Multiple Particle Tracking MPT

After cellular uptake by endocytosis, the gene carrier must traverse the expansive and molecularly crowded cytoplasm to reach the nucleus. Unfortunately, the biophysical and biological mechanisms underlying the intracellular transport of gene carriers remain largely unknown, which limits our ability to make rational modifications to gene carriers for improved gene delivery. Nuclear translocation is a critical bottleneck in gene delivery [201 -203]; however, it is unclear whether this limitation is owed to difficulty in gene carrier transport to the nucleus through the crowded cytoplasm, to the transport through the nuclear membrane, or to something else completely.

Quantitative investigations of the intracellular transport of synthetic gene carriers are currently undocumented. Confocal microscopy and electron microscopy (EM) have been used often to qualitatively study intracellular trafficking of nonviral systems [85,116,215-219], allowing the locations of complexes to be determined, over time. FRAP has recently been used to quantify overall rates of DNA molecules in the cytoplasm [196]. With these ensemble transport techniques, however, information such as the rates of individual particle movements, the mode of transport (such as random versus directed or active), and the trajectory and directionality of the transport remains elusive.

Using MPT [170], our group recently showed that the intracellular transport of PEI/DNA nanocomplexes involves active (i.e., nonrandom) transport to the perinuclear space of Cos-7 cells within 30 minutes [118]. The implication is that reaching the nucleus is not a rate-limiting step in PEI-mediated gene delivery, at least in Cos-7 cells. Comparison with other cell types and DNA vectors is currently under way to determine whether intracytoplasmic transport to the nucleus is a critical barrier to efficient gene delivery. Actively transported complexes moved several orders of magnitude faster [as measured by mean square displacement (MSD)] than complexes undergoing random thermal motion. For example, many actively transported complexes can travel 10 mm in less than one minute, whereas the average diffusive PEI/DNA nanocomplex (Dove = 0.0008 mm2/s), would take 8.7h to travel the same 10 mm.

Actively transported PEI/DNA nanocomplexes exhibited an average velocity of 0.2 mm/sec [118], a value on the same order of magnitude as motorprotein driven motion. Transport was revealed to be microtubule dependent, because both active transport and perinuclear accumulation were abolished upon microtubule depolymerization. Experiments utilizing MPT to quantify the other intracellular barriers to gene delivery are under way.

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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