Mechanism of plasmid delivery by hydrodynamic tail vein injection. II. Morphological studies.

BACKGROUND: The efficient delivery of plasmid DNA (pDNA) to hepatocytes by a hydrodynamic tail vein (HTV) procedure has greatly popularized the use of naked nucleic acids. The hydrodynamic process renders onto the tissue increased physical forces in terms of increased pressures and shear forces that could lead to transient or permanent membrane damage. It can also trigger a series of cellular events to seal or reorganize the stretched membrane. Our goal was to study the uptake mechanism by following the morphological changes in the liver and correlate these with the fate of the injected plasmid DNA. METHODS: We utilized both light microscopic (LM) and electron microscopic (EM) techniques to determine the effect of the HTV procedure on hepatocytes and non-parenchymal cells at various times after injection. The LM studies used paraffin-embedded livers with hematoxylin and eosin (H&E) staining. The immune-EM studies used antibodies labeled with sub-nanometer gold particles followed by silver enhancement to identify the location of injected pDNA at the subcellular level. The level of overall damage to liver cells was estimated based on alanine aminotransferase (ALT) release and clearance. RESULTS: Both the LM and EM results showed the appearance of large vesicles in hepatocytes as early as 5 min post-injection. The number of vesicles decreased by 20-60 min. Plasmid DNA molecules often appeared to be associated with or inside such vesicles. DNA could also be detected in the space of Disse, in the cytoplasm and in nuclei. Non-parenchymal cells also contained DNA, but HTV-induced vesicles could not be observed in them. CONCLUSIONS: Our studies suggest an alternative or additional pathway for naked DNA into hepatocytes besides direct entry via membrane pores. It may be difficult to prove which of these pathways lead to gene expression, but the membrane pore hypothesis alone appears insufficient to explain why expression happens preferentially in hepatocytes. Further study is needed to delineate the importance of each of these putative pathways and their interrelationship in enabling oligonucleotide (siRNA) activity and pDNA expression.

Mechanism of plasmid delivery by hydrodynamic tail vein injection. I. Hepatocyte uptake of various molecules.

BACKGROUND: The hydrodynamic tail vein (HTV) injection of naked plasmid DNA is a simple yet effective in vivo gene delivery method into hepatocytes. It is increasingly being used as a research tool to elucidate mechanisms of gene expression and the role of genes and their cognate proteins in the pathogenesis of disease in animal models. A greater understanding of its mechanism will aid these efforts and has relevance to macromolecular and nucleic acid delivery in general. METHODS: In an attempt to explore how naked DNA enters hepatocytes the fate of a variety of molecules and particles was followed over a 24-h time frame using fluorescence microscopy. The uptake of some of these compounds was correlated with marker gene expression from a co-injected plasmid DNA. In addition, the uptake of the injected compounds was correlated with the histologic appearance of hepatocytes. RESULTS: Out of the large number of nucleic acids, peptides, proteins, inert polymers and small molecules that we tested, most were efficiently delivered into hepatocytes independently of their size and charge. Even T7 phage and highly charged DNA/protein complexes of 60-100 nm in size were able to enter the cytoplasm. In animals co-injected with an enhanced yellow fluorescent protein (EYFP) expression vector and fluorescently labeled immunoglobulin (IgG), hepatocytes flooded with large amounts of IgG appeared permanently damaged and did not express EYFP-Nuc. Hepatocytes expressing EYFP had only slight IgG uptake. In contrast, when an EYFP expression vector was co-injected with a fluorescently labeled 200-bp linear DNA fragment, both were mostly (in 91% of the observed cells) co-localized to the same hepatocytes 24 h later. CONCLUSIONS: The appearance of permanently damaged cells with increased uptake of some molecules such as endogenous IgG raised the possibility that a molecule could be present in a hepatocyte but its transport would not be indicative of the transport process that can lead to foreign gene expression. The HTV procedure enables the uptake of a variety of molecules (as previous studies also found), but the uptake process for some of these molecules may be associated with a more disruptive process to the hepatocytes that is not compatible with successful gene delivery.