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Polyplex formation (complexation) and gene release fromthe polyplexes(decomplexation) are major events in polymeric gene delivery; however,the effect of the decomplexation rate on transfection has been rarelyinvestigated. This study employed mixed polymers of poly(L-lysine) (PLL: MW ∼7.4 kDa) and reducible PLL (RPLL) (MW ∼6.7kDa) to design decomplexation rate-controllable PLL100–xRPLLx/pDNA complexes(PRLxpolyplexes). The transfection efficiencyof a model gene (luciferase) in MCF7 and HEK293 cell lines increasedwith increasing x(RPLL content) in the PRLxpolyplexes until peaking at x=2.5 and 10, respectively, after which point transfection efficiencydeclined rapidly. In MCF7 cells, PRL2.5polyplex produced3 or 223 times higher gene expression than PLL or RPLL polyplexes,respectively. Similarly, the transfection efficiency of PRL10polyplex-transfected HEK293 cells was 3.8 or 67 times higher thanthat of PLL or RPLL polyplexes, respectively. The transfection resultswere not apparently related to the particle size, surface charge,complexation/compactness, cellular uptake, or cytotoxicity of thetested polyplexes. However, the decomplexation rate varied by RPLLcontent in the polyplexes, which in turn influenced the gene transfection.The nuclear localization of pDNA delivered by PRLxpolyplexes showed a similar trend to their transfection efficiencies.This study suggests that an optimum decomplexation rate may resultin high nuclear localization of pDNA and transfection. Understandingin decomplexation and intracellular localization of pDNA may helpdevelop more effective polyplexes. [ABSTRACT FROM AUTHOR]

Abstract: Although polymers, polyplexes, and cells are exposed to various extracellular and intracellular pH environments during polyplex preparation and polymeric transfection, the impact of environmental pH on polymeric transfection has not yet been investigated. This study aims to understand the influence of environmental pH on polymeric transfection by modulating the pH of the transfection medium or the culture medium. Changes in the extracellular pH affected polymeric transfection by way of complex factors such as pH-induced changes in polymer characteristics (e.g., proton buffering capacity and ionization), polyplex characteristics (e.g., size, surface charge, and decomplexation), and cellular characteristics (e.g., cellular uptake, cell cycle phases, and intracellular pH environment). Notably, acidic medium delayed endocytosis, endosomal acidification, cytosolic release, and decomplexation of polyplexes, thereby negatively affecting gene expression. However, acidic medium inhibited mitosis and reduced dilution of gene expression, resulting in increased transfection efficiency. Compared to pH 7.4 medium, acidic transfection medium reduced gene expression 1.6–7.7-fold whereas acidic culture medium enhanced transfection efficiency 2.1–2.6-fold. Polymeric transfection was affected more by the culture medium than by the transfection medium. Understanding the effects of extracellular pH during polymeric transfection may stimulate new strategies for determining effective and safe polymeric gene carriers. [Copyright &y& Elsevier]

Abstract: The use of genetically modified islets is a potential strategy for overcoming pitfalls that currently plague islet transplantation. This study employed functional polymeric vectors specifically designed to transfect insulin-secreting cells and results were compared to various non-viral vectors. The evaluation included transfection efficiency, experimental condition effects, gross morphological observation, cytotoxicity, apoptosis, gene distribution in treated islets, insulin secretion function and time-dependent gene expression pattern. Observations from this study suggest that 1) the experimental conditions for islet transfection should be optimized, 2) the cytotoxicity of sulfonylurea containing vectors differs between the RINm5F cell line and primary pancreatic islets, 3) the non-viral vectors were primarily located in the peripheral region of an islet where the initial cell toxicity/apoptosis was apparent, 4) the genetic modification of pancreatic islets with genes for secretory proteins is more feasible than for residing proteins, and 5) the gene construct selection may prolong the gene expression period and oscillating pattern as demonstrated in this study. This study provides some fundamental background information that will aid in further designing polymeric gene vectors for the optimal manipulation of pancreatic islets prior to transplantation. [Copyright &y& Elsevier]

Regnase-1 is an endoribonuclease that regulates the stability of target genes. Here, we investigated whether Regnase-1 plays a regulatory role in the pathophysiology of atopic dermatitis, a chronic inflammatory skin disease. Regnase-1 levels were decreased in skin and serum of atopic dermatitis patients and mice. Regnase-1 +/- mice exhibited more severe atopic dermatitis symptoms than wild-type mice in a house dust mite allergen-induced atopic dermatitis model. Regnase-1 deficiency led to the global changes in gene expression related with innate immune and inflammatory responses, in particular chemokines. The skin Regnase-1 level had an inverse relationship with chemokine expression when we analyzed samples of atopic dermatitis patients and Regnase-1-deficient mice, suggesting that potentiated chemokine production contributes to the augmented inflammation at lesion sites. Subcutaneous administration of recombinant Regnase-1 to mice significantly ameliorated atopic dermatitis-like skin inflammation with reduced chemokine production in a house dust mite-induced atopic dermatitis NC/Nga mouse model. These results indicate that Regnase-1 plays an essential role in maintaining skin immune homeostasis as a regulator of chemokine expression. Modulating Regnase-1 activity may be an efficient therapeutic strategy for treating chronic inflammatory diseases, including atopic dermatitis. [Display omitted] • Regnase-1 expression is decreased in skin and serum of atopic dermatitis patients. • Regnase-1 deficiency is linked to aggravated inflammation in atopic dermatitis. • Regnase-1 deficiency exacerbates chemokine expression in the lesional skin. • Recombinant Regnase-1 ameliorates atopic dermatitis symptoms and chemokine production. • Regnase-1 is an efficient therapeutic target for maintaining skin immune homeostasis. [ABSTRACT FROM AUTHOR]

• Gene complexation affinity of polycation controls tightness and size of polyplex. • Smaller, tighter polyplex delivered more pDNA into nucleus than larger, looser one. • Larger polyplex had slower cytosolic and nuclear transport of pDNA than smaller one. • Too tighter polyplex made poor gene expression due to poor pDNA release in nucleus. • Tightness and size of polyplex strongly influence polymeric gene transfection. The delivery of plasmid DNA (pDNA) using polycations has been investigated for several decades; however, obstacles that limit efficient gene delivery still hinder the clinical application of gene therapy. One of the major limiting factors is controlling pDNA binding affinity with polymers to control the complexation and decomplexation of polyplexes. To address this challenge, polycations of α-poly(L -lysine) (APL) and ε-poly(L -lysine) (EPL) were used to prepare variable complexation/decomplexation polyplexes with binding affinities ranging from too tight to too loose and sizes ranging from small to large. APL-EPL/ATP-pDNA polyplexes were also prepared to compare the effects of endosomolytic ATP on complexation/decomplexation and the sizes of polyplexes. The results showed that smaller and tighter polyplexes delivered more pDNA into the cells and into the nucleus than the larger and looser polyplexes. Larger polyplexes exhibited slower cytosolic transport and consequently less nuclear delivery of pDNA than smaller polyplexes. Tighter polyplexes exhibited poor pDNA release in the nucleus, leading to no improvement in transfection efficiency. Thus, polyplexes should maintain a balance between complexation and decomplexation and should have optimal sizes for effective cellular uptake, cytosolic transport, nuclear import, and gene expression. Understanding the effects of complexation/decomplexation and size is important when designing effective polymer-based electrostatic gene carriers. [ABSTRACT FROM AUTHOR]