Abstract
Recently, much effort has been expended on the development of non-viral gene delivery systems based on polyplexes of nucleic acids with various cationic polymers. Natural polysaccharide derivatives are promising carriers due to their low toxicity. In this work, chitosan was chemically modified by a reaction with 4-formyl-n,n,n-trimethylanilinium iodide and pyridoxal hydrochloride and subsequent reduction of the imine bond with NaBH4. This reaction yielded three novel derivatives, n-[4-(n’,n’,n’-trimethylammonium)benzyl]chitosan chloride (TMAB-CS), n-[(3-hydroxy-5-(hydroxymethyl)-2-methyl-4-pyridine)methyl]chitosan chloride (Pyr-CS), and n-[4-(n’,n’,n’’-trimethylammonium)benzyl]-n-[(3-hydroxy-5-(hydroxymethyl)-2-methyl-4-pyridine)methyl]chitosan chloride (PyrTMAB-CS). Their structures and degrees of substitution were established by 1H NMR spectroscopy as DS1 = 0.22 for TMAB-CS, DS2 = 0.28 for Pyr-CS, and DS1 = 0.21, DS2 = 0.22 for PyrTMAB-CS. Dynamic light scattering measurements revealed that the new polymers formed stable polyplexes with plasmid DNA encoding the green fluorescent protein (pEGFP-N3) and that the particles had the smallest size (110–165 nm) when the polymer:DNA mass ratio was higher than 5:1. Transfection experiments carried out in the HEK293 cell line using the polymer:DNA polyplexes demonstrated that Pyr-CS was a rather poor transfection agent at polymer:DNA mass ratios less than 10:1, but it was still more effective than the TMAB-CS and PyrTMAB-CS derivatives that contained a quaternary ammonium group. By contrast, TMAB-CS and PyrTMAB-CS were substantially more effective than Pyr-CS at higher polymer:DNA mass ratios and showed a maximum efficiency at 200:1 (50%–70% transfected cells). Overall, the results show the possibility of combining substituent effects in a single carrier, thereby increasing its efficacy.
Highlights
The task of gene therapy or other promising trends in modern medicine is to treat diseases by introducing nucleic acids that can affect gene expression in affected cells [1]
The transfection efficiency of CS is influenced by several factors, such as MW and degree of acetylation (DA)
CS with a low molecular weight (MW = 3000–4000) has better solubility and provides a higher level of gene expression compared to high molecular weight CS [26]; it is more degraded by hydrolytic enzymes and the intestinal bacterial flora [27]
Summary
The task of gene therapy or other promising trends in modern medicine is to treat diseases by introducing nucleic acids that can affect gene expression in affected cells [1]. Their use is further complicated by the complexities involved in industrial virus production. A further complication was exemplified by the famous Gelsinger case in 1999, in which an 18-year-old patient died from an unexpectedly severe immune reaction four days after injection with a modified adenovirus into the liver [4]. These drawbacks have resulted in increased interest in the use of non-viral vectors in gene therapy
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