WO2019090783A1

WO2019090783A1 - Networked cationic polymer and preparation thereof

Info

Publication number: WO2019090783A1
Application number: PCT/CN2017/110731
Authority: WO
Inventors: Tuo Jin; Shun Chen; Jia Feng
Original assignee: Shanghai Jiao Tong University
Priority date: 2017-11-13
Filing date: 2017-11-13
Publication date: 2019-05-16

Abstract

Provided are a networked cationic polymer and preparation thereof. Said networked cationic polymer has a backbone that involves aromatically conjugated imine linkages and possesses sufficient network cavity to load nucleic acids intra-molecularly. Said networked cationic polymer can be synthesized by reacting a linear or branched multi-amine bearing molecule or oligomer with an aromatic dialdehyde through Shiff-base reaction, and dropping the corresponding product to an aqueous solution of un-treated linear or branched multi-amine bearing molecule or oligomer under pre-determined pH. Said networked cationic polymer may be used to pack or absorb nucleic acids to form a uni-molecular polyplex. Said uni-molecular polyplex may be encapsulated by assembling a tri-block copolymer membrane on surface.

Description

UNI‐MOLECULAR POLYPLEX

FIELD OF THE INVENTION

This invention pertains to a structure design and a synthetic method of cationic polymers useful as gene (DNA and RNA) carriers degradable to endogenous monomers a safety‐known species.

BACKGROUND OF THE INVENTION

While the ability of nucleic acids (RNA/DNA) to express, silence, edit, and regulate genes hasshown great promises as a new class of medicines, turning these polynucleotides from therapeuticactives to practical drugs is retarded by the lack of feasible delivery carriers [1, 2] . Although the viralability to deliver their genetic materials into host cells in animals has evidenced existence of thechemical mechanisms for constructing sufficiently functional carriers in nature, decades longresearch efforts have yet to reach a successful synthetic carrier for systemic delivery. A critical shortcomingof the synthetic carriers reported to date is their undefined structures by dynamic assembly involvingarbitrary numbers of polymer molecules for which sufficient and precise functionalization comparable to virusesbecomes difficult. For example, chemically conjugating all the functional components to cationiclipids or polymers, the two typical materials for packing negatively charged nucleic acids, associatedwith complicated synthetic processes and inefficient siRNA encapsulation [3, 4] . Covalently attachingfunctional agents to already formed lipoplexes or polyplexes through the so‐called click reactionsexposed nucleic acids to reactive hazards [5, 6] . Electrostatic adsorption of cell‐targeting poly‐anionicelectrolytes to the surface of the cationic nanoparticles resulted in replacement of loaded nucleicacids due to the same anionic polyelectrolyte nature [7] . Nano‐encapsulation involved organicsolvents and dynamic particle size distribution [8, 9] . Although polymeric dendrimers, dendrimer‐likemicelles, and ring‐overlaid peptide fibers possess defined core and surface structure, they packnucleic acids by their surface charges through inter‐molecular complexation involving arbitrarynumbers of polymer molecules and lead to undefined particle size and surface [10, 11] . Polyplexes formedfrom cationic polymers conjugated with cyclodextrin at the side chain may allow PEG and celltargeting agents to “plug” in through a hydrophobic tag without exposing nucleic acids to chemicalreactions and organic solvents [12] . A microfluidic process further ensured assembling such polyplexesof 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 andshapes, they lack the ability to facilitate nucleic acid escape from the lysosomal degradation, anessential step of delivering RNA/DNA to the sites of action [14] . It is also highly challenging that apractically druggablesynthetic carrier system must be simple in structure and easy to be formulatedin addition to the capability to accomplish all the inter‐and intra‐cellular delivery tasks. This invention isaimed to create a broadly applicable nucleic acid packing material that meets the essential criteriadiscussed above simultaneously.

BRIEF DESCRIPTION OF THE INVENTION

To address these challenges above, we devleoped athermodynamically self‐regulating process to synthesize a structurally defined networked cationicpolymer which is capable to pack sufficient copies of polynucleotides inside its intra‐molecularcavity. In addition, this nucleic acid‐carrying macromolecule may be further functionalized bywrapping with a protective surface membrane which can immobilize cell‐targeting agents inprecisely optimized population for inter‐cellular recognition. The mechanistic rationales of thechemical 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 regulatedcondensation of linear and branched amino group‐bearing reactants through aromatically (imidazolering for example) conjugated imine linkages. The imidazole ring possesses a pKa (5.9) slightly higher than theendosome pH (5.8) , which ensures the aromatic poly‐imine linkages to break in response to thecellular environment. As the reaction progresses, sufficient Zeta potential is generated around thegrowing networked polymer to prohibit additional cationic reactants to further approach and lead toself‐termination of the polymerization. More interestingly, when appropriate amount of siRNA are added gradually tothe polymer solution, the anionic polynucleotides are adsorbed electrostatically inside the network cavity of eachmolecule of the cationic polymer to cause the networked polymer to collapse to apolyplex particle. The excess positive charges drive the flexible polymer backbone to wriggle to thesurface of the particle and locate the electrostatically adsorbed siRNA into the center of thenetwork structure. The remaining surface charges prevent the uni‐molecular polyplexes from aggregating to larger particles. Finally, a rationally designed tri‐blockcopolymer consisting a carboxyl saccharide guiding block, a hydrophobic central block and a stericstabilization PEG block is allow to adsorb at the cationic polyplex surface electrostatically and to align to amembrane. 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 carboxylsaccharide as well as the hydrophobicity of the central block drive the tri‐block copolymer to align to auni‐lamella membrane [15, 16] . Selected cell‐targeting agents may be conjugated to the distal end of thePEG block and immobilized on the particle surface in optimized population. We name this core‐shellstructured nano‐particulate as “polywraplex” [15] .

The networked cationic polymer of convergent molecular size was synthesized by condensinglinear oligo‐spermine‐imidazole‐4, 5‐imine and branched low molecular weight polyethylene imine (PEI‐1800) through the same imidazole imine linkage. The oligo‐spermine‐imidazole‐4, 5‐imine, synthesized by titrating excess amount of imidazole‐4, 5‐diformaldehyde inspermine, possessed two unreacted aldehydes at the two ends to react with the primary amine groupsof the branched PEI‐1800 to form imidazole‐4, 5‐imine linkages. The average chain length of oligospermine‐imidazole‐4, 5‐imine as determined by the molar ratio of imidazole‐4, 5‐diformaldehydeover spermine, and in the present study, three ratios, 5/3, 5/4, and 5/5 were used. For the successivepolymerization, the molar ratio of the reactants was 5/1 of imidazole‐4, 5‐diformaldehydeinvolved in oligo‐spermine‐imidazole‐4, 5‐imine synthesis over PEI‐1800 because the danglingformaldehydes of the former and the primary amines of the later reacted with each other.

DETAILED DEXSCRIPTION OF FIGURES AND TABLES

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-NMRspectra 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, confirmingthe conversion of the aldehyde tothe 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 DEXCRIPTION OF THE INVENTION

The present invention discloses a nucleic acid‐delivering cationic polymer each molecule of which may pack sufficient copies of RNA or DNA inside its network cavity intra‐molecularly and form a condensed nanometer‐sized particle, named polyplex or uni‐molecular polyplex. The backbone of the networked RNA/DNA packing polymer is formed through aromatically conjugated imine linkages which allow the polymer to degrade readily in response to pH dropping below or close to the pKa of the nitrogen‐containing aromatic ring. Another feature of the nucleic acid packing cationic polymer is its uniform and precisely customizable size. Since each molecule of the cationic polymer may condense nucleic acids to form a polyplex particle, the size of the gene carrying polyplex particle can be precisely predesigned. The excess cationic charges of the formed uni‐molecular polyplex may guide to a self‐assembly of tri‐block copolymer membrane on its surface, which offers an opportunity to immobilize required functional components on the particle surface. The mechanisms and rationales of the series of interlocked thermodynamically self‐regulated processes to assemble the said synthetic carrier of nucleic acids are summarized schematically in Figure 1. The features of this nucleic acid delivering material are further described in details below.

Design of the uni‐molecular polyplex

The so‐called “uni‐molecular polyplex” represents a complex particles formed from a single molecule of cationic polymer and multiple molecules of anionic nucleic acids. The cationic polymer should possesses a three dimensional structure and sufficient network cavity to load nucleic acids inside intra‐molecularly. The networked cationic polymer should be extended due to its intra‐molecular repulsion to allow the molecules of nucleic acids to enter, but collapse to smaller and denser particles after electrostatic interaction with the anionic nucleic acids. The excess cationic charges and the flexibility of the polymer backbone ensues the nucleic acids be packed in the central region of the polymer. Moreover, the backbone of the networked cationic polymer should be degradable in response to the cellular environment, for example the lowered pH. To achieve this property, the polymer backbone should contain pH responsive structures or linkages such as esters, phosphates, and especially aromatically conjugated imines. The aromatic rings are best to possess a pKa below 8, and ideally between 4 and 6.5. Some nitrogen‐containing heterogeneous aromatic rings may fall in this category. Imidazole ring is a good example for its pKa of 5.9, right above the pH of endosomes. Therefore, imidazole‐4, 5‐imine is one of the ideal linkage for incorporating into the backbone of the networked cationic polymer.

Zeta potential regulated polymerization of networked cationic polymer

To form a networked cationic polymer, branched and linear multi‐amine bearing building blocks should be involved. To use Zeta potential to regulate the polymer size, the reaction to conjugate the branched and linear reactants must be carried out in a solvent wherein the amine groups can be protonated and charged. To have the polymer backbone to degrade in response to the cellular pH, the linkage between the branched and linear reactants must be stable under neutral condition but instable under acidic buffer. To meet these criteria, we used Shiff‐base reaction link the branched and linear multi‐amine reactants through an aromatically conjugated imine linkage in water. The branched and the linear multi‐amine bearing reactants should have primary amines at each end of their chains. As an example, branched low molecular weight polyethylene imine, PEI‐1.8K, and a linear spermine imidazole‐4, 5‐imine may be condensed to the networked cationic polymer in an aqueous solution. As the polymerization is progressing, a diffuse double layer is formed around the growing networked cationic polymer. Sufficient Zeta potential will be resulted from the diffuse double layerwhen the polymer reaches certain size that prohibits additional cationic reactants to approach and terminates the polymerization (Figure 2) . The length the linear reactant may also be a factor determining the size, as well as the cavity volume and mesh size of the network cationic polymer. The synthetic process, the length of the linear oligomer, such as spermine oligomer, is determined by the ratio of the aromatic di‐or bis‐aldehydes over the multi‐amine bearing monomer. The typical range of the ratio is between 2/1 to 1/1.

Formation of uni‐molecular polyplexes

The term “uni‐molecular polyplex” refers to polyplex formed from a single molecule of cationic polymer, inside of which multiple nucleic acids are packed. When anionic nucleic acids such as siRNAs were added gradually into a solution of the networked cationic polymer, the electrostatically stretched polymer backbones shrank and encapsulated the adsorbed nucleic acids to form a tightly packed polyplex from one molecule cationic polymer (Figure 1) . The net positive charges drove the flexible polymer backbones to creep to the polyplex surface. The strategy of Zeta potential guided polymerization of networked cationic polymer and encapsulation by single polymer molecule enables assembly of size‐tailorablepolyplexes. The pH responsive backbones degrade in response to the target cell environment and release the nucleic acids packed in the polyplexes.

Surface modification of uni‐molecular polyplex

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. A con‐venient method had been reported to assemble a tri‐block copolymer membrane around each cationic polyplex core [15] . 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 unila‐lamella membrane thermodynamically on the surface of poly‐plexes when presented 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 purpose to assemble a surface anchored uni‐lamella membrane around each polyplex particle may offer an effective protection for the nucleic acid core against enzymatic degradation, pre‐phagocytic leaking due to replacement by anionic electrolytes, and moreover a flexibility to immobilize required functional components, such as cell‐targeting agents. For the later, the distal end of the PEG block of the tri‐block copolymer may best be conjugated with a click reaction head such as an azide group or an active ester (such as a highly selective pentafluorophenol ester) .

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

NTWcatPLMpolyplexes 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/mLethidium 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 networked cationic polymer whose backbone involves aromatically conjugated linkages and possesses sufficient network cavity to load nucleic acids intra‐molecularly.
The networked cationic polymer of claimed in claim 1, wherein the aromatically conjugated imine linkagesinvolve a nitrogen‐containing heterogeneous aromatic ringto respond to pH changes.
The networked cationic polymer of claimed in claim 2, wherein the nitrogen‐containing heterogeneous aromatic ring possesses a pKa below 8, with the best below 6.5.
The networked cationic polymer of claimed in claim 2, wherein the nitrogen‐containing heterogeneous aromatic ring may be imidazole, pyridine, pyrimidine, or pyrazole.
The best functioning nitrogen‐containing heterocyclic aromatic rings of claim 4 is imidazole.
The networked cationic polymer of claimed in claim 1, wherein the basic building blocks linked by the aromatically conjugated imine linkages are branched and linear multi‐amine‐bearing moleculesor oligomers possessing at least three amino groups.
The linear multi‐amine‐bearing moleculesor oligomers of claim 6 are oligomers of spermine, spermidineor other multi‐amine‐bearing linear.
The branched multi‐amine‐bearing moleculesor oligomers of claim 6 is low molecular weight polyethylene imine or other branched multi‐amine‐bearing molecules
Both the linear multi‐amine‐bearing moleculesor oligomers and the branched multi‐amine‐bearing moleculesor oligomers of claim 6have primary amines at each or most of ends which may react with aldehyde through Shiff‐base reaction.
Each of the networked cationic polymer of claimed in claim 1 may be used to pack or absorb nucleic acids intra‐molecularly into its network cavity to form a uni‐molecular polyplex.
The uni‐molecular polyplex of claim 10 may be encapsulated by assembling a tri‐block copolymer membrane at its surface, wherein the copolymer consists a carboxyl saccharide block to guid the surafe adsorption, a hydrophobic central block to align an isolation layer, and a polyethylene glycol block to offer steric stability.
The distal end of the PEG block of the tri‐block copolymer may be conjugated with a conjugated with a click reaction head such as an azide group or an active ester to immobilize selected cell‐targeting modelcules.
A method to synthesize the networked cationic polymer of claim 1 comprises the steps of

a) having the linear or the branched multi‐amine bearing molecule or oligomer to react with an aromatic di‐or bis‐aldehyde by which ends of the molecule or oligomer are conjugated dangling aromatic aldehyde;

b) dropping the product formed in step a) to an aqueous solution of un‐treated branched or linear multi‐amine bearing molecule or oligomer under pre‐determined pH.

c) Separating and purifying the targeted product of step b) .
The method of claim 13, wherein the desired size of the networked cationic polymer is achieved by adjusting the pH of the aqueous solutions of the reactants.
The method of claims 14, wherein the pH of the aqueous solutions of the reactants is adjusted to be between 8 and 11for targeted molecular size of the networked cationic polymer.
The method of claims 13, wherein the desired average molecular size of the networked cationic polymer is achieved by adjusting the salt concentration of the aqueous solutions of the reactants.
The method of claim 13, wherein the salt is selected from inorganic compounds, comprising sodium chloride.
The method of claim 13, wherein the desired network cavity and network mesh size is achieved by adjusting the length of the linear muti‐amine bearing reactant.
The method of claim 18, wherein the linear multi‐amine bearing oligomer of desired length is synthesized by adjusting the molar ratio of the aromatic di‐or bis‐aldehyde to the multi‐amine bearing monomer between 2/1 to 1/1.
The method of claim 13, wherein the desired network cavity and network mesh size is achieved by adjusting the molar ratio of the branched multi‐amine bearing molecule to the linear multi‐amine bearing reactant.