Abstract
Gene transfection is a valuable tool for analyzing gene regulation and function, and providing an avenue for the genetic engineering of cells for therapeutic purposes. Though efficient, the potential concerns over viral vectors for gene transfection has led to research in non-viral alternatives. Cationic polyplexes such as those synthesized from chitosan offer distinct advantages such as enhanced polyplex stability, cellular uptake, endo-lysosomal escape, and release, but are limited by the poor solubility and viscosity of chitosan. In this study, the easily synthesized biocompatible and biodegradable polymeric polysorbate 80 polybutylcyanoacrylate nanoparticles (PS80 PBCA NP) are utilized as the backbone for surface modification with chitosan, in order to address the synthetic issues faced when using chitosan alone as a carrier. Plasmid DNA (pDNA) containing the brain-derived neurotrophic factor (BDNF) gene coupled to a hypoxia-responsive element and the cytomegalovirus promotor gene was selected as the genetic cargo for the in vitro transfection-guided neural-lineage specification of mouse induced pluripotent stem cells (iPSCs), which were assessed by immunofluorescence staining. The chitosan-coated PS80 PBCA NP/BDNF pDNA polyplex measured 163.8 ± 1.8 nm and zeta potential measured −34.8 ± 1.8 mV with 0.01% (w/v) high molecular weight chitosan (HMWC); the pDNA loading efficiency reached 90% at a nanoparticle to pDNA weight ratio of 15, which also corresponded to enhanced polyplex stability on the DNA stability assay. The HMWC-PS80 PBCA NP/BDNF pDNA polyplex was non-toxic to mouse iPSCs for up to 80 μg/mL (weight ratio = 40) and enhanced the expression of BDNF when compared with PS80 PBCA NP/BDNF pDNA polyplex. Evidence for neural-lineage specification of mouse iPSCs was observed by an increased expression of nestin, neurofilament heavy polypeptide, and beta III tubulin, and the effects appeared superior when transfection was performed with the chitosan-coated formulation. This study illustrates the versatility of the PS80 PBCA NP and that surface decoration with chitosan enabled this delivery platform to be used for the transfection-guided differentiation of mouse iPSCs.
Highlights
Gene transfection represents a valuable tool for analyzing gene/protein regulation and function; it provides a means to treat genetic disorders or acquired diseases by delivering therapeutic genetic material or regulatory elements into the patient’s cells [1,2,3].Gene transfection can be mediated broadly by biological, chemical, or physical means [4]; viral vectors represent the most commonly employed biological method for stable and sustainable gene transfection, problems associated with insertional mutagenesis, immunogenicity, package size limitation, and hazards to laboratory personnel are wellknown drawbacks of this method [5,6,7]
The average diameter (Dav) of the polysorbate 80 (PS80) polybutylcyanoacrylate nanoparticles (PBCA NP) was the smallest which was measured at 120.5 ± 1.2 nm and was close to electrical neutrality with a zeta potential of −2.0 ± 0.1 mV
We have demonstrated in our previous study the potential of PS80 PBCA NP as an anionic carrier for mouse induced pluripotent stem cells (iPSCs) transfection
Summary
Gene transfection represents a valuable tool for analyzing gene/protein regulation and function; it provides a means to treat genetic disorders or acquired diseases by delivering therapeutic genetic material or regulatory elements into the patient’s cells [1,2,3].Gene transfection can be mediated broadly by biological, chemical, or physical means [4]; viral vectors represent the most commonly employed biological method for stable and sustainable gene transfection, problems associated with insertional mutagenesis, immunogenicity, package size limitation, and hazards to laboratory personnel are wellknown drawbacks of this method [5,6,7]. Physical methods utilize various forms of mechanical forces to deliver genes into cells bypassing various extra- and intracellular barriers and circumvents endocytosis, yet these methods are still technically demanding, challenging to scale and limited by poor cell viability [8]; chemical methods make use of polymers or lipids as non-viral vectors to carry genetic materials in the form of polyplexes and lipoplexes, respectively, these methods may be less likely to cause insertional mutagenesis, immunogenicity, and injuries to cells, and is less restricted by package size, but may be associated with a lower transfection efficiency than viral vectors for transfecting primary cells, progenitor cells, and stem cells [9].
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