| Nanoparticles |
The heterogeneous populations of nanoparticles, within any particular sample The size, polydispersity and charge of nanoparticles depend on the measurement techniques and hydration state Assembly and disassembly of nanoparticles are dynamic processes that change during storage, blood circulation and tissue and cellular distribution Changing one of the nanoparticle characteristic (e.g. size) affects others Premature release of the linked fluorophore: The stability of the link used to label the nanoparticles in the blood and tissues and the effect of fluorophores on the stability and pharmacokinetics of nanoparticles are important considerations Impure fluorophore results in tracking two populations (free and conjugated dyes)
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| Cellular |
The cellular processes are dynamic and interchangeable and inhibition of specific endocytic pathways can inhibit other pathways Nanoparticle efficacy varies in different cell lines, cell cycles, growth media, and passage number
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| In vivo |
Pharmacokinetics depend on the animal model and disease status; different tumors and different sizes of the same tumor respond differently to therapy and may accumulate different amounts of the injected nanocarriers Physiological factors: age, sex, weight, etc.
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| Instrumentations |
The use of two fluorophores: unresolved emission spectra lead to misinterpretation Confocal microscopy:
Three-dimensional imaging is essential Apparent co-localization may be obtained from structures in close proximity without real co-localization in the same organelle Difficult to differentiate between cellular binding and uptake Background, detector saturation and spectral overlap should be avoided
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