ABSTRACT
Targeted drug delivery to specific subcellular compartments of brain cells is challenging despite their importance in the treatment of several brain-related diseases. Herein, we report on shape-directed intracellular compartmentalization of nanoparticles in brain cells and their ability to deliver therapeutic molecules to specific organelles. Iron oxide (Fe3O4) nanoparticles with different morphologies (spheres, spindles, biconcaves, and nanotubes) were synthesized and coated with a fluorescent carbon layer derived from glucose (Fe3O4@C). In vivo studies showed that the Fe3O4@C nanoparticles with biconcave geometry localized predominantly in the nuclei of the brain cells, whereas those with nanotube geometry were contained mostly in the cytoplasm. Remarkably, a small-molecule activator of histone acetyltransferases delivered into the nuclei of the brain cells using nanoparticles with biconcave geometry showed enhancement in enzymatic activity by a factor of three and resulted in specific gene expression (transcription) compared with that of the molecule delivered to the cytoplasm using nanotube geometry.
Subject(s)
Benzamides/administration & dosage , Brain/metabolism , Drug Delivery Systems , Metal Nanoparticles/administration & dosage , p300-CBP Transcription Factors/metabolism , Animals , Benzamides/chemistry , Cell Line , Cell Line, Tumor , Female , Ferric Compounds/chemistry , Glucose/chemistry , Metal Nanoparticles/chemistry , Mice, Inbred BALB C , Nanotubes/chemistryABSTRACT
In this report, we demonstrate glucose-derived carbon nanospheres to be an emerging class of intracellular carriers. The surfaces of these spheres are highly functionalized and do not need any further modification. Besides, the intrinsic fluorescence property of carbon nanospheres helps in tracking their cellular localization without any additional fluorescent tags. The spheres are found to target the nucleus of the mammalian cells, causing no toxicity. Interestingly, the in vivo experiments show that these nanospheres have an important ability to cross the blood-brain barrier and localize in the brain besides getting localized in the liver and the spleen. There is also evidence to show that they are continuously being removed from these tissues over time. Furthermore, these nanospheres were used as a carrier for the membrane-impermeable molecule CTPB (N-(4-chloro-3-trifluoromethylphenyl)-2-ethoxybenzamide), the only known small-molecule activator of histone acetyltransferase (HAT) p300. Biochemical analyses such as Western blotting, immunohistochemistry, and gene expression analysis show the induction of the hyperacetylation of histone acetyltransferase (HAT) p300 (autoacetylation) as well as histones both in vitro and in vivo and the activation of HAT-dependent transcription upon CTPB delivery. These results establish an alternative path for the activation of gene expression mediated by the induction of HAT activity instead of histone deacetylase (HDAC) inhibition.
Subject(s)
Cell Membrane/metabolism , Cell Nucleus/metabolism , Fluorescent Dyes/pharmacology , Gene Expression Regulation , Genetic Techniques , Genetic Therapy/methods , Genetic Vectors/genetics , Nanotubes, Carbon/chemistry , HeLa Cells , Histones/metabolism , Humans , Liver/metabolism , Microscopy, Fluorescence/methods , Nanospheres/chemistry , Spleen/metabolismABSTRACT
We have analyzed single nucleotide polymorphisms (SNPs) in the XPC, XPA, and XPG genes of the nucleotide excision repair (NER) pathway in the Indian population. In the XPC gene we observed nine polymorphisms in the coding region, four polymorphisms in the intronic region, and two polymorphisms in the 5' untranslated region (UTR). In the XPA gene we observed one frequent SNP (allele frequency 0.48) within the 5' UTR at the 1665 position in a large proportion of the sample. In addition, we observed three novel heterozygous polymorphisms (a C to A transversion at position 1523 and a G to A transition at positions 1418 and 1458, with an allele frequency of 0.004) within the promoter region. In silico PCR analysis demonstrated that all three novel polymorphisms lie within a putative CpG island and that the variation at position 1418 falls within the potential GATA1/2/3 transcription factor(s) binding site and also within the negative control element. We performed a gel retardation assay with HeLa cell nuclear extract with an oligonucleotide encompassing this region. One of the alleles found at position 1458 of the XPA gene showed a significant change in protein-DNA interaction. In the XPG gene we found five polymorphisms in the coding region and one each in the 5' UTR of exon 1 and in intron 13.
