Controlled preparation of cholesterol-bearing polycations with pendent l-lysine for efficient gene delivery

Abstract

A new series of cholesterol-bearing poly(methacrylate)s with pendant cationic (l-)-lysine (Chol-PHML) was synthesized via reversible addition–fragmentation chain transfer (RAFT) polymerization, followed by Boc-deprotection. Their structures were characterized by nuclear magnetic resonance (NMR) spectroscopy and gel permeation chromatography (GPC). Then, the average particle sizes and zeta potentials of the Chol-PHML/pDNA polyplexes were examined using dynamic light scattering (DLS), and their plasmid DNA (pDNA)-binding ability was analyzed using agarose gel retardation assay. In vitro cytotoxicity of the polycations was determined using a cell counting kit-8 (CCK-8) assay and lactate dehydrogenase (LDH) kits in HeLa cells. The polyplex’s gene transfection effectiveness was investigated using a luciferase assay. The results revealed that the Chol-PHML series could efficiently bind/load pDNA with a high binding affinity (N/P < 3.0) and subsequently form nanoparticles with the sizes of 90–200 nm and zeta potentials of ∼+40 mV in an aqueous solution. The in vitro cytotoxicity and gene transfection efficiency considerably depended on the Chol-PHML vector’s molecular weight. The optimal Chol-PHML₂₇ exhibited a substantially higher gene transfection efficiency and lower cytotoxicity than those of the linear structural PLL (15 K–30K) and PHML₂₆ control. This study presents a simple method for synthesizing biocompatible and controllable cationic gene vectors by introducing hydrophobic cholesterol moieties, which will function as a promising foundation for gene delivery.

Graphical Abstract

Keywords:

• Cholesterol
• gene delivery
• lysine
• pDNA
• RAFT polymerization

Disclosure statement

The authors declare no conflict of interest.

Data availability statement

Some or all data, models, or code generated or used during the study are available from the corresponding author by request.

Additional information

Funding

We gratefully acknowledge the financial support from Natural Science Foundation of Jiangsu Province (No: BK20190113), Jinling Institute of Technology (No: jit-b-201828, jit-fhxm-202114), and National Natural Science Foundation of China (No: 51902145).