Polyethylenimine-grafted copolymer of poly(L-lysine) and poly(ethylene glycol) for gene delivery

Purpose: PEI is an effective gene carrier but high-molecular-weight PEI is nonbiodegradable and cytotoxic; PLL is biodegradable but lacks proton-buffering capacity, has low transfection efficiency, and is also cytotoxic. A vector combining the complementary strengths of PEI and PLL is needed.

Hypothesis: If low-molecular-weight linear PEI (IPEI) is grafted onto a PEG-b-PLL diblock copolymer, then the resulting ternary copolymer PEG-b-PLL-g-IPEI (PPI) will retain PLL biodegradability, gain PEI-like proton buffering and endosomal escape, reduce cationic cytotoxicity, condense pDNA, and mediate efficient gene transfection. Folate conjugation should further enhance delivery to folate receptor–positive cancer cells, including delivery of the therapeutic TRAIL gene.

Aims: Synthesize and characterize PEG-b-PLL-g-IPEI copolymers with varying IPEI content. - Evaluate cytotoxicity, proton buffering, pDNA condensation, protection, and polyplex size/zeta potential. - Assess reporter gene transfection in multiple cell lines and folate-targeted delivery in FR-positive vs FR-negative cells. - Test therapeutic TRAIL gene delivery, TRAIL expression, caspase-3 activation, apoptosis, and cell growth inhibition.

Delivery system: Platform: Biodegradable ternary copolymer gene delivery vector. - Polymer chemistry: PEG-b-PLL diblock copolymer grafted with low-molecular-weight linear PEI (IPEI, \(M_n\) ~645 or 2200) via CDI activation, yielding PEG-b-PLL-g-IPEI (PPI). PPI variants differed in IPEI content: PPI1 ~18.9%, PPI2 ~29.0%, PPI3 ~56.6%. - Targeting ligand: Folate conjugated to PPI via PEG linker (FA-PPI) for folate receptor–positive cells. - Payload: pEGFP-N3 reporter plasmid and pCMVTRAIL therapeutic plasmid. - Polyplex formation: Electrostatic complexation at various N/P ratios; polyplexes characterized for size, zeta potential, and DNA protection.

Approach: In vitro only: No in vivo animal experiments. - Cell lines: HepG2, U251, BHK-21, 293T, and Bel 7402. 293T and Bel 7402 were FR-positive; HepG2 was FR-negative. - Cytotoxicity: MTT assay in Bel 7402 cells over 0.1–100 µg/mL polymer, 48 h. - Transfection: pEGFP-N3 at N/P 5, 10, and 20; EGFP-positive cells quantified by flow cytometry and visualized by fluorescence microscopy. - Targeting: FA-PPI3/pEGFP vs PPI3/pEGFP in FR-positive and FR-negative cells, with free folate competition. - Therapeutic gene: FA-PPI3/pCMVTRAIL in Bel 7402 cells; TRAIL expression and caspase-3 cleavage by Western blot; apoptosis by TUNEL; viability by MTT.

Key methods: FTIR, \(^1\)H NMR, and GPC for copolymer synthesis and composition. - Acid-base titration for proton buffering capacity. - Agarose gel retardation and DNase I protection assays for pDNA condensation/stability. - DLS for particle size and zeta potential. - AFM for polyplex morphology. - Flow cytometry and fluorescence microscopy for EGFP transfection. - Western blot for TRAIL and caspase-3. - TUNEL assay for apoptosis. - MTT assay for cytotoxicity and cell viability.

Key results: Cytotoxicity: At 100 µg/mL, PPL left 41% cell viability, while PPI3 left 78%; PPI1 and PPI2 left 51% and 56%, respectively. At 22 µg/mL nitrogen concentration, viability was 23% for PPL vs 78% for PPI3. - Buffering: PPI3 showed prominent proton buffering from pH 9 to 3; PPI1 overlapped with PPL. - DNA condensation: PPL fully retarded pDNA at N/P 2; PPI required N/P 4 for complete retardation; PPI3 showed less retardation at N/P 2. PPI2/PPI3 polyplexes at N/P 8–25 had zeta potential < +5 mV and size <200 nm. - Transfection: PPI2 and PPI3 mediated 13.32–26.64% EGFP-positive cells, about 5–10-fold higher than PPL, whose highest efficiency was only 2.97%. PPI3 was highest in all three cell lines. PPI1 was lower (5.42–11.42%). - Folate targeting: FA-PPI3/pEGFP gave higher transfection than PPI3/pEGFP in FR-positive 293T and Bel 7402 cells, but not in FR-negative HepG2 cells. - TRAIL therapy: FA-PPI3/pTRAIL induced 50.76% apoptosis, vs 23.54% for PPI3/pTRAIL, 32.8% for PPL/pTRAIL, and 8.3% for FA-PPI3/pEGFP. Cell viability after FA-PPI3/pCMVTRAIL was 36.26%, compared with 77.68% for FA-PPI3/pEGFP and 79.26% for naked pCMVTRAIL.

Interpretation: The authors conclude that grafting low-molecular-weight linear PEI onto PEG-b-PLL produces a biodegradable vector with reduced cytotoxicity, useful proton buffering, efficient pDNA condensation, and improved transfection. Folate targeting further increases gene delivery to FR-positive cancer cells, and FA-PPI-mediated TRAIL delivery causes apoptosis and growth inhibition. They present PPI as a promising nonviral gene delivery system combining low toxicity, biodegradability, and high transfection efficiency.

Limitations: Purely in vitro; no in vivo biodistribution, circulation, tumor targeting, efficacy, or toxicity data. - TRAIL therapeutic effect was demonstrated only in cultured Bel 7402 cells. - Folate targeting was validated only in cell culture, not in animal tumor models. - No direct comparison with a clinical/commercial gold-standard vector such as branched PEI 25 kDa or Lipofectamine. - Long-term biodegradation, clearance, and immunogenicity were not evaluated. - PPI transfection still required relatively high N/P ratios, and efficiency remained moderate in absolute terms.