WO2018053795A1

WO2018053795A1 - Cationic polymer of network structure for packing nucleic acids intra-molecularly

Info

Publication number: WO2018053795A1
Application number: PCT/CN2016/099849
Authority: WO
Inventors: Tuo Jin; Shun Chen
Original assignee: Shanghai Jiao Tong University
Current assignee: Shanghai Jiao Tong University
Priority date: 2016-09-23
Filing date: 2016-09-23
Publication date: 2018-03-29
Anticipated expiration: 2019-03-23

Abstract

This invention demonstrated a unique cationic polymer that possesses a number features essential for systemic delivery of nucleic acids simultaneously. First, this polymer was synthesized by Zeta potential-regulated condensation of linear and branched amino group bearing oligomers through imidazole-4, 5-imine linkages, wherein the polymer diameter can be precisely adjusted from tens to hundreds nanometers by Zeta potential determinates such NaCl concentration. Most interestingly, the polymer was structured with sufficient network cavity to pack plenty copies of siRNA intramolecularly to form a polyplex of single polymer molecule. In addition, the polymer backbone degraded readily to non-toxic species and released nucleic acids in response to cellular environment due to the pKa (5.9) of its aromatically conjugated imine linage. Finally, the polyplex of single polymer molecule can be further encapsulated by a self-assembled a unilamella membrane of rationally designed triblock copolymer to which selected functional components such as cell targeting agents can be immobilized in optimized population. The lack of medicinally feasible synthetic carriers remains as the standing-hurdle blocking nucleic acids to covert from therapeutic actives to practical medicines. The present invention may serve as a "universal" vehicle to equip with variety of delivery functions for its defined structure and compatible flexibility.

Description

Cationic Polymer of Network Structure for Packing Nucleic Acids Intra-molecularly

Field of the Invention

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.

Background of the Invention

While the ability of nucleic acids (RNA/DNA) to express， silence， edit， and regulate genes has shown great promises as a new class of medicines， turning these polynucleotides from therapeutic actives to practical drugs is retarded by the lack of feasible delivery carriers [1， 2] . Although the viral ability to deliver their genetic materials into host cells in animals has evidenced existence of the chemical mechanisms for constructing sufficiently functional carriers in nature， decades long research efforts have yet to reach a successful system for systemic delivery. A critical shortcoming of the synthetic carriers reported to date is their undefined structures by dynamic assembly involving arbitrary numbers of molecules for which sufficient and precise functionalization like viruses becomes difficult. For example， chemically conjugating all the functional components to cationic lipids or polymers， the two typical materials for packing negatively charged nucleic acids， associated with complicated synthetic processes and inefficient siRNA encapsulation [3， 4] . Covalently attaching functional agents to already formed lipoplexes or polyplexes through the so-called click reactions exposed nucleic acids to reactive hazards [5， 6] . Electrostatic adsorption of cell-targeting poly-anionic electrolytes to the surface of the cationic nanoparticles resulted in replacement of loaded nucleic acids due to the same anionic polyelectrolyte nature [7] . Nano-encapsulation involved organic solvents and dynamic particle size distribution [8， 9] . Although 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. Although the precise nano-architectures of 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.

Summarv of the Invention

To address these challenges in developing gene-packing materials， we figured out a thermodynamically self-regulating process to synthesize a structurally defined networked cationic polymer which is capable to pack sufficient copies of polynucleotides inside its intra-molecular cavity. In addition， 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.

First， 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. As the reaction progresses， 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.

It should be especially noted that this Zeta potential regulated polymerization is not limited to particular reaction. For any polymerization reaction that generate increasing Zeta potential along with molecular growth of the product， 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. In another words， the linear and branched amino group bearing reactants (molecules or oligomers) 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. For example， the ends of linear and branched reactants (molecules or oligomers) complex to each other through multiple hydrogen bonds such as those in double helix RNA or DNA. In the case of non-degradable linkages between the linear and branched amino group-bearing reactants， the pH-responsive degradability is achieved by pH-responsive degradable structure within the reactants.

When appropriate amount of siRNA are added gradually to the polymer solution， 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.

Finally， 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. We name this core-shell structured nano-particulate as “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. Unlike the reported gene-packing 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.

The rationales of the thermodynamically favored assembly process of the easily functionalizing synthetic carrier system are schematically described in Figure 1.

Brief Description of Drawinos

Figure 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.

Figure 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.

Figure 5. Gel retardation assay of polyplexes prepared with NTWcatPLM and siRNA. M＝marker， N＝naked siRNA， 1～5 means mass ratios of NTWcatPLM to siRNA ＝ 1∶1， 2∶1， 3∶1， 4∶1， and 5∶1 respectively.

Figure 6. ¹H-NMR spectra of NTWcatPLM and related reactants. The chemical shift (over δ10.10) assigned for aldehyde protons disappeared and those assigned for imine protons (δ7.54) groups appeared after the reaction， confirming the conversion of the aldehyde to the imine bonds (-C＝N-) .

Figure 7. FT-IR spectra of NTWcatPLM and related reactants. Formation of the networked cationic polymer through imidazole-4， 5-imine linkages was confirmed by the disappearance of the -HC＝O stretching of the carbonyl groups of imidazole-4， 5-dialdehydes (1670cm^-1) and the appearance of the -HC＝N-stretching of the newly formed imidazole-4， 5-imine linkages (1633cm^-1) .

Detailed Description of the Invention

The structure of the polymer

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) . Normally， the larger mesh size， the lower charge density. The mesh size is also an important factor for the size of nucleic acids which is feasible to be adsorbed inside the network structure of the cationic polymer. The longer of the linear reactant as well as the lower degree of branching of the branched reactant， the larger mesh size and the better accessibility for larger polynucleotides to adsorb inside could be achieved.

To synthesize the aromatically conjugated imine linkages， 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. These 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.

It should be especially noted that 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. In the case of non-degradable linkages between the linear and branched amino group-bearing reactants， the pH-responsive degradability is achieved by pH-responsive degradable structure within the reactants. As an example in the present invention， imidazole-4， 5-imine is involved in the linear reactant， oligo-spermine imidazole-4， 5-imine.

Synthetic process

As mentioned above， 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. As the polymerization progress， 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.

Since size of the 3D networked cationic polymer is determined by Zeta potential generated around the formed macromolecule， various 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. In an example of 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. For this polymer， 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.

Besides the length of the linear reactant， the degree of branching of the branched reactant may also be used to adjust polymer backbone density， Zeta potential and polymer size. In addition， 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.

For merely adjusting the polymer size， pH， especially salt concentration， are factors easier to use. 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. As an example of the present invention， NaCl was added in the reaction system to adjust the final polymer size.

It should be especially noted that 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. For any polymerization reaction that generate increasing Zeta potential along with molecular growth of the product， 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. In another words， 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. For example， the ends of linear and branched reactants complex to each other through multiple hydrogen bonds such as those in double helix RNA or DNA. In the case of non-degradable linkages between the linear and branched amino group-bearing reactants， the pH-responsive degradability is achieved by pH-responsive degradable structure within the reactants. As an example in this invention， imidazole-4， 5-imine is involved in the linear reactant， oligo-spermine imidazole-4， 5-imine.

Packing nucleic acids inside a single molecule of networked cationic polymer

The network structure of the cationic polymer is fully extended in water due to the intra-molecular repulsion between its cationic charges. When nucleic acids are gradually added， the anionic polynucleotides should adsorb onto the networked cationic polymer electrostatically. At the beginning， 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. In the meanwhile， 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.

As the nucleic acid to polymer ratio increases， the size of the formed polyplex particle， i.e. the gene-packing polymer， is getting smaller and smaller at reaches a minimum. Above this critical ratio， 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.

Functionalizing nucleic acids packing cationic polymer with self-assembly membrane

For systemic delivery， 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. We have reported a convenient method to assemble a tri-block copolymer mem-brane around each cationic polyplex core [15] . This self-assembly method may be applied perfectly to the networked gene-packing cationic polymer. 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.

Applications of networked cationic polymer

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.

For packing macromolecules of substantially large molecular weight， for example over 500KD， the networked cationic polymer may be assembled in the presence of the macromolecule to be packed. In this case， the non-covalent complexation discussed above should be used to form the networked cationic polymer.

Examples

The invention is illustrated by the following examples， the purpose of which is to facilitate the understanding of the principle of the invention without， however， limiting its scope as outlined in the claims.

Example 1

This example represents a typical prior art of cross-link polymer， and is given for reference only.

PEI 1.8K， spermine and imidazole-4， 5-dicarbodialdehyde (IM) ware dissolved in ddH2O with 0.01mmol/mL respectively. 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℃. We abbreviate this networked cationic polymer to NTWcatPLM hereafter.

To confirm the synthesis of the polymer， ¹H-NMR and FT-IR were used. ¹H-NMR spectrum was obtained in D₂O with 0.03％ (v/v) tetramethylsilane (TMS) as internal standard using a Varian Mercury Plus 400 MHz spectrometer. FT-IR spectrum was recorded in a KBr pellet using a Bruker Optics FT-IT spectrometer in the range of 400 ～ 4000 cm^-1.

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) .

Example 2

To investigate the influence， 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.

Example 3

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

a) dissolving the cationic polymer in an aqueous solution with pH adjusted between 7 and 9；

b) dissolving a nucleic acid to be loaded in aqueous solution；

c) adding the nucleic acid solution of b) into the cationic polymer solution of a) gradually.

Briefly， 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) .

Example 4

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 1×10⁴ 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 μg/mL. After incubation for 4h， 20μL of MTT (5mg/mL) solution was added into each well and was allowed to react for 6h at 37℃. Then the medium of each well was replaced with 150μL 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.

Example 5

RNA silence experiments were also performed on SMMC7721 stably expressing luciferase cell lines. Cells were seeded into 48-well plates at a density of 1×10⁵ 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 μg/mL， Solarbio， China) ， washed the cells by 1×PBS (Hyclone， USA) twice， and added 250μL medium without serum at each well. 50μL 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℃ in a 5％CO₂ 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.

Expression of luciferase was measured according to the instruction. The cells were washed twice with PBS and lysed with lysis buffer (1×， Promega) . Cell debris was removed by centrifugation at 12,000 rpm for 3 mins (Eppendorf 5810R Centrifuge， Germany) and 20μL of the supernatant add 20μL substrate solution (Luciferase Assay System， Promega) . The luminescence was measured by Single Tube Luminometer (Berthold Detection Systems GmbH) . The total protein concentrations in cell lysates were determined using Micro BCATM Protein Assay Kit (Thermo Scientific Pierce) . Luciferase activity was evaluated by relative light units (RLUs) per protein concentrations (mg) .

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Claims

A cationic polymer of network structure that possesses sufficient internal cavity to adsorb nucleic acids and other multi-anionic agents intra-molecularly.
The cationic polymer of claim 1， wherein the polymeric backbone involves aromatically conjugated imine linkages.
The cationic polymer of claim 2， wherein the aromatically conjugated imine linkages are degradable in response to pH changes.
The cationic polymer of claim 3， wherein the pH-responsively degradable aromatically conjugated imine linkage possesses a pKa below 8.
The cationic polymer of claim 4， wherein the pH-responsively degradable aromatically conjugated imine linkage possesses a pKa between 3 and 6.
The cationic polymer of claim 5， wherein the pH-responsively degradable aromatically conjugated imine linkage is imidazole-4， 5-imine.
The cationic polymer of claim 5， wherein the pH-responsively degradable aromatically conjugated imine linkage is selected from 2， 5-pyridinediimine， 2， 6-pyridinediimine， 2， 3-pyridinediimine， and 2， 5-pyridazinediimine.
The cationic polymer of claim 1， wherein the network structure is formed from linkages between linear and branched amino group bearing molecules.
The cationic polymer of claim 8， wherein the linkages between linear and branched amino group bearing molecules is aromatically conjugated imines.
The cationic polymer of claim 8， wherein the linkages between linear and branched amino group bearing molecules is imidazole-4， 5-imines.
The cationic polymer of claim 8， wherein the linkages between linear and branched amino group bearing molecules is specific multi-hydrogen bonds.
The cationic polymer of claim 8， wherein the linkages between linear and branched amino group bearing molecules is other covalent binds.
The cationic polymer of claim 8， wherein aromatically conjugated imine is involved inside the structure of the linear or the branched amino group bearing molecules.
The cationic polymer of claim 1， wherein the network structure possesses sufficient cavity to pack tens to thousands copies of nucleic acids.
A method to synthesize or assemble the cationic polymer of network structure of claim 1， comprising the steps of

a) preparing the linear or branched amino group bearing molecule (or oligomer) which has di- or bis-aldehyde at each of its chain ends；

b) allowing the linear or branched amino group bearing molecules to react with each other in an aqueous medium or a water containing medium.
The method of claim 15， wherein the length of the linear amino group bearing molecule or oligomer is prepared to desired length for adjusting the mesh size of the network structure of the cationic polymer of claim 1.
The method to synthesize or assemble the cationic polymer of network structure of claim 15， wherein the linear or branched amino group bearing molecule (or oligomer) are conjugated with multi-hydrogen bonds forming agents at each of its chain ends.
The method to synthesize or assemble the cationic polymer of network structure of claim 15， wherein the molecular size of the polymer is adjusted by varying pH of the reaction system.
The method to synthesize or assemble the cationic polymer of network structure of claim 15， wherein the molecular size of the polymer is adjusted by adding salt of predetermined concentration into the reaction system.
The method to synthesize or assemble the cationic polymer of network structure of claim 18， wherein the pH of the reaction system is selected between 9 and 12.
The method to synthesize or assemble the cationic polymer of network structure of claim 19， wherein the salt is NaCl.
The method to synthesize or assemble the cationic polymer of network structure of claim 21， wherein the NaCl concentration varies between 0 and 30 mM.
The cationic polymer of network structure of claim 1， wherein nucleic acids may be condensed inside the network cavity.
The cationic polymer of network structure of claim 23， wherein the nucleic acids are siRNA.
The method to synthesize or assemble the cationic polymer of network structure of claim 15， wherein the di- or bis-aldehyde at each of the chain ends of the linear or branched amino group bearing reactants is selected from imidazole-4， 5-dialdehyde， 2， 5-pyridinedialdehyde，2， 6-pyridinedialdehyde， 2， 3-pyridinedialdehyde， and 2， 5-pyridazinedialdehyde.