Abstract
Using natural-based lipids to construct biocompatible, controllable and efficient nanocarriers and elucidating their structure–function relationships, was regarded as an important area for creating sustainable biomaterials. Herein, we utilized two natural steroids: cholesterol and diosgenin (bearing different hydrophobic tails) as the building blocks, to synthesize a series of natural steroid-based cationic random copolymers PMA6Chol-r-PDMAEMA and PMA6Dios-r-PDMAEMA via RAFT polymerization. The results demonstrated that the steroid-r-PDMAEMA copolymers could efficiently bind pDNA (N/P < 3.0) and then form near-spherical shape (142–449 nm) and positively-charged (+11.5 to +19.6 mV) nanoparticles. The in vitro cytotoxicity and gene transfection efficiency greatly depend on the steroid hydrophobic tail structures and steroid/PDMAEMA block ratios. Optimum transfection efficiency of the (Chol-P1/pDNA and Dios-P3/pDNA) nanoplexes could reach to 18.1–31.2% of the PEI-25K/pDNA complex. Moreover, all of the steroid-r-PDMAEMA/Cy3-pDNA nanoplexes have an obvious “lysosome localization” effect, indicating the steroid structures do not remarkably influence the intracellular localization behaviors of these nanoplexes.
Highlights
IntroductionIntroducing hydrophobic moieties into cationic polymers could improve plasmid (pDNA) binding/loading affinity and nanoplex stability by facilitating the formation of “hydrophobic core–hydrophilic shell” structure, they could increase membrane fusion and cellular uptake, which gave rise to enhanced gene transfection.[15]
Cationic polymers as non-viral gene carriers have been intensively studied in recent decades, due to their controllable structure and function, excellent siRNA/pDNA condensing capability, as well as low immunogenicity.[1,2] many cationic polymer carriers have been developed to realize efficient gene transfection, some defects, such as low solution stability, poor serum compatibility, high cytotoxicity and low cellular uptake, have still largely restricted their practical applications.[3]
Based on the above background, to explore the effects of steroid hydrophobic moieties on gene transfection, in this work, we prepared series of new steroid-based cationic random copolymers, including PMA6Chol-rPDMAEMA (Chol-P1, P2, P3) and PMA6Dios-r-PDMAEMA (DiosP1, P2, P3), via reversible addition fragmentation transfer (RAFT) polymerization of cholesterol-based monomer MA6Chol with the DMAEMA monomer. Their structures were characterized by nuclear magnetic resonance (NMR) and gel permeation chromatography (GPC). pDNA binding affinities of the assynthesized steroid-r-PDMAEMA copolymers were determined by agarose gel retardation assay, the average particle sizes, zeta potentials and morphologies of steroid-r-PDMAEMA/pDNA nanoplexes were analyzed by dynamic laser scattering (DLS) and transmission electron microscope (TEM), respectively
Summary
Introducing hydrophobic moieties into cationic polymers could improve plasmid (pDNA) binding/loading affinity and nanoplex stability by facilitating the formation of “hydrophobic core–hydrophilic shell” structure, they could increase membrane fusion and cellular uptake, which gave rise to enhanced gene transfection.[15]. We synthesized a series of cationic cholesterol-disul de (CHOSS) lipids with low cytotoxicity and high gene transfection efficiency in COS-7 cells, their pDNA nanoplexes showed an interesting “perinucleic localization” effect.[47] apart from cholesterol, other steroid-based cationic gene carriers were rarely prepared and studied, the structural dependence of their self-assembly properties, cytotoxicity, gene transfection capability, endocytosis and intracellular trafficking/localization, were still remain obscure.[48] Noteworthy, most of the steroidal compounds have similar four-ring core skeletons but different hydrophobic tails, which made them good model molecules for further investigation of the structure–function relationships. Based on the above background, to explore the effects of steroid hydrophobic moieties on gene transfection, in this work, we prepared series of new steroid-based cationic random copolymers (steroid-r-PDMAEMA), including PMA6Chol-rPDMAEMA (Chol-P1, P2, P3) and PMA6Dios-r-PDMAEMA (DiosP1, P2, P3), via RAFT polymerization of cholesterol-based monomer MA6Chol (or diosgenin-based monomer MA6Dios) with the DMAEMA monomer Their structures were characterized by nuclear magnetic resonance (NMR) and gel permeation chromatography (GPC). Intracellular localization of the PMA6Chol4-rPDMAEMA190 (Chol-P1)/Cy3-pDNA and PMA6Dios18-rPDMAEMA180 (Dios-P3)/Cy3-pDNA nanoplexes was observed by uorescence microscopy
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