Bioreducible Polyspermine as Less Toxic and Efficient Gene Carrier

Purpose: Bioreducible polymers are attractive as gene carriers because they can reduce cytotoxicity compared with non-degradable cationic polymers such as polyethyleneimine (PEI). However, the safety and transfection efficiency of spermine-based bioreducible polyspermines had not been directly compared with linear PEI (lPEI), a current non-viral gold standard. The study aimed to develop low-molecular-weight spermine-originated bioreducible polyspermines (BPSs) that mimic linear PEI structurally while introducing intracellular degradability.

Hypothesis: If low-molecular-weight spermine-based bioreducible polyspermines are synthesized with disulfide linkages, then they will form nanosized, positively charged pDNA complexes, exhibit strong endosomal buffering capacity, degrade in thiol-rich intracellular environments, and provide transfection efficiency comparable to lPEI25K and bPEI25K with significantly lower cytotoxicity.

Aims: Synthesize BPSs with different molecular weights by varying oxidative polymerization time. - Characterize buffering capacity, pDNA condensation, particle size, zeta potential, and reductive degradability. - Evaluate cytotoxicity and in vitro transfection efficiency in multiple cell lines. - Compare BPS performance against branched PEI25K (bPEI25K) and linear PEI25K (lPEI25K).

Approach: In vitro only. Cell lines: HEK293 (human embryonic kidney), MCF7 (breast adenocarcinoma), HeLa (cervix adenocarcinoma). - Cytotoxicity: MTT assay; polymer concentrations 0–400 µg/mL; polyplexes at various weight ratios (WRs). - Transfection: Luciferase reporter assay; BPS/pDNA WRs 2–20; lPEI25K/pDNA N/P 15; bPEI25K/pDNA N/P 5. - No in vivo model. No targeting ligand. No long-term expression or biodistribution.

Key methods: Synthesis confirmation: ¹H NMR. - Buffering capacity: Acid-base titration from pH 7.4 to 5.1. - Particle size and zeta potential: Dynamic light scattering (DLS). - pDNA condensation and bioreducibility: Agarose gel electrophoresis with and without DTT. - Cytotoxicity: MTT assay. - Transfection efficiency: Luciferase assay normalized to protein content.

Key results: Buffering capacity: BPS1 = 65%, BPS2 = 50%, BPS3 = 47%; lPEI25K = 56%, bPEI25K = 58%. - Particle size / charge: BPS/pDNA complexes were <200 nm at WR 10:1; BPS1 and BPS2 formed ~100 nm particles at WR ≥20. Zeta potentials were positive, >20 mV at WR ≥2. - Bioreducibility: DTT treatment released pDNA from BPS polyplexes, confirming disulfide degradation; lPEI25K and bPEI25K showed no DNA release. - Cytotoxicity: BPSs were less toxic than both PEIs. In HEK293 cells, BPS death rate ≤30% at 50 µg/mL; bPEI25K caused ~80% death at 20 µg/mL; lPEI25K caused ~50% death at 50 µg/mL. Similar trends in MCF7 and HeLa. - Transfection: BPS/pDNA transfection was comparable to lPEI25K/pDNA (N/P 15) and bPEI25K/pDNA (N/P 5) in MCF7 and HeLa. In HEK293, BPS/pDNA showed ~3-fold higher transfection at WR 15:1. Differences among BPS1, BPS2, and BPS3 were not significant.

Interpretation: The authors claim that BPSs show significantly reduced cytotoxicity and comparable transfection efficiency relative to lPEI25K and bPEI25K. They conclude that reducible polymers capable of mimicking linear PEI structure and function, such as BPS, are a sound strategy for gene delivery.

Limitations: In vitro only: No in vivo validation, biodistribution, or therapeutic efficacy. - No targeting ligand: Delivery relies on nonspecific electrostatic interactions. - Serum-free transfection: Transfection was performed in serum-free medium for 4 h; serum stability was not systematically assessed. - Modest advantage: Transfection was generally comparable to PEI, not clearly superior, except in HEK293 cells. - No mechanistic imaging: Endosomal escape and intracellular trafficking were inferred from buffering and bioreducibility, not directly visualized. - No long-term expression or safety data. - DOI/link missing in the supplied file.