  • R 2 is an alkylene group of dC 4 , preferably, R 2 is an alkylene group of dC 2
  • R 3 and R 4 are each independently As the alkyl group of dC 4 , preferably, R 3 and each are independently a methyl group or an ethyl group, and further preferably, R 3 and R 4 are simultaneously a methyl group or an ethyl group.
  • the bromine-functional polyester may be various macromolecular polyesters capable of functioning as an initiator for atom transfer radical polymerization, preferably, the number average molecular weight of the bromine-functional polyester. It is 500-30000, and more preferably 1000-15000.
  • the bromine-functional polyester is preferably a homopolymer of ⁇ -(2-bromo-methylpropionate)-caprolactone, ⁇ - a copolymer of (2-bromo-methylpropionate)-caprolactone and a lactone and a copolymer of ⁇ -(2-bromo-methylpropionate)-caprolactone and a lactide or a plurality of, wherein the lactone is ⁇ -hydroxybutyl ester and/or ⁇ -hydroxypentyl ester, the lactide is glycolide and/or lactide, and further preferably, the bromine functional group is aggregated
  • the ester is a homopolymer of ⁇ -(2-bromo-methylpropionate)-caprolactone.
  • the amphiphilic comb-shaped graft copolymer is subjected to a process of preparing the nano-particles in the process of preparing the ternary composite, since the amphiphilic comb-shaped graft copolymer has Hydrophilic side chain and hydrophobic main chain, therefore, after being dissolved in an organic solvent, it is slowly added dropwise to the aqueous phase system, and after the organic solvent is completely evaporated, the pH of the polymer solution is adjusted to the middle with dilute hydrochloric acid. Sexually, a hydrophobic core, a nanoparticle of a hydrophilic outer shell is obtained.
  • the prepared polymer solution is preferably sterilized by filtration through a 0.22 micron Millipore sterile membrane and stored at 4 ° C until use. It is particularly clear that since the amphiphilic comb-shaped graft copolymer and other cationic polymers are both polymeric and require a certain processing step to become granular, they are still classified in the present application as In the category of cationic polymers. However, since the particles in the nanometer scale are formed in advance, the diameter of the particles can be determined.
  • the particle diameter range of the positively charged nanoparticles described in the present application includes the amphiphilic comb-shaped graft copolymer.
  • the particle diameter of the formed nanoparticles, the nanoparticles formed by the amphiphilic comb-shaped graft copolymer in the present invention preferably have a diameter of 30 to 360 nm.
  • the conditions of the atom transfer radical polymerization are well known to those skilled in the art, such as a nitrogen gas, a polymerization temperature of 30 to 70 ° C, and a polymerization time of 10 to 18 hours.
  • the nucleic acid of the present invention may be an oligonucleotide and/or a polynucleotide, which is a concept well known in the art, polymerized from a nucleomonomer, according to the essence of the present invention
  • the nucleic acid may be a nucleic acid of any length, and preferably the nucleic acid has a molecular weight of lxl0 3 - lxl0 8 , further preferably 5xl0 3 - lxl0 7 .
  • the nucleic acid may be a ribonucleic acid or a deoxyribonucleic acid, and may be a single-stranded nucleic acid, a double-stranded nucleic acid, a triple-stranded nucleic acid, or a hybrid stranded nucleic acid composed of a ribonucleic acid and a deoxyribonucleic acid. Any basic nucleotide unit can be applied to the present invention.
  • the B component is preferably a ribonucleic acid, further preferably a double-stranded ribonucleic acid, in particular a double-stranded ribonucleic acid having a single-stranded region of 1-10 nucleotides in length, ie, double-stranded ribose
  • the nucleic acid has a single-stranded region of 1-10 nucleotides in length downstream of the 5' end or 3' end of one of the strands, and the single-stranded region may be ribonucleic acid or Deoxyribonucleic acid, the most typical structure that satisfies the above characteristics, is a small interfering nucleic acid sequence.
  • the B component may be a partially chemically modified ribonucleic acid, and the partially modified chemically modified ribonucleic acid may be modified in various ways in the art, such as ribose modification, base. At least one of a modification and a modification of a phosphodiester bond.
  • the ribose modification refers to a modification of the 2'-OH in the nucleotide pentose sugar, that is, introducing a certain substituent at the hydroxyl position of the ribose, for example, a 2'-fluoro modification, as shown in the formula (3); 2'-oxymethyl modification, as shown in formula (4); 2'-oxyethylene methoxy modification, as shown in formula (5); 2,4'-dinitrophenol modification, as in formula ( 6); locked nucleic acid (LNA), as shown in formula (7); 2'-amino modification, as shown in formula (8) - deoxy modification, as shown in formula (9).
  • a 2'-fluoro modification as shown in the formula (3)
  • 2'-oxymethyl modification as shown in formula (4)
  • 2'-oxyethylene methoxy modification as shown in formula (5)
  • 2,4'-dinitrophenol modification as in formula ( 6)
  • locked nucleic acid (LNA) locked nucleic acid
  • the base modification refers to modification of a base of a nucleotide, for example, a 5'-bromouracil modification, as shown in the formula (10); 5'- The iodouracil modification is as shown in the formula (11); the N-methyluracil modification is as shown in the formula (12); and the 2,6-diaminoguanidine modification is as shown in the formula (13).
  • the modified group contained in the partially chemically modified ribonucleic acid may be various groups for chemical modification of nucleic acids in the art, for example, the modified group contained in the partially chemically modified ribonucleic acid may be fluorine. At least one of a thiol group, a thio group, an alkoxy group, an alkyl group, and an acyl group, the modifying group is not limited to the above, and these groups may be modified according to the principles of the present invention. On the same or different nucleotides.
  • nucleic acids containing non-natural bases and nucleic acids subjected to various modifications can be achieved by ionizing a negatively charged group in an aqueous solution and combining it with the Y component by electrostatic interaction. Therefore, the nucleic acid in the present invention is not limited to a nucleic acid formed of a natural base.
  • the copolymer may be a block copolymer and/or a graft copolymer.
  • the block copolymer is preferably at least one of an AB type, an ABA type, and a BAB type block copolymer.
  • the number average molecular weight of the copolymer is preferably from 1,000 to 400,000, further preferably from 2,000 to 200,000; wherein the AB type, ABA type and BAB type block copolymers are well-known concepts in the art, and AB type block copolymerization
  • the diblock copolymer consisting of A and B, the ABA type block copolymer and the BAB type block copolymer are triblock copolymers composed of A and B.
  • A is polyglutamic acid embedding.
  • the number average molecular weight of A is preferably from 1,000 to 300,000, further preferably from 5,000 to 100,000; and B is a polyethylene glycol block and/or a polyethylene glycol block having a terminal group at the end, And the number average molecular weight of B is preferably from 200 to 30,000, more preferably from 1,000 to 20,000.
  • the copolymer may also be a graft copolymer
  • the graft copolymer is preferably a graft copolymer containing a polyglutamic acid main chain and a side chain
  • the number average molecular weight of the polyglutamic acid main chain is preferably from 1,000 to 300,000, further preferably from 5,000 to 150,000; and the number average molecular weight of each side chain is preferably from 200 to 30,000, more preferably from 1,000 to 10,000, most preferably It is 1000-5000.
  • the polyethylene glycol having a targeting group at one end is a polyethylene glycol whose terminal is modified by a targeting substance
  • the targeting is conventionally defined in the art, and refers to Limiting therapeutic effects, drug effects, and the like to a particular target cell, tissue, or organ without affecting the function of other normal cells, tissues, or organs, such as covalent bonds and therapeutic effects
  • a drug effect or a component having other functions is linked to achieve confinement of the attached component to a particular target cell, tissue or organ to which the targeting group, i.e., a targeting substance, is attached The group formed.
  • the manner of the connection may be various ways of achieving the above object, as long as the targeting group formed by the targeting substance does not affect the biological activity of the connected component, and the connected component does not affect the target.
  • the locating function of the directional group, at the same time, the targeting group and the component to be linked can be combined in a stable form during preparation and use, and therefore, different targeting substances can be based on the respective Structural feature selection is linked to the therapeutic or drug-effect component in different ways.
  • many of the targeted groups are present due to the wide application of targeting groups in research and clinical applications.
  • the compounds have been commercialized, and various polyethylene glycols having a targeting group at the end as used in the present invention are commercially available. These commercially available compounds having a targeting group can satisfy the above requirements for a compound having a targeting group.
  • the selection of the targeting substance depends on the cell, tissue or target as the target.
  • Organs Currently, a variety of substances have been developed that target different cells, tissues and organs, including small molecule compounds, short peptides with targeting effects, such as RGD short peptides that specifically target endothelial cells (fine -Glycine-aspartate), aptamers, etc., which are defined by the well-known definitions in the art, ie, by specific factor cell enrichment phylogenetic (SELEX) techniques for specific cells, proteins
  • SELEX specific factor cell enrichment phylogenetic
  • the targeting substance may be any of the above-mentioned targeting substances, and preferably, the targeting substance is folic acid, mannose, galactose, a short peptide having a guiding effect, and an aptamer.
  • the targeting substance is folic acid, mannose, galactose, a short peptide having a guiding effect, and an aptamer.
  • a targeting ability to a specific target such as an organ, a tissue, a cell, etc.
  • the present invention provides a liquid containing a ternary composite, characterized in that the liquid containing the ternary composite contains water and the above ternary composite.
  • the ternary composite further contains other ions, such as various forms of phosphate ions, alkali metal ions, etc. in the phosphate buffer, and the pH of the liquid containing the ternary complex is 5.5-8. It is preferably 6.0-7.4, further preferably 7.2-7.4.
  • the concentration of the ternary complex in the liquid containing the ternary complex is not particularly limited in the present invention, and in the application for the purpose of cell transfection, preferably, the minimum concentration required to satisfy the cell transfection is the lower limit.
  • the upper limit of the total amount of the ternary composite which can be contained in the liquid of a specific volume is further preferably, and the concentration of the ternary complex in the liquid containing the ternary composite is 0.001 to 4.0 g/liter, more preferably 0.01-3 g / liter, most preferably 0.02-1 g / liter; when used in cell transfection experiments, because of the lower amount of nucleic acid required, a lower concentration of the liquid containing the ternary complex can be directly prepared.
  • the prepared liquid containing the ternary complex may also be diluted, and the concentration of the liquid containing the ternary complex is preferably 0.001 to 0.25 mg of nucleic acid/ml, further preferably 0.005 to 0.1, based on the concentration of the nucleic acid. Milligram nucleic acid / ml.
  • the ternary complex in the liquid containing the ternary composite is granular and has an average particle diameter of 20 to 500 nm, further preferably 50 to 380 nm, and most preferably 50. -300 nm.
  • the present invention provides a method for preparing a liquid containing a ternary composite, characterized in that the method comprises
  • An aqueous solution of the substance Y is mixed with an aqueous solution of the substance D, and the resulting mixture is mixed with an aqueous solution of the substance M; wherein the substance Y is a carrier capable of forming a positively charged group in the aqueous solution; the substance D is a nucleic acid
  • the M substance is a copolymer capable of forming a negatively charged group in an aqueous solution.
  • the copolymer capable of forming a negatively charged group in an aqueous solution may be various copolymers capable of forming a negatively charged group in an aqueous solution in the art, in order to form a finally formed ternary composite.
  • the liquid of the substance has better biological activity.
  • the copolymer capable of forming a negatively charged group in an aqueous solution is a copolymer of polyglutamic acid and polyethylene glycol and/or polyglutamic acid. A copolymer formed with polyethylene glycol having a targeting group at its end.
  • the concentration of each substance in the aqueous solution of the substance Y, the substance D and the substance M can be varied within a wide range, and the selection of the concentration of each substance in the preparation process depends on the concentration of the desired final product.
  • the concentration of the Y substance in the aqueous solution of the substance Y is from 0.001 to 1 g/liter, more preferably from 0.005 to 0.5.
  • the gram/liter is most preferably 0.01 to 0.1 g/liter;
  • the concentration of the substance D in the aqueous solution of the substance D is 0.001 to 1 g/liter, further preferably 0.005 to 0.1 g/liter, and most preferably 0.01 to 0.04 g.
  • the concentration of the substance M in the aqueous solution of the substance M is from 0.001 to 1 g/liter, further preferably from 0.01 to 0.5 g/liter, and most preferably from 0.02 to 0.3 g/liter.
  • the mixing conditions are conventional mixing conditions, and the temperature and time may be varied within a wide range.
  • the temperature and time of the two mixing may be the same or different, preferably the same, and the temperature may be, for example, 4 ° C -50 °C, preferably 15 ° C to 40 ° C, the time may be 5 minutes or more, and in view of the balance between time saving and mixing effect, the mixing time is preferably 10 minutes to 60 minutes, further preferably 15 to 30 minutes.
  • the aqueous solution of the substance Y is prepared by dissolving the amphiphilic comb-shaped graft copolymer with an organic solvent and slowly adding it dropwise.
  • the pH is adjusted to 6.5-7.5 with dilute hydrochloric acid, and more preferably 7.0-7.4.
  • the aqueous solution of the substance Y is a buffer solution of the substance Y, and more preferably, the buffer solution is a phosphate buffer solution, and the pH of the phosphate buffer solution is preferably 7- 7.4.
  • the aqueous solution of the substance D is also preferably a buffer solution of the substance D. More preferably, the buffer solution is a phosphate buffer solution, and the pH of the phosphate buffer solution is preferably 7-7.4, further preferably The aqueous solution of the substance Y and the aqueous solution of the substance D are each a phosphate buffer solution, and the pH is 7.2-7.4.
  • the Y substance is preferably a nanoparticle and/or a cationic polymer, both the nanoparticle and the cationic polymer contain a group z, the group Z being capable of binding hydrogen ions and forming the positively charged group group.
  • the ratio of the number of moles of the group z, the number of moles of nucleotides in the nucleic acid, and the number of moles of the carboxyl group in the copolymer is the same as described above, and will not be described herein.
  • the number of moles of the group z, the number of moles of nucleotides in the nucleic acid, and the number of moles of carboxyl groups in the copolymer cause the zeta potential of the ternary complex in the liquid containing the ternary complex It is -20 mV to 30 mV, and further preferably -15 mV to 20 mV.
  • the definition of the group Z the selection of the nanoparticles, various descriptions of the cationic polymer, the definition, type and molecular weight of the nucleic acid, and the type and specific description of the copolymer are the same as the foregoing, and will not be described herein. .
  • the present invention provides a liquid containing a ternary composite which is obtained by the above-described method for preparing a liquid containing a ternary composite.
  • the ternary complex and the liquid containing the ternary complex provided by the present invention can be applied to cell transfection, disease prevention, diagnosis, and gene therapy as a nucleic acid delivery method, similar to other vectors or delivery methods. At the same time, it can also be used in the preparation of a cell transfection reagent for the preparation of a medicament for the prevention, diagnosis or gene therapy of diseases. For example, by transfecting a ternary complex of the present invention and/or a liquid containing a ternary complex into a living body cell, the substance D in the ternary complex is specific to the genetic information to be detected and separated from the body.
  • the final diagnosis can be obtained by detecting gene defects or abnormalities, and thus can be applied in the preparation of diagnostic drugs for diseases. Since the ternary complex of the present invention and/or the liquid containing the ternary complex can efficiently deliver siRNA, it can also be used for the preparation of gene therapy drugs. Moreover, since the ternary complex and/or the ternary complex-containing solution of the present invention have both low toxicity and high transfection efficiency, they can be better applied to the above than the prior art carriers and methods. Among various fields. Hereinafter, the contents of the present invention will be described in more detail by way of examples.
  • the prepared 5 L ternary complex-containing liquid (concentration of 1 ⁇ ⁇ /50 ⁇ and ⁇ ⁇ 5x nucleic acid loading buffer was thoroughly mixed, and then 0.8% by weight of agarose (containing 0.5 g/mL of bromination) Ethigen) gel electrophoresis was carried out, the voltage was 120V, the electrophoresis time was 40 minutes, and the DNA electrophoresis pattern was observed under ultraviolet light and photographed.
  • the ternary composite particle size, nanoparticle particle size and Zeta potential measurement were measured by Brookhaven's BI 90 Plus/Zetaplus laser particle size analyzer.
  • the measurement temperature was 25 °C, the angle was 90 °, and the incident light wavelength was 618 nm.
  • the transmission electron microscope used was a Dutch JEM-100CX II transmission electron microscope.
  • the copper mesh was first immersed in the sample solution to be tested, and then dyed with a 0.1% by weight phosphotungstic acid solution. After about 3 minutes, the excess liquid was filtered off with filter paper and air-dried at room temperature. It was then observed by transmission electron microscopy and the morphology of the particles in the system was taken.
  • the NMR spectrometer used was a Bruker 400M NMR spectrometer. Specific calculation methods reference, Boulmedais F., Frisch B., Etienne 0., Lavalle Ph., Picart C., Ogier J., Voegel JC., Schaaf P., Egles C. Polyelectrolyte multilayer films with pegylated polypeptides as a new Type of anti-microbial protection for biomaterials, Biomaterials, 2004, 25, 2003-2011.
  • nucleotide sequence of EGFP-N1 is shown in SEQ ID NO: 1.
  • cytotoxicity test The cytotoxicity of the polymer is detected by the MTT method (tetrazolium salt colorimetric method), and the specific method is as follows:
  • Luciferase protein detection was carried out after further incubation for 48 hours in a 5 vol% C0 2 incubator at 37 ° C, and the total protein concentration was determined using a BCA (Pierce, USA) kit.
  • the inhibition efficiency (i.e., gene silencing efficiency) of the siRNA was calculated by comparison with the blank control group.
  • the N/P/C value is obtained by the following method: calculating the total number of moles of the group Z in the Y substance according to the structural formula of the Y substance combined with the weight of the Y substance, for a commercially available transfection reagent, according to the reagent The information provided by the box is calculated to obtain an N value; based on the weight of the added substance D combined with the molecular weight of the nucleotide in the substance D, the total number of moles of nucleotides in the substance D is calculated to obtain a P value; The weight is combined with the content of the polyglutamic acid moiety in the M substance, and the number of moles of the carboxyl group in the substance D is calculated to obtain a C value, and then an N/P/C value is obtained.
  • a ternary complex of a desired N/P/C value and/or a liquid containing a ternary complex can be obtained by changing the ratio of the Y substance, the D substance, and the M substance.
  • the parameters of the different M substances used are listed in Table 1, and the M substances described in the present invention. Both are self-made, among which the preparation method of graft copolymer is referred to the literature (Boulmedais F., Frisch B., Etienne 0., Lavalle Ph., Picart C., Ogier J., Voegel JC, Schaaf P., Egles C.
  • EGFP-N1 plasmid DNA (purchased from Invitrogen, the number of nucleotides of plasmid DNA was 4.7 kb) was used in PBS (composition of NaCl 137 mmol/L, KC1 2.7 mmol/L, Na 2 HP0 4 4.3 mmol/L, KH 2 P0 4 1.4mmol/L, pH 7.4) Dissolved (concentration lg/5 ( ⁇ L), 50 ⁇ PEI (25kDa, purchased from Sigma-Aldrich, 0.0222 mol N atom per gram of polymer) A solution of PBS (same composition and pH value as above) (1.35 g/5 ( ⁇ L) was added dropwise to 50 ⁇ of EGFP-N1 plasmid in PBS, and shaken while adding, and allowed to mix thoroughly, room temperature 25 ° Place C for 20 minutes; add 5 ( ⁇ L of M substance (M substance is Ml, see Table 1 for details) of the aqueous solution (1.31 ⁇
  • EGFP-N1 plasmid DNA was used in PBS (composition of NaCl 137 mmol/L, KC1 2.7 mmol/L, Na 2 HP0 4
  • EGFP-N1 plasmid DNA was dissolved in PBS (same as in Example 1) at a concentration of ⁇ g/10 ( ⁇ L), and 50 ⁇ M of hydrazine (same as in Example 1) of PBS (same as in Example 1) was dissolved.
  • the liquid P4-P7 containing the ternary complex was prepared according to the method described in Example 1, except that the PEI was replaced by chitosan (CS, 50 KDa, the degree of deacetylation was 95%, purchased from Shandong Aokang Biotechnology Co., Ltd. Company, nitrogen atom The content is 0.0061 mol / gram of chitosan) or chitosan quaternary ammonium salt (NCS, made by the literature method (M. Thanou, BI Florea, M. Geldof, et al., Quatemized chitosan oligomers as novel gene delivery vectors in epithelial Cell lines, Biomaterials, 2002, 23 (1): 153-159.
  • the liquid P8-P10 containing the ternary complex was prepared according to the method described in Example 1, except that PEI was replaced with a cationic liposome (N, [N-[l-(2,3-Dioleoyloxy)propyl]] - ⁇ , ⁇ , ⁇ -trimethylammonium methylsulfate (DOTAP), purchased from Sigma-Aldrich, containing 0.0013 mol of N atom per gram of Lip), the choice of M substance is shown in Table 2, and according to the various The N/P/C value determined the ratio of the three substances added, wherein the EGFP-N1 plasmid was added in the same amount as in Example 1.
  • DOTAP cationic liposome
  • the liquid P11 containing the ternary complex was prepared according to the method described in Example 1, except that the PEI was replaced with Transfectam reagent (Tra, operated according to the kit instructions, purchased from Promega, containing 0.005 lmol per gram of Tra Charge), the selection of the M substance is shown in Table 2, and the ratio of the three substances added is determined according to various N/P/C values in Table 2, wherein the amount of the EGFP-N1 plasmid is added and Example 1 the same.
  • the liquid P12-P14 containing the ternary complex was prepared according to the method described in Example 1, except that PEI was replaced with a polyamide-amine dendrimer (PAMAM, N atom content of 0.0177 mol/g PAMAM, The number average molecular weight was 14270 g/mol, purchased from Sigma-Aldrich Co., Ltd., and the selection of the M substance was as shown in Table 2, and the ratio of the three substances added was determined according to various N/P/C values in Table 2.
  • the amount of the EGFP-N1 plasmid added was the same as in Example 1.
  • the liquid P15-P17 containing the ternary complex was prepared according to the method described in Example 1, except that PEI was replaced with poly- ⁇ -amino ester (PBAEs, self-made according to literature methods (DM Lynn, R. Langer, Degradable Poly). (P-amino esters): Synthesis, Characterization, and Self- Assembly with Plasmid DNA, Journal of the American Chemical Society, 2000, 122 (44): 10761-10768.), per gram of polymer
  • the number of moles of N (the number of N atoms in the repeating unit) I (the molecular weight of the repeating unit), the number average molecular weight is 14000 g / mol), the selection of the M substance is shown in Table 2, and according to each of Table 2
  • the N/P/C value determined the ratio of the three substances added, wherein the EGFP-N1 plasmid was added in the same amount as in Example 1.
  • the liquid P18-P20 containing the ternary complex was prepared according to the method described in Example 1, except that PEI was replaced with N,N-dimethylaminoethyl methacrylate (PDMAEMA, N atom content was 0.0064 mol/ Gram PDMAEMA (containing one N atom per DMAEMA repeat unit), according to the literature (J.-Y. Cherng, P. van de Wetering, H.
  • This preparation example was used to prepare an aqueous solution of a polycaprolactone grafted polydimethylaminoethyl methacrylate (PCL-g-PDMAEMA) copolyester.
  • PCL-g-PDMAEMA polydimethylaminoethyl methacrylate
  • the polymer solution was slowly added dropwise to 10 mL of deionized water under stirring. Tetrahydrofuran was slowly volatilized by stirring in a fume hood at room temperature. After the solvent was completely evaporated, the polymer solution was adjusted to pH 7.2 with dilute hydrochloric acid. The prepared polymer solution was sterilized by filtration through a 0.22 micron Millipore sterile membrane and stored at 4 ° C until use.
  • PCL-g-PDMAEMA 1, PCL-g-PDMAEMA2, PCL-g-PDMAEMA3, PCL-g-PDMAEMA4 was prepared as described above, and PCL-g-PDMAEMA was present in the form of nanoparticles in an aqueous solution, four copolymers.
  • the structural properties and aqueous solution nanoparticle size are listed in Table 3.
  • the liquid P21-P25 containing the ternary complex was prepared according to the method described in Example 1, except that the PEI was replaced by PCL-g-PDMAEMAl, and the selection of the M substance was as shown in Table 2, and according to Table 2
  • the various N/P/C values determined the ratio of the three substances added, wherein the EGFP-N1 plasmid was added in the same amount as in Example 1.
  • the liquid P26-P32 containing the ternary complex was prepared according to the method described in Example 1, except that the PEI was replaced with a different PCL-g-PDMAEMA, and the selection of the Y substance and the M substance was as shown in Table 2, The ratio of the three substances to be added was determined in accordance with the various N/P/C values in Table 2, wherein the EGFP-N1 plasmid was added in the same amount as in Example 1.
  • the liquid P33-P35 containing the ternary complex was prepared according to the method described in Example 1, except that the PEI was replaced with PCL (15000)-g-PEI (800yD/M-8 (made according to the literature method).
  • PCL (15000)-g-PEI 800yD/M-8 (made according to the literature method).
  • Qiu, YH Bae Self-assembled polyethylenimine-graft-poly ([epsilon]- caprolactone) micelles as potential dual carriers of genes and anticancer drugs, Biomaterials, 2007, 28(28): 4132-4142.
  • the number average molecular weight is 20000, graft ratio: 25.0%
  • PCL(5000)-b-PEI(2000)/D/M-10 made by literature method (P. Xu, EA Van Kirk, Y.
  • Example 36 Suriano, et al., One-Pot Synthesis of Weil-Defined Amphiphilic and Adaptative Block Copolymers via Versatile Combination of "Click” Chemistry and ATRP, Macromolecular Rapid Communications, 20 07, 28(22): 2151-2158.), the number average molecular weight is 7000 g/mol, the graft ratio is 28.6%), and the ratio of the three substances added is determined according to the N/P/C value of Table 2, wherein The amount of the EGFP-N1 plasmid added was the same as in Example 1.
  • Example 36 The amount of the EGFP-N1 plasmid added was the same as in Example 1.
  • the liquid P36 containing the ternary complex was prepared according to the method described in Example 1, except that the M substance was a polyethylene glycol end with graft copolymerization of a target group modified by folic acid and polyglutamic acid. (M-19, purchased from Beijing Keykai Technology Co., Ltd.), Y substance is PCL-g-PDMAEMA2, and the proportion of the three substances added is determined according to the N/P/C value in Table 2, wherein, EGFP-N1 The amount of the plasmid added was the same as in Example 1.
  • Example 37-40 The amount of the plasmid added was the same as in Example 1.
  • the liquid P37-P40 containing the ternary complex was prepared according to the method described in Example 36, except that the M substance was a polyethylene having a targeting group modified by mannose, galactose, RGD, Aptamer at one end.
  • Graft copolymer of diol and polyglutamic acid, PGA-g-PEG-G in Table 1 represents polyglutamic acid-g-polydiethanol graft copolymer with terminal galactose modification, PGA-g- PEG-S represents a polyglutamic acid-g-polydiethanol graft copolymer whose end is a mannose modification.
  • the selection of the Y substance is shown in Table 2.
  • the ratio of the three substances added was determined according to the N/P/C value in Table 2, wherein the EGFP-N1 plasmid was added in the same amount as in Example 1.
  • polyethylene glycol with a targeting group modified by mannose, galactose and RGD at one end was purchased from Beijing Keykai Technology Co., Ltd.
  • Polyethylene glycol having a targeting group modified by Aptamer at one end was obtained by the following method.
  • SEQ ID NO: 2 The base sequence of Aptamer is shown in SEQ ID NO: 2, which is 5'-ACC TGG GGG AGT ATT GCG GAG GAA GGT GTC ACA(A)i 0 -3', in order to connect Aptamer to the end of polyethylene glycol.
  • nucleic acid having the amino acid sequence modified at the 3' end, ie, the structure is 5'-ACC TGG GGG AGT ATT GCG GAG GAA GGT GTC ACA(A)i 0 -NH 2 -3' (Beijing Key Kai Technology Co., Ltd., reacted with a polyethylene glycol modified at the end of the carboxyl group (Beijing Key Kai Technology Co., Ltd.), prepared by reference method Aptamer-PEG (S. Dhar, FX Gu, R.
  • the liquid P41-P43 containing the ternary complex was prepared according to the method described in Example 1, except that the PEI was replaced by PCL-g-PDMAEMAl, and the D substance was siR Al.
  • the selection of the M substance was as shown in Table 2. And determine the ratio of the three substances added according to the various N/P/C values in Table 2, wherein 50 ⁇ l of siRNA 1 having a concentration of 1 ⁇ ⁇ /50 ⁇ was added.
  • the nucleotide sequence of the siRNA1 is shown in SEQ ID NO: 3 and SEQ ID NO: 4.
  • Antisense strand 5'-UUUCCUUCCAAAACAAdTdT-3' (SEQ ID NO: 4).
  • a liquid containing a ternary complex, P44-P46, was prepared as described in Example 41, except that
  • PCL-g-PDMAEMAl was replaced by cationic gold nanoparticles (C-Au).
  • C-Au cationic gold nanoparticles
  • the choice of M material is shown in Table 2.
  • the ratio of the three substances added was determined according to the N/P/C values in Table 2.
  • cationic gold nanoparticles are compounded with carboxylated gold nanoparticles and PEK25KD) (S. Guo, Y. Huang, Q.
  • the gold nanoparticles have a particle diameter of 10-40 nm, and the N atom content can be determined by titrating PEI with trinitrobenzenesulfonic acid. , the content of N atoms is 0.0099 mol / gram of cationic gold nanoparticles).
  • Test example 1-4
  • the liquid of the composite was prepared according to the method described in Example 21 except that the M substance was not added to obtain a liquid DPI containing a binary complex having an N/P value of 10:1. Comparative example 2-8
  • a solution containing a binary complex was prepared according to the method described in Comparative Example 1, and the N/P value was adjusted to obtain a liquid DP2-DP8 containing a binary complex, wherein the N/P value of DP2 was 0.5:1, DP3
  • the N/P value is 1:1
  • the N/P value of DP4 is 2:1
  • the N/P value of DP5 is 4:1
  • the N/P value of DP6 is 5:1
  • the N/P value of DP7 is 20.
  • DP8 has an N/P value of 30:1.
  • the liquid of the composite was prepared in the same manner as in Example 42 except that the substance M was not added, and the liquid DP9 containing the binary complex was obtained.
  • the DP9 had an N/P value of 10:1. Test the comparison 1-5
  • PEI and B Lipofectamine (Lipofectamine 2000, Invitrogen), the same amount of EGFP-N1 was transfected as a control experiment, and the results are shown in Fig. 4, wherein the cell activity of the Lipofectamine control group was set to 100, The relative activities of the cells in the other groups (including the experimental group) were calculated. The cell viability of the PEI control group was 90.4, and the cell activities of the DPI and DP6-DP8 control groups were 60.2, 83.1, 18.3, and B 12.4, respectively. Test comparison 14
  • the gene silencing efficiency of DP9 was examined, and the same amount of siRNA1 was transfected as a control group using the prior art, that is, using the conventional transfection reagent Lipofectamin 2000 (Invitrogen), and the results are shown in Fig. 5, wherein Lipofectamin 2000 The gene silencing efficiency was 50.2, and the gene silencing efficiency of DP9 was 60.6.
  • Table 1 shows the composition and structure of the different M substances used in the examples, the number average molecular weight of the M substances, the graft ratio, the number average molecular weight of the PGA chains, and the number average molecular weight of the PEG chains.
  • Table 2 shows the composition and structure of the different M substances used in the examples, the number average molecular weight of the M substances, the graft ratio, the number average molecular weight of the PGA chains, and the number average molecular weight of the PEG chains.
  • the transfection efficiency is the transfection efficiency of the EGFP-N1 plasmid when the D substance is the EGFP-N1 plasmid
  • the gene silencing efficiency is the inhibition efficiency of the siRNA1 against the target gene when the substance D is siRNA1.
  • the ternary complex-containing liquid of the present invention has a high cell transfection efficiency or R A interference activity (i.e., gene silencing efficiency of siRNA1).
  • Table 3 reflects the number average molecular weight of the main chain of the different comb-shaped graft copolymers, the number average molecular weight of the side chain, the number of PDMAEMA branches per large chain, and the average particles of the nanoparticles formed by the amphiphilic comb-shaped graft copolymer. diameter.

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Abstract

Cette invention concerne un complexe ternaire comprenant les constituants A, B, C, le constituant A étant un véhicule à charge positive, le constituant B étant un acide nucléique à charge négative et le constituant C étant un copolymère à charge négative. Un liquide contenant ce complexe ternaire et un procédé de préparation correspondant font également l'objet de cette invention. Ce complexe ternaire et le liquide contenant ce complexe ternaire peuvent améliorer l'efficacité de transfection et accroître la toxicité simultanément, pouvant ainsi être utilisés dans la transfection cellulaire, la prophylaxie et le diagnostic de maladies et la thérapie génique.
PCT/CN2011/075735 2010-11-22 2011-06-14 Complexe ternaire, liquide contenant un complexe ternaire, procédé de préparation et utilisation correspondante WO2012068870A1 (fr)

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CN103255174B (zh) * 2013-05-02 2015-10-28 天津大学 以聚乙二醇接枝的透明质酸为外层的三元复合物及三元复合物的液体与应用
CN110129367B (zh) * 2019-05-28 2020-04-07 苏州博特龙免疫技术有限公司 一种阳离子型转染试剂及其制备方法和应用

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CN101755048A (zh) * 2007-06-05 2010-06-23 日东电工株式会社 用于递送核酸的peg-pei共聚物
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