Monitoring intracellular degradation of exogenous DNA using diffusion properties

Abstract

Artificial nonviral gene vectors have the potential to improve the safety of gene therapy; however, such artificial vectors are less efficient for gene expression due to the existence of various barriers to delivery of exogenous DNAs to the nucleus in the living cell. Here we describe the degradation activities of cytoplasmic nucleases, which are involved as a barrier to efficient exogenous gene expression. The size and structure of degraded DNA were monitored by fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) in solution and in the cytoplasm of living cells. Differences in degradation by endo-and exonucleases were confirmed by differences in the size and structure of DNA. Moreover, we confirmed the influence of the exonuclease degradation in cytoplasm on the expression rate of DNA transfection with a cationic lipid. Based on a comparison of in vitro and cell measurements, we conclude that cytoplasmic degradation by exonucleases can be a considerable barrier against efficient gene delivery.

Targeted drug delivery to tumors: Myths, reality and possibility

“…In that sense, intracellular targeting is as important as systemic targeting. Thus, understanding mechanisms of intracellular localization is critical in developing some drug delivery systems [25], and various experimental tools have been developed recently to study intracellular trafficking of the drug carriers [26–29]. An improved understanding of intracellular trafficking mechanisms will likely allow for effective targeting to specific locations within targeted cells required to optimize the efficacy of certain drugs.…”

Intracellular partitioning of cell organelles and extraneous nanoparticles during mitosis

“…The acidic environment of the endosomes and presence of degrading enzymes such as lipases will result in the degradation of most liposomes and polymers [102], where the nondegradable polymer backbones or cationic lipids are exocytosed or kept within the lysosomes. DNA molecules which do reach the cell cytoplasm are rapidly degraded by cytoplasmic nucleases and their cytoplasmic residence time should therefore be kept minimal [103].…”

Synthesis and Characterization of Glycol Chitosan DNA Nanoparticles for Retinal Gene Delivery

“…Because the NPs must spend some time in the nuclease rich cytosol, DNase resistance is a key feature of effective delivery vehicles. [2, 18] To assess the ability of GCS NPs to protect genetic cargo from nucleases, NPs or uncompacted pDNA were incubated with DNase I and then chitosanase (which hydrolyzes beta-1,4-linkages and releases the DNA from the NP) (Fig. 1C).…”

“…[1] The most basic gene therapy is the direct delivery of plasmid DNA (pDNA), but the efficacy of this approach is limited by the fact that pDNA is inefficiently taken into most cells and nuclei, and is unstable in the cytosol due to degradation by nucleases. [2] As a result, significant effort has been directed toward identifying effective, biocompatible agents to facilitate delivery of therapeutic DNAs to the target sites.…”