Loading and triggered disassembly of vault protein nanocapsules. Lisa Ellen Goldsmith

ISBN: 9781109057928

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NOOKstudy eTextbook

227 pages


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Loading and triggered disassembly of vault protein nanocapsules.  by  Lisa Ellen Goldsmith

Loading and triggered disassembly of vault protein nanocapsules. by Lisa Ellen Goldsmith
| NOOKstudy eTextbook | PDF, EPUB, FB2, DjVu, audiobook, mp3, RTF | 227 pages | ISBN: 9781109057928 | 9.23 Mb

Vaults are hollow, ribonucleoprotein capsules (72.5 x 41 nm) comprised primarily of 96 self-assembled copies of one 97 kDa protein. The biological function for these nanocapsules, which are ubiquitous intracellular components of eukaryotes, isMoreVaults are hollow, ribonucleoprotein capsules (72.5 x 41 nm) comprised primarily of 96 self-assembled copies of one 97 kDa protein. The biological function for these nanocapsules, which are ubiquitous intracellular components of eukaryotes, is unknown- yet they may prove useful for material entrapment and drug delivery due to their large accessible lumen and ability to be taken up by mammalian cell lines.

Studies presented here investigate two key areas in the development of vaults as drug delivery vehicles: (1) active loading of the vault capsule interiors, and (2) solution conditions for triggered material release.-Initial studies of solution conditions that may trigger vault conformational change revealed a ∼60% increase in intrinsic fluorescence intensity and more than a 2-fold increase in fluorescence quenching by acrylamide at pH 3.4, compared to initial pH 6.5. TEM offered visual conformation of rapid vault disassembly at its waist into halves upon exposure to pH below 4.0, as suggested by QCM studies of immobilized vaults.

Such low pH conditions are physiologically relevant in the endosomes and lysosomes of cells, and may allow for controlled release of encapsulated contents.-If vaults are to serve as protective compartments for the transport of drugs, it is necessary to target material to the vault nanocapsule interior in order to optimize drug loading. The mINT (minimal interaction domain) protein is known to enter and associate with the vault interior via transient vault openings.

Using attached gold nanoparticles, studies presented here established the utility of mINT as a protein shuttle with which to load vaults with desired species. Loading of vaults with 6-His-tagged protein or peptide therapeutics (using 6-His-GFP as a model fluorescent protein) as well as with a functional small-molecule drug, doxorubicin, were achieved.

Encapsulation of gold, GFP, and doxorubicin was verified by parallel SDS-PAGE and western blot, fluorescence, and TEM. Effective delivery of encapsulated doxorubicin to HeLa (human cervical cancer) cells, at the current level of internal drug loading via covalent attachment to the mINT shuttle, could not be verified. Therefore, alternate strategies of increasing loading were explored, including attachment of drug-loaded dendrimers (highly branched polymers) to the mINT protein.



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