ABSTRACT
In the present study it is shown that poloxamer 188, added before or immediately after an electrical pulse used for electroporation, decreases the number of dead cells and at the same time does not reduce the number of reversible electropores through which small molecules (cisplatin, bleomycin, or propidium iodide) can pass/diffuse. It was suggested that hydrophobic sections of poloxamer 188 molecules are incorporated into the edges of pores and that their hydrophilic parts act as brushy pore structures. The formation of brushy pores may reduce the expansion of pores and delay the irreversible electropermeability. Tumors were implanted subcutaneously in both flanks of nude mice using HeLa cells, transfected with genes for red fluorescent protein and luciferase. The volume of tumors stopped to grow after electrochemotherapy and the use of poloxamer 188 reduced the edema near the electrode and around the subcutaneously growing tumors.
Subject(s)
Antineoplastic Agents/pharmacokinetics , Drug Delivery Systems/methods , Electroporation/methods , Poloxamer/administration & dosage , Animals , Bleomycin/pharmacokinetics , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival , Cisplatin/pharmacokinetics , Flow Cytometry , HeLa Cells , Hemolysis/drug effects , Humans , Hydrophobic and Hydrophilic Interactions , Jurkat Cells , Luciferases/genetics , Luciferases/metabolism , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Mice , Mice, Nude , Porosity/drug effects , Propidium , Spectrometry, Fluorescence , Whole Body Imaging , Xenograft Model Antitumor Assays , Red Fluorescent ProteinABSTRACT
The combination of RNA interference (RNAi) with the tetracycline-controlled transcription activation (tet) system promises to become a powerful method for conditional gene inactivation in cultured cells and in whole organisms. Here, we tested critical sequence elements that originated from miRNA mR-30 for optimal efficiency of RNAi-based gene knockdown in mammalian cells. Rationally designed miRNAs, expressed conditionally via the tet system, led to an efficient knockdown of the expression of both reporter genes and the endogenous mitotic spindle protein TPX2 in HeLa cells. Quantitative studies of the tet-controlled gene inactivation revealed that the residual expression of the target gene is an intrinsic attribute of all cells that cannot be eliminated either by increasing the miRNA to target mRNA ratio or by simultaneous expression of miRNAs targeting different sequences within the transcript. The kinetic analysis of the reversibility of the miRNA mediated knockdown suggests that the recovery of target gene expression is primarily driven by cell division. Our miRNA design provides a useful tool for conditional gene inactivation in combination with the RNA-polymerase II based tet system. The identified characteristics of the conditional RNAi-mediated knockdown need to be considered for its application in cell culture or in vivo.
