{'fraction_non_alphanumeric': 0.01393884892086331, 'fraction_numerical': 0.0017985611510791368, 'mean_word_length': 5.3210227272727275, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Study of the internalization and subcellular trafficking of model antisense compounds in Hep G2 cells

Introduction Antisense therapy has the potential to specifically treat a wide variety of diseases on the cellular level by inhibiting undesired protein synthesis. This potential has stimulated a significant amount of research in this area but problems limit antisense in reaching its potential. One of the greatest obstacles of antisense therapy is delivery of the oligonucleotide to its target. The antisense oligonucleotide must reach its complementary mRNA in the cytoplasm or nucleus to be effective. To do this, the oligonucleotides must pass one or more biological membranes, but their charge and/or size should prevent this. The goal of this research is to develop methods to study the internalization and subcellular trafficking of compounds into cells. From this basis, we can systematically change the delivery system to improve cytoplasmic or nuclear delivery of oligonucleotides or other therapeutic agents. The oligonucleotides and model compounds are delivered via water soluble polymers (see Figure 1). The polymer contains targeting moieties which results in internalization by receptor mediated endocytosis. The oligonucleotides and target compounds are covalently attached to the polymer backbone by nondegradable or degradable spacers. The degradable spacers are composed of a tetrapeptide sequence, GlyPhe-Leu-Gly, which is cleaved by enzymes in the lysosomes allowing release of the oligonucleotide or model compound in the lysosome. This will allow us to determine whether the model compound can escape when released in the lysosome. Internalization and subcellular trafficking are monitored by confocal fluorescent microscopy. The oligonucleotide or other target compound is labeled with a fluorescent tag such as fluorescein to allow detection. Confocal microscopy has the advantage over conventional microscopy in that light from outside the focal plane is excluded from the detector. This allows one to scan the cell looking at thin slices at different depths (in the z-direction). With a bright sample, slices as thin as 1 μm are easily obtained. Fluorescein Oligonucleotide NH HPMA z y x C CH3