Synthetic Approaches for Nucleic Acid Delivery: Choosing the Right Carriers

If the inherited chemical and physical properties of formulation materials (lipids, polymers, peptides/proteins) are systematically correlated with their nucleic acid delivery functions—including condensation/protection, cellular internalization, endosomal escape, cargo release, and nuclear trafficking/entry—then researchers can rationally select or design the appropriate carrier for a given nucleic acid payload and therapeutic application, overcoming the multiple cellular barriers that limit non-viral gene delivery.

• Primary Aim: To compare different formulation materials (lipids, polymers, peptides/proteins) and analyze the correlation between their material properties and nucleic acid delivery functions.

- Secondary Aims (framed as questions): 1. How do different types of formulation materials achieve nucleic acid condensation and protection? 2. How do different formulation materials facilitate the cellular internalization of nucleic acids? 3. What is the mechanism used by each type of formulation material to achieve endosomal escape? 4. What are the strategies leading to nucleic acid release from the carriers? 5. How do different formulation materials carry nucleic acids toward the nucleus? 6. What are the strategies used for the active entry of DNA into the nucleus?

• Ultimate Goal: To summarize the properties of different types of formulation materials and the prospects for the development of next-generation gene delivery vehicles.

Component: Lipids; Examples Discussed: Cationic: DOTAP; Neutral/helper: DOPE, cholesterol, DOPC; Anionic: CHEMS; pH-sensitive/ionizable: DOSPA (in Lipofectamine), DLin-MC3-DMA (in FDA-approved LNP for siRNA); PEG-lipid conjugates

Component: Polymers; Examples Discussed: Polyethyleneimine (PEI: linear and branched), chitosan (natural polysaccharide), PLGA, PEG, poly-L-lysine (PLL), poly(amidoamine) (PAMAM), cyclodextrin (CD)-PEI, PEI-stearic acid, PEI-salicylamide, poly(disulfide amide) (PDSA)

Component: Peptides/Proteins; Examples Discussed: Cell-penetrating peptides (CPPs: TAT, R8, penetratin, MPG, CADY), nuclear localization signals (NLS: SV40 PKKKRKV), fusogenic peptides (HA2, INF7, E5, GALA, KALA), melittin (and CDM-modified melittin), protamine, histidine-rich peptides

Component: Artificial Viruses / VLPs; Examples Discussed: Spherical parapoxvirus-like nanococoons (K3C6SPD/pDNA), filamentous TMV-like artificial viruses, rod-shaped TMV mimics, filamentous nanoribbons for siRNA, virus-like particles (VLPs) from JC polyomavirus, vesicular stomatitis virus, Archeoglobus fulgidus

Component: Payloads; Examples Discussed: pDNA (plasmid DNA), mRNA, siRNA, miRNA, ODN (oligodeoxynucleotide), sgRNA, sgRNA (CRISPR)

Component: Targeting Ligands; Examples Discussed: Folic acid (FL), transferrin (TF), epidermal growth factor (EGF), oligo-arginines (R1, R4, R8), dynein-binding peptides, NLS peptides

Component: Stimuli-Responsive Elements; Examples Discussed: pH-sensitive lipids, redox-responsive disulfide bonds (GSH-triggered), pH-triggered polymer chain length shortening

• Compares three major classes of formulation materials: lipids, polymers, and peptides/proteins.
• Discusses natural and synthetic sources.
• Covers the full delivery pathway: condensation → cellular internalization → endosomal escape → cargo release → nuclear trafficking → nuclear entry.
• Includes a summary table (Table 1) of representative studies with formulation material, nucleic acid, cell type, size, surface charge, and reference.
• Proposes an "ideal carrier" model (Figure 8) incorporating dynein-binding peptides for directed nuclear trafficking and NLS-modification for nuclear pore complex docking.

• Live cell imaging / confocal microscopy: For visualizing intracellular trafficking and endosomal escape.
• Single-Particle Tracking (SPT) and Multiple Particle Tracking (MPT): For quantifying directed vs. random motion in cytoplasm.
• Raster Image Correlation Spectroscopy (RICS) and iMSD analysis: For analyzing polyplex motion.
• 3D Single-Particle Tracking: For lipoplex motion.
• PCR (polymerase chain reaction): For quantifying plasmid copies in the nucleus.
• Electron microscopy: For visualizing polyplex/nuclear entry.
• Fluorescence correlation spectroscopy: For DNA mobility in live cells.
• Transfection efficiency assays: Luciferase, GFP, and YFP reporter gene expression.
• Silencing assays: siRNA-mediated knockdown (e.g., anti-luciferase).
• Dynamic light scattering (DLS) / Zeta potential: For size and surface charge characterization (as reported in Table 1).

• Internalization efficiency: Lipofectamine-based lipoplexes achieved ~45% internalization efficiency in A549 cells, compared with ~10% for adenovirus.
• Nuclear delivery bottleneck: Of ~2,000 to 100,000 plasmid copies delivered into the cytoplasm by polyplexes and lipoplexes, only 1% to 10% were delivered into the nucleus.
• NLS enhancement: SV40 NLS-conjugated lipoplexes showed a 3-fold increase in nuclear accumulation and 10- to 11-fold enhanced transgene expression. NLS-PEI/cyclodextrin/DNA enhanced transgene expression in both dividing and non-dividing cells, with nuclear entry observed within 6 hours (vs. perinuclear accumulation only for controls).
• Endocytic pathway determinants: For PEG-PCL nanoparticles, surface oligo-arginine length dictated endocytic pathway: non-modified and R1 entered via clathrin-mediated endocytosis; R4 via lipid-raft dependent endocytosis; R8 via both lipid-raft dependent endocytosis and macropinocytosis. Surface ligand density did not influence pathway but did influence the number of internalized polyplexes.
• Ligand-mediated uptake enhancement: EGF-modified polyplexes increased cellular uptake from 20% to 50%; CPP decoration showed 10-fold cellular uptake improvement.
• Trafficking velocities: Clathrin-mediated vesicles: ~0.7 μm/s; PEI polyplexes in HuH-7 cells: ~0.65 μm/s; caveolae-dependent PEG-PLL polyplexes: 0.09–0.11 μm/s; macropinocytosed R8-modified lipoplexes: 0.21 ± 0.19 μm/s.
• Direct fusion vs. endocytosis: Lipoplexes (DOTAP:DOPE:cholesterol 11:2:7) entered HeLa cells mainly via direct membrane fusion for siRNA delivery; however, direct fusion accounts for only a small population, with the majority entering via endocytosis.
• Cholesterol effect: Lipoplexes containing cholesterol (DC-cholesterol/DOPE/DNA) showed successful endosomal escape, whereas control (DOTAP/DOPC/DNA) without cholesterol was trapped.
• Artificial virus properties: K3C6SPD/pDNA co-assembled into spherical nanococoons (~70 nm, +25 mV) that protected DNA from enzymatic digestion. Filamentous TMV-like artificial viruses formed from chimeric coat proteins and pDNA/mRNA. Glucose-GSGSGKKKKKKKKKGSGGSKWKEWKWKWKWEWG/siRNA complexes were ~70 nm with ~0 mV surface charge.
• Cargo release balance: Lipoplexes release nucleic acids easily (advantageous for cytoplasmic-acting siRNA/mRNA) but provide poor protection for nuclear-acting pDNA. Polyplexes are more stable but require responsive chemistry (pH-triggered, redox-triggered) for cargo release.

• Lipids share chemical structures with cellular membranes, enabling membrane fusion and easy nucleic acid release—ideal for cytoplasmic-acting nucleic acids (siRNA, mRNA)—but their small molecular structure compromises lipoplex stability and gene protection for nuclear delivery.
• Polymers form stable polyplexes due to high molecular weight, but their macromolecular structure makes membrane interaction difficult; protonation of amino groups is critical for endosomal escape via the sponge effect. Responsive chemistry (pH/redox-triggered) allows smart control of nucleic acid release.
• Peptides/Proteins are biologically derived, can form ordered "capsid"-like artificial viruses, facilitate endosomal escape, and direct nuclear trafficking and entry—bridging the gaps of lipids and polymers.

The authors propose that a combination of different formulation materials is a promising approach to develop multifunctional artificial viruses. Understanding the correlation between material properties and nucleic acid delivery capabilities paves the way for next-generation gene delivery vehicles. They also emphasize that there is still a big gap between translational studies and clinical applications.

• No primary experimental data; conclusions are synthesized from existing literature.
• No systematic search strategy, inclusion/exclusion criteria, or meta-analysis.
• No head-to-head comparison of different carriers under standardized conditions; comparisons are drawn across studies with varying cell lines, nucleic acid types, and assay conditions.
• Limited clinical translation data; most cited studies are in vitro or preclinical.
• The review acknowledges that a "direct correlation has not been established from individual studies" between formulation material properties and intracellular nucleic acid delivery.

Limitations of the field highlighted by the authors:

• Endosomal escape remains a major bottleneck: Most synthetic nucleic acid complexes enter cells via endocytosis and end up in lysosomes for enzymatic digestion.
• Nuclear entry mechanism unclear: After NLS-guided docking on the nuclear pore complex (NPC), the subsequent step of nuclear entry of DNA or intact complex "remains to be addressed."
• NLS burial issue: If NLS is used as the DNA condensation agent, complexation with DNA may bury the NLS, reducing importin-binding capability.
• Uncontrollable cargo release: For polyplexes, nucleic acid release relying on polyanionic exchange with endogenous biopolymers is uncontrollable.
• VLP production limitations: Tedious and time-consuming cloning, hard to control cargo encapsulation, and low production yield of effective VLPs.
• Cytotoxicity: Cationic lipids and polymers have the potential to induce cytotoxicity and particle instability; PEGylation and neutral lipid incorporation are used to mitigate but not eliminate this.
• No large-animal validation: Most studies are in cell lines or mouse models; scalability and long-term safety remain unproven.