ε-Poly-L-Lysine/Plasmid DNA Nanoplexes for Efficient Gene Delivery In Vivo

Purpose: Non-viral gene delivery vectors are safer than viral vectors but often suffer from lower transfection efficiency and cytotoxicity. ε-Poly-L-lysine (ε-PLL) is a naturally occurring, FDA GRAS cationic polymer with low molecular weight and potential for low toxicity, but its use as a pDNA nanocarrier for efficient in vivo gene delivery was largely unexplored.

Hypothesis: If ε-PLL is used as a cationic polymer to form polyplexes with plasmid DNA, then it will provide improved transfection efficacy and safety compared to synthetic poly-L-lysine (PLL) and the commercial reagent SuperFect, due to its low molecular weight, ability to form near-neutral polyplexes, and non-synthetic origin.

Aims: Develop and characterise ε-PLL/pDNA polyplexes at various N/P ratios. - Optimise size, zeta potential, and DNA condensation. - Evaluate DNase protection, serum stability, and haemocompatibility. - Compare in vitro transfection, cellular uptake, and cytotoxicity with PLL and SuperFect in MCF-7, HeLa, and HEK-293 cells. - Assess in vivo toxicity and gene expression in mice and tumour-bearing rats.

Approach: In vitro: MCF-7 (breast carcinoma), HeLa (cervical carcinoma), HEK-293 (embryonic kidney) cell lines. - In vivo toxicity: Swiss albino mice; single intravenous dose 5 mg/kg of ε-PLL/pDNA, PLL/pDNA, or SuperFect/pDNA; n = 5 per group. - In vivo gene expression: Female Sprague Dawley rats with DMBA-induced tumours; intratumoral injection of free pDNA or polyplexes (30 µL equivalent to 0.1 µg plasmid); n = 5 per group; analysis 48 h post-injection. - Controls: PBS; free pDNA; untreated cells. - No survival study, no tumour growth inhibition study, no biodistribution.

Key methods: Physicochemical characterisation: DLS (size, zeta potential); SEM and TEM. - DNA binding: Agarose gel retardation; heparin displacement; ethidium bromide exclusion. - Stability: DNase I protection (absorbance at 260 nm and gel); serum stability (10% FBS). - Haemocompatibility: Haemolysis assay; SEM of RBCs. - In vitro transfection/uptake: CLSM; fluorescence spectrophotometry for EGFP. - Cytotoxicity: MTT assay. - In vivo toxicity: Plasma ALT, AST, BUN, creatinine; histology of liver, kidney, spleen, lungs; spleen weight. - In vivo gene expression: CLSM of tumour sections; fluorescence spectrophotometry of tumour homogenates.

Key results: Physicochemical properties: ε-PLL/pDNA at N/P 50 formed ~194 nm particles with ~+10.9 mV zeta potential. PLL and SuperFect formed larger particles; SuperFect ~800 nm and ~+50 mV. - In vitro transfection: ε-PLL/pDNA was 3.5-, 3.79-, and 4.79-fold higher than PLL/pDNA and 1.60-, 1.53-, and 1.79-fold higher than SuperFect/pDNA in MCF-7, HeLa, and HEK-293 cells, respectively. - In vivo gene expression: ε-PLL/pDNA showed 19.3-fold, 6.03-fold, and 1.47-fold increases compared to free pDNA, PLL/pDNA, and SuperFect/pDNA, respectively. - Safety: <8% haemolysis and >98% cell viability in vitro. In vivo, ε-PLL/pDNA caused no significant changes in ALT, AST, BUN, or creatinine; PLL increased ALT, AST, BUN, creatinine; SuperFect significantly increased AST and caused minor spleen enlargement. Histology showed no noticeable organ changes for ε-PLL, while PLL and SuperFect caused observable kidney, liver, spleen, and lung changes.

Interpretation: The authors claim that ε-PLL/pDNA polyplexes provide enhanced transfection efficiency and better tolerability than PLL and SuperFect, both in vitro and in vivo. ε-PLL is proposed as a low-cost, non-toxic, naturally occurring non-viral vector for gene delivery. The ease of preparation and stability in DNase and serum support further exploration. Targeted polyplexes could be developed by functionalising ε-PLL amine groups, though N/P ratios would need re-optimisation.

Limitations: In vitro cell lines only: MCF-7, HeLa, HEK-293; no primary or patient-derived cells. - In vivo models: Mice for toxicity, rats for gene expression; no survival or tumour inhibition endpoints. - No biodistribution or pharmacokinetics. - No targeting ligand: Delivery relies on local intratumoral injection. - Small sample sizes: n = 3 for in vitro; n = 5 for in vivo groups. - No long-term toxicity or repeated-dose data. - Mechanism not fully resolved: Endosomal escape and intracellular trafficking not directly visualised. - No comparison with viral vectors. - No clinical translation data. - Targeted functionalisation mentioned as future work, not demonstrated.