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WO2018053795A1 - Polymère cationique de structure de réseau pour le conditionnement intra-moléculaire d'acides nucléiques - Google Patents

Polymère cationique de structure de réseau pour le conditionnement intra-moléculaire d'acides nucléiques Download PDF

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WO2018053795A1
WO2018053795A1 PCT/CN2016/099849 CN2016099849W WO2018053795A1 WO 2018053795 A1 WO2018053795 A1 WO 2018053795A1 CN 2016099849 W CN2016099849 W CN 2016099849W WO 2018053795 A1 WO2018053795 A1 WO 2018053795A1
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cationic polymer
polymer
network structure
linear
amino group
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Tuo Jin
Shun Chen
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Shanghai Jiao Tong University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • This invention pertains to a structure design and a synthetic method of a cationic polymer useful as nucleic acid (DNA and RNA) carriers for in vitro and in vivo delivery.
  • RNA/DNA nucleic acids
  • polymeric dendrimers, dendrimer-like micelles, and ring-overlaid peptide fibers possess defined core and surface structure, they pack nucleic acids by their surface charges through inter-molecular complexation involving arbitrary numbers of polymer molecules and lead to undefined particle size and surface [10, 11] .
  • Polyplexes formed from cationic polymers conjugated with cyclodextrin at the side chain may allow PEG and cell targeting agents to “plug” in through a hydrophobic tag without exposing nucleic acids to chemical reactions and organic solvents [12] .
  • a microfluidic process further ensured assembling such polyplexes of uniform size [13] . This insertion method, however, does not offer charge neutralization and coreisolation.
  • nucleic acids may achieve defined sizes and shapes, they lack the ability to facilitate nucleic acid escape from the lysosomal degradation, an essential step of delivering RNA/DNA to the sites of action [14] . It is also highly challenging that a practically druggable synthetic carrier system must be simple in structure and easy to be formulated in addition to the capability to accomplish all the inter-and intra-cellular delivery tasks. This invention is aimed to create a broadly applicable nucleic acid packing material that meets the essential criteria discussed above simultaneously.
  • this nucleic acid carrying macromolecule may be further functionalized by wrapping with a protective surface membrane which can immobilize cell-targeting agents in precisely optimized population for inter-cellular recognition.
  • the mechanistic rationales of the chemical art for assembling this bio-delivery material are described schematically in Figure 1.
  • the size-defined networked cationic polymer is synthesized by a Zeta potential regulated condensation of linear and branched amino group-bearing reactants (molecules or oligomers) through aromatically (imidazole ring for example) conjugated imine linkages to form a networked polymer.
  • the imidazole ring possesses a pKa (5.9) slightly higher than the endosome pH (5.8) , which ensures the aromatic poly-imine linkages to break in response to the cellular environment.
  • Zeta potential is generated around the growing networked polymer.
  • the Zeta potential increases with the increase in the polymer size, and finally reached a level sufficient to prohibit additional cationic reactants to further approach and lead to self-termination of the polymerization. Since the polymerization may be terminated by the increasing Zeta potential, various Zeta potential determinates, such as pH, salt concentration, and charge density, may be used to adjust the polymer molecular weight and size. The said charge density may be determined by the length of the linear oligomer, for which longer oligomer will lead to larger mesh size of the networked polymer and less charge density.
  • this Zeta potential regulated polymerization is not limited to particular reaction.
  • the size of the resulted polymer can be regulated by Zeta potential, as well as Zeta potential determinates, such as charge density, pH and salt concentration.
  • This Zeta potential regulated size-determination may also be applied to the process of super-molecule assembly.
  • the linear and branched amino group bearing reactants molecules or oligomers
  • the ends of linear and branched reactants complex to each other through multiple hydrogen bonds such as those in double helix RNA or DNA.
  • the pH-responsive degradability is achieved by pH-responsive degradable structure within the reactants.
  • the anionic polynucleotides are adsorbed inside the network cavity of each molecule of the cationic polymer electrostatically to cause the adsorbent molecule to collapse to a polyplex particle.
  • the excess positive charges drive the flexible polymer backbone to wriggle to the surface of the particle and locate the electrostatically adsorbed siRNA into the center of the network structure of the polymer.
  • the remaining surface charges prevent the polyplexes formed from single polymer molecule from aggregating to larger particles.
  • a rationally designed tri-block copolymer consisting a carboxyl saccharide guiding block, a hydrophobic central block and a steric stabilization PEG block is allow to adsorb at the cationic polyplex surface electrostatically to form a membrane by self-assembly. While the tri-block copolymer tends to aggregate to a micelle by itself, the strong charge-charge interaction between the cationic polyplex and the multi-anionic carboxyl saccharide and the hydrophobicity of the central block drive the tri-block copolymer to align to a uni-lamella membrane [15, 16] .
  • Selected cell-targeting agents may be conjugated to the distal end of the PEG block and immobilized on the particle surface in optimized population.
  • the surface population of the cell-targeting agents may be fine tuned by varying the ratio of tri-block copolymer conjugated with and without the targeting moieties.
  • this core-shell structured nano-particulate was named “polywraplex” [15] .
  • Cationic polymers or cationic lipids are the two most convenient and broadly applied gene-packing materials, which form nanoparticles with polynucleotides through electrostatic complexation involving arbitrary numbers of the polymer molecules.
  • the numbers of the cationic polymers involved in formation of the nano-particulate delivery systems vary randomly as a function of pH and concentration of the materials.
  • the networked cationic polymer of the present invention condense nucleic acids intra-molecularly into each polyplex particle formed from a single molecule of the polymer. Since the polymer size is customizable by Zeta potential determinates, the polyplex it forms will possesses well-defined size and structure.
  • thermodynamically favored assembly process of the easily functionalizing synthetic carrier system are schematically described in Figure 1.
  • FIG. 1 Schematic description of thermodynamically self-regulated assembling of a synthetic carrier of nucleic acids consisting a polyplex core formed from a single-molecule of pH-responsive networked cationic polymer and a uni-lamella shell of rationally designed tri-block copolymer.
  • the spermine-imidazole oligomer was syn-thesized at the imidazole-4, 5-dialdehyde to spermine ratio of 5/3, 5/4 and 5/5, respectively; and the NTWcatPLM was synthetized by titrating the polyspermine oligomer into PEI-1.8K in the molar amount of 1/5 of that of imidazole-4, 5-dialdehyde.
  • Figure 2 Molecular size and Zeta potential of the networked cationic polymer as functions of reaction parameters of Zeta potential-regulated polymerization.
  • A Time of the condensation reaction;
  • B Ration of linear/branched reactors and length of the oligo-spermine-imidazole-4, 5-imine;
  • C pH of reaction medium; or
  • D NaCl concentration in reaction medium.
  • Figure 3 Packing siRNA inside a single molecule of networked cationic polymer, followed by self-assembly of tri-block copolymer shell.
  • A Diameter and Zeta potential of polyplexes as a function of nucleic acid to polymer ratio during siRNA titration;
  • B and
  • C Transmission electron microscopic images of networked cationic polymer, 410 nm in diameter, before and after adding siRNA at 1/10 weight ratio;
  • D Diameter and Zeta potential of polyplexes formed from single molecule of networked cationic polymer before and after assembly of tri-block copolymer membrane.
  • FIG. 4 Cytotoxicity and gene silencing efficiency of the networked cationic polymer as siRNA delivery materials.
  • A Viability of SMMC7721 cells treated with the networked cationic polymer and PEI-25K, respectively, as examined by MTT assay.
  • B Silence of luciferase gene in SMMC7721 cells stably expressing luciferase treated with anti-luciferase siRNA naked and delivered by PEI-25K and networked cationic polymer at various polymer to siRNA ratios.
  • FIG. 1 Gel retardation assay of polyplexes prepared with NTWcatPLM and siRNA.
  • M marker
  • N naked siRNA
  • the present invention discloses a cationic polymer which is structured with a networked backbone polymerized through aromatically conjugated imine linkages.
  • An example of the aromatic ring is imidazole which possesses a pKa of 5.9, slightly lower than the pH inside the s of cells. Other aromatic rings possessing different pKa may be used to construct this polymer for different usages.
  • the network structure of the polymer is formed by condensing linear and branched amino group-bearing reactants through the aromatically conjugated imines, such as imidazole-4, 5-diimine.
  • the mesh size of the network structure of the polymer is determined by the length of linear reactant, such as oligo-sermine-imidazole-4, 5-diimine, and the degree of branching of the branched reactant, such as low molecular weight polyethylene imine (PEI) .
  • linear reactant such as oligo-sermine-imidazole-4, 5-diimine
  • degree of branching of the branched reactant such as low molecular weight polyethylene imine (PEI) .
  • PEI low molecular weight polyethylene imine
  • aromatic di-or bis-aldehydes other than imidazole-4, 5-dialdehyde are those with a pKa below 8.0, prefer between 3.0 and 6.0, comprising terephthalaldehyde, isophthalaldehyde, phthaldialdehyde, 2, 5-pyridinedicarboxaldehyde, 2, 6-pyridinedicarboxaldehyde, 2, 3-pyridinedicarboxaldehyde, 2, 5-pyridazinedicarboxaldehyde.
  • aromatically conjugated di-or bis-aldehydde are used to form aromatically conjugated imine linkages, comprising imidazole-4, 5-diimine 2, 5-pyridinediimine, 2, 6-pyridiimne, 2, 3-pyridinediimine, and 2, 5-pyridazinediimine.
  • this Zeta potential regulated polymerization is not limited to particular covalent bond such as aromatic imines. It can be any covalent bond or non-covalent specific bonds, such as multiple hydrogen bonds.
  • the pH-responsive degradability is achieved by pH-responsive degradable structure within the reactants.
  • imidazole-4, 5-imine is involved in the linear reactant, oligo-spermine imidazole-4, 5-imine.
  • the cationic polymer of the present invention is synthesized by condensing linear and branched multi-amino group bearing oligomers through aromatically conjugated imine linkages.
  • the imine linkages are formed by the reaction between aldehyde and primary imine at the ends of the linear and the branched reactants, respectively.
  • the polymerization reaction is carried out in water or water-containing solvents for which the amino groups can be protonated and generate cationic charges.
  • networked cationic polymer is formed, and the cationic charges of protonated amino groups turn to be Zeta potential around the formed macromolecule.
  • the Zeta potential is getting higher and higher along with the growth of the polymer, and finally reaches level sufficient to prohibit additional cationic reactant to approach.
  • the polymerization is therefore self-terminated by the increasing Zeta potential.
  • Zeta potential determinates may be used to manipulate molecular weight of the cationic polymer.
  • Useful Zeta potential determinates comprise charge density (or amino group population) , pH and salt concentration.
  • charge density regulation in the present invention the chain length of the linear reactant, such as oligo-spermine imidazole-4, 5-imine, is used to adjust charge density. The longer chain of the linear reactant, the larger mesh size of the network structure of the polymer is formed. Accordingly, density of the polymer backbone, which is parallel to the density of the cationic charges, is lowered.
  • the size has to grow larger to generate sufficient Zeta potential to terminate the reaction.
  • the advantage of using chain length of the linear reactant to regulate the polymer size is that the mesh size may be adjusted at the same time. Sufficient mesh size of the polymer’s network structure is essential for loading higher molecular weight polynucleotides inside the gene-packing polymer.
  • the degree of branching of the branched reactant may also be used to adjust polymer backbone density, Zeta potential and polymer size.
  • degree of branching of the branched reactant determines the intra-molecular cross-linking density. For example, if the branched reactant has only three branches, each has a primary amine at the end, the formed polymer should be a spherical surface in shape.
  • pH especially salt concentration
  • Elevating pH will reduce the population of protonated amino groups, and thus result in lowering of the Zeta potential and enlargement of polymer size.
  • Adding salt concentration to the reaction system may directly suppress Zeta potential of a particle, such as the growing polymer, and result in larger size of the final product of the polymerization reaction.
  • NaCl was added in the reaction system to adjust the final polymer size.
  • this Zeta potential regulated polymerization is not limited to particular reaction, but can be achieved through any condensation reaction or even non-reactive but specific interaction between the linear and branched reactants.
  • the size of the resulted polymer can be regulated by Zeta potential, as well as Zeta potential determinates, such as charge density, pH and salt concentration. This Zeta potential regulated size-determination may also be applied to the process of super-molecule assembly.
  • the linear and branched amino group bearing reactants can be cross-linked through non-covalent linkage, wherein the non-covalent linkages should be specific to each other between the ends of the linear reactant and those of the branched reactant.
  • the ends of linear and branched reactants complex to each other through multiple hydrogen bonds such as those in double helix RNA or DNA.
  • the pH-responsive degradability is achieved by pH-responsive degradable structure within the reactants.
  • imidazole-4, 5-imine is involved in the linear reactant, oligo-spermine imidazole-4, 5-imine.
  • the network structure of the cationic polymer is fully extended in water due to the intra-molecular repulsion between its cationic charges.
  • the anionic polynucleotides should adsorb onto the networked cationic polymer electrostatically.
  • the adsorbed polynucleotide is at the surface of the polymer, and center of the positive charges and that of the negative charges are separated.
  • the separated charge centers generate an electromotive force that drives the adsorbed polynucleotide to move towards the positive charge center.
  • the flexible backbone of the cationic polymer wiggles to the surface of the nucleic acid-packing polymer under the intra-molecular repulsion between the excess cationic charges.
  • the adsorbed polynucleotides cause collapse of the network structure of the cationic polymer due to strong electrostatic interaction as evidenced by the drastic shrinkage of the extended 3D networked polymer and retained Zeta potential.
  • the retained Zeta potential after packing anionic polynucleotides is attributed to the retained charge density at the surface of the shrunk particle.
  • the size of the formed polyplex particle i.e. the gene-packing polymer
  • the diameter of formed polyplex particle rebounds. It is reasonable that the particle reaches its minimum size, the shrinking internal cavity of the network structure of the polymer is filled, and additional nucleic acids are adsorbed at the surface of the cationic polymer and caused inter-particulate aggregation.
  • the size-optimized pH-responsive polyplexes should be covered by a protective shell, which helps to neutralize their net charges and prevent from pre-phagocytic degradation.
  • a protective shell which helps to neutralize their net charges and prevent from pre-phagocytic degradation.
  • the tri-block copolymer consists a carboxyl saccharide block to guide to polyplex surface, a hydrophobic central block to form an isolation layer, and a steric stabilization PEG block to pro-long in vivo circulation.
  • This tri-block copolymer formed micelles in aqueous solution alone, but aligned to a unilamella membrane on the surface of polyplexes thermodynamically when the charged particles present in the solution.
  • the hydrophobic central block is essential for the surface alignment and for preventing the multi-anionic charges of the saccharide block from inter-penetrating into the polyplex core to replace the poly-anionic nucleic acids.
  • Cell targeting moieties may be conjugated to the distal end of the PEG block and mixed with the targeting agent free block copolymers in optimized ratio for selective cell targeting.
  • the polymers embodying in the present invention are applicable in several aspects. First it can be used to pack plasmids, RNAi, CRISPR/Cas9, and multiple miRNA for gene expression, gene silencing, gene editing, and gene regulation. The above applications can be achieved in cellular level or in experimental animals, pets, farming animals, or even human.
  • This networked cationic polymer may also be used to pack anionic fluorescent indicators, anionic diagnostic agents, and anionic proteins.
  • the networked cationic polymer may be assembled in the presence of the macromolecule to be packed.
  • the non-covalent complexation discussed above should be used to form the networked cationic polymer.
  • This example represents a typical prior art of cross-link polymer, and is given for reference only.
  • the polymer (named as PSIM) was synthesized by dropping IM aqueous solution slowly into spermine solution with stirring. After dropping, the chemical reaction continued stirring over night and the PSIM solution was prepared. Then dropping the PSIM solution slowly into PEI 1.8K aqueous solution with stirring. The final product was got after lyophilization and then stored in -80°C. We abbreviate this networked cationic polymer to NTWcatPLM hereafter.
  • Particle size and zeta potential were determined by dynamic light scattering (DLS) using a Brookhaven Instruments Corporation 90 Plus Particle Size Analyzer. The morphology of polyplexes was examined by transmission electron microscope (TEM) .
  • TEM transmission electron microscope
  • PEI solutions were pretreated with HCl (0.1M) , or NaOH (0.1M) , or NaCl. And then PSIM solution was slowly into PEI aqueous solution with stirring. The following steps were in the same as Example 1.
  • NTWcatPLM polyplexes were prepared by adding NTWcatPLM aqueous solution to siRNA solution with various mass ratios carefully. Polywraplex was formed as follow.
  • the polyplexes are prepared with three steps, including
  • the triblock copolymer mPEG45-PCL20-maltotriose-COO - was added to the polyplex solution at the predetermined mass ratios.
  • Polyplexes of dendrimer 8G were prepared with the same process as the control.
  • Particle size and zeta potential were determined by dynamic light scattering (DLS) using a Brookhaven Instruments Corporation 90 Plus Particle Size Analyzer. The morphology of polyplexes was examined by transmission electron microscope (TEM) at mass ratio of 20 ⁇ 1. Gel retardation assay were prepared at various mas ratio and electrophoresed on a 1% (w/v) agarose gel pretreated with 0.5mg/mL ethidium bromide in 1 ⁇ Tris-acetate-EDTA (TAE) buffer at 110V. The gel was analyzed on a UV illuminator (Tanon 2500 Gel Image System) .
  • Toxicity of NTWcatPLM was measured by the MTT assay in comparison with PEI 25 KDa.
  • SMMC7721 stably expressing luciferase cells were seeded in a 96-well plate at a density of 1 ⁇ 10 4 cells/well and incubated for 24 hours, then treated with a series of concentrations of NTWcatPLM and PEI 25 KDa solutions from 10 to 500 ⁇ g/mL. After incubation for 4h, 20 ⁇ L of MTT (5mg/mL) solution was added into each well and was allowed to react for 6h at 37°C. Then the medium of each well was replaced with 150 ⁇ L of DMSO and the plates were incubated for 10min at room temperature. Absorbance at 570nm was measured with Microplate Reader (3M, USA) , and reference at 630nm. The MTT value of untreated cells was considered as 100%cell viability. All transfection and toxicity assays were performed in triplicate.
  • RNA silence experiments were also performed on SMMC7721 stably expressing luciferase cell lines.
  • Cells were seeded into 48-well plates at a density of 1 ⁇ 10 5 cells/well 24h before transfection. When the Cells were cultured to 90%confluence in Dulbecco’s Modified Eagle’s Medium (DMEM, Hyclone, USA) containing 10%FBS (Hyclone, USA) and 1%penicillin/streptomycin (Stock 10,000 U/mL, 10,000 ⁇ g/mL, Solarbio, China) , washed the cells by 1 ⁇ PBS (Hyclone, USA) twice, and added 250 ⁇ L medium without serum at each well.
  • DMEM Dulbecco’s Modified Eagle’s Medium
  • polyplexes with different polymer/siRNA mass ratios ranging from 5 to 30 were gently overlaid into the wells. Each well contains 500ng siRNA.
  • the plates were incubated at 37°C in a 5%CO 2 incubator for 4h. After incubation, the transfection medium was replaced with 0.5 mL fresh complete medium. The plates were incubated for 48h under the same conditions as previously.
  • luciferase activity was evaluated by relative light units (RLUs) per protein concentrations (mg) .

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Abstract

La présente invention concerne un polymère cationique unique qui possède un certain nombre d'éléments essentiels pour une administration systémique d'acides nucléiques simultanément. Tout d'abord, ce polymère a été synthétisé par condensation régulée par potentiel zêta d'oligomères portant des groupes amino linéaires et ramifiés par l'intermédiaire de liaison imidazole-4,5-imine, le diamètre du polymère pouvant être ajusté avec précision de plusieurs dizaines à centaines de nanomètres par des facteurs déterminants du potentiel zêta tels que la concentration en NaCl. De manière plus intéressante, le polymère a été structuré avec une cavité de réseau suffisante pour conditionner de nombreuses copies d'ARNsi intramoléculairement pour former un polyplexe d'une molécule de polymère unique. De plus, le squelette polymère se dégrade facilement en espèces non toxiques et en acides nucléiques libérés en réponse à un environnement cellulaire en raison du pKa (5,9) de sa liaison imine à conjugaison aromatique. Enfin, le polyplexe de molécule de polymère unique peut en outre être encapsulé par une membrane unilamellaire auto-assemblée de copolymère triséquencé conçu de manière rationnelle sur laquelle des composants fonctionnels sélectionnés tels que des agents de ciblage cellulaire peuvent être immobilisés dans une population optimisée. L'absence de supports synthétiques médicalement possibles constitue un obstacle permanent empêchant que les acides nucléiques passent de principes thérapeutiques actifs à des médicaments utilisables dans la pratique. La présente invention peut servir de véhicule "universel" à équiper de diverses fonctions d'administration en raison de sa structure définie et d'une flexibilité compatible.
PCT/CN2016/099849 2016-09-23 2016-09-23 Polymère cationique de structure de réseau pour le conditionnement intra-moléculaire d'acides nucléiques Ceased WO2018053795A1 (fr)

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Cited By (2)

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US12344709B2 (en) 2018-04-27 2025-07-01 Genedit Inc. Cationic polymer and use for biomolecule delivery
US12415891B2 (en) 2018-09-25 2025-09-16 The University Of Tokyo Amphiphilic poly (amino acid), block copolyer using the amphiphilic poly (amino acid), and complex including the amphiphilic poly (amino acid) or the block copolymer and nucleic acid

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