Nucleic Acid Therapeutics Using Polyplexes: A Journey of 50 Years (and Beyond)

> If polyplexes are engineered with dynamic, multifunctional domains—shielding, targeting, endosomolytic, and nuclear import functions—and with precise, sequence-defined cationic cores, then they can protect nucleic acid cargo, overcome biological barriers, and deliver it to the required intracellular site of action, improving therapeutic efficacy.

Feature: System; Description: Polyplexes: electrostatic complexes of nucleic acids with cationic polymers

Feature: Cationic polymers; Description: DEAE-dextran, poly-L-lysine (PLL), poly-L-ornithine, poly-L-arginine, polyethylenimine (PEI; linear and branched), PAMAM dendrimers, chitosan, cyclodextrin conjugates, biodegradable poly(β-amino esters), pseudodendritic oligoethylenimines, sequence-defined oligo(ethanamino)amides

Feature: Payloads; Description: pDNA, mRNA, siRNA, miRNA, antisense oligonucleotides, splice-switching oligonucleotides, aptamers, immunostimulatory CpG oligonucleotides, poly(I:C)

Feature: Shielding domains; Description: PEG, pHPMA, hydroxyethyl starch (HES), hyaluronic acid, polysarcosine

Feature: Targeting ligands; Description: Transferrin, folate, EGF, RGD peptides, GalNAc, aptamers, antibodies, other receptor ligands

Feature: Endosomolytic functions; Description: Proton sponge polymers (PEI, PAMAM), histidine residues, INF7 peptide, melittin, KALA/EALA peptides, pH-responsive hydrophobic domains

Feature: Dynamic linkages; Description: pH-labile acetals, pyridylhydrazones, bioreducible disulfides

Feature: Nuclear import/retention; Description: NLS peptides, histone H3 tail peptides, microtubule-associated transport peptides

• In vitro cell culture studies and structure–activity relationship analyses.
• In vivo animal models from cited literature.
• Human clinical trials summarized in Table 3, including melanoma, cystic fibrosis, bladder cancer, ovarian cancer, solid tumors, HIV, and inherited TTR amyloidosis.
• Disease contexts include cancer, genetic diseases, infections, and other acquired or inherited disorders.

No primary experimental data are presented.

• Physicochemical characterization: dynamic light scattering, zeta potential, electron microscopy, SAXS, FRET.
• Intracellular trafficking: live-cell fluorescence microscopy, confocal microscopy, spinning disk confocal microscopy.
• Functional assays: transfection efficiency, gene silencing, reporter gene expression, cytotoxicity.
• Structure–activity relationships: sequence-defined oligomer synthesis by solid-phase peptide synthesis, systematic variation of charge, topology, and functional domains.
• Clinical readouts: target mRNA silencing in tumor biopsies, serum protein knockdown, immune responses.

• No efficient solution yet exists for intranuclear delivery of polyplexes and DNA formulations.
• First-generation polymers such as PEI and PAMAM are nondegradable and show molecular-weight-dependent cytotoxicity.
• The “PEG dilemma”: shielding improves circulation but can reduce cellular uptake and transfection; targeting ligands can increase immunogenicity and opsonization.
• Endosomal escape remains a major limitation; the proton sponge hypothesis is debated and does not fully explain PEI/PAMAM activity.
• Polyplex stability in blood is limited by electrolyte/protein interactions, complement activation, aggregation, and premature dissociation.
• Clinical translation is still limited, with only a small number of completed human trials.
• The review is not systematic and provides no meta-analysis or quantitative comparison across all studies.