Structure Dependence of Lysosomal Transit of Chitosan-Based Polyplexes for Gene Delivery

Abstract

Chitosan-based polyplexes are known to traffic through lysosomes for a relatively long time, independent of the degree of deacetylation (DDA) and the number average molecular weight (Mn) of the polymer, even though both of these parameters have profound effects on polyplex stability and transfection efficiency. A better understanding of the lysosomal barrier is paramount to the rational design of vectors capable of overcoming obstacles to transgene expression. The aim of the present study was to investigate if lysosomal transit affects chitosan-based polyplex transfection efficiency in a structure-dependent (DDA, Mn) manner. Toward this end, we analyzed the effects of intracellular trafficking modifying agents on transfection efficiency and intracellular vesicular trafficking of polyplexes with different structural properties and stabilities or nucleic acid binding affinity. The use of agents that modify endosome/lysosome acidification and transit processes by distinct mechanisms and their effect on cell viability, polyplex uptake, vesicular trafficking, and transfection efficiency revealed novel and strong chitosan structure-dependent consequences of lysosomal transit. Inhibiting lysosomal transit using chloroquine significantly increased the efficiency of unstable polyplexes, while having minimal effects for polyplexes with intermediate or high stability. In parallel, specifically inhibiting the acidification of vesicles abrogated transfection for all formulations, suggesting that vesicular acidification is essential to promote transfection, most probably by facilitating lysosomal escape. These results provide novel insights into the structure-performance relationship of chitosan-based gene delivery systems.

Chitosan/Hyaluronic Acid Nanoparticles: Rational Design Revisited for RNA Delivery

“…(B) The endosomal escape would logically increase with both chitosan molecular weight and DD, be it due to either proton sponge effect (higher concentration of “free” counterions) or membrane poration (higher membrane damage, which also causes higher chitosan cytotoxicity). Larger endosomolytic activity has been reported for small chitosans (in chitosan/pDNA complexes, A/P = 5), but we believe this to be due to the larger chitosans being more difficult to liberate from binary polyplexes at low A/P ratio, and therefore being comparatively less available for membrane poration. Since the present study used ternary polyplexes (HA presence likely facilitating decomplexation) and high A/P ratios (≥9), the chitosan in our nanoparticles is likely to increase its endosomolytic activity with both molecular weight and DD, i.e., when it is also a better RNA binder.…”

Novos sistemas de delivery, uso tecnológico e reposicionamento da cloroquina e hidroxicloroquina: Uma revisão integrativa

“…Por ocasionar o escape do agente terapêutico das vesículas lisossomais, a CQ, quando presente no meio de cultivo celular, aumentou a taxa de eficiência da transfecção de poliplexos de quitosana-enxerto-poli (ɛ-caprolactona) trimetilada (TMCg-PCL) demonstrando um potencial das nanopartículas de TMC-g-PCL / DNA como um transportador eficiente para a entrega de genes (Tang et al, 2014). Além disso, estas podem fornecer novos resultados sobre a relação estrutura -desempenho de sistemas de entrega de genes baseados em quitosana (Thibault et al, 2016). Além disso, em um estudo que associa as propriedades favoráveis do peptídeo Tat do HIV-1 com as dos sistemas lipídicos para entrega de DNA, combinou-se o lipídio FuGENE HD (FH) com a sequência peptídica Tat modificada com resíduos de histidina e cisteína (mTat) e observou-se, mais uma vez, que o tratamento com cloroquina, associado à endocitose dependente de energia, aumentou significativamente a eficiência de transfecção com mTat / FH (Yamano et al, 2011).…”

Nanotechnology for Cancer Therapy Based on Chemotherapy

“…Since multiple genetic mutations contribute to tumorigenesis and genetic heterogeneity leads to MDR, therapeutic genes, besides chemotherapeutic agents, have emerged in cancer treatment by precise regulating the specific gene expression, such as MicroRNAs (miRNAs), small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs) and so on [ 14 , 107 , 108 ]. Among them, miRNAs are small noncoding RNAs, which play an effect on mRNA translation and various biological processes because a miRNA targets multiple mRNAs [ 109 ].…”