Nanoscale functionalities for biopharmaceutical drug delivery
published: Jan. 18, 2008, recorded: October 2007, views: 915
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One of our recent research challenges has been exploitation of the active principle of protein aggregation, involving metal coordination of specifically designed protein analogues. Specifically designed analogues of tumour necrosis factor alpha (TNF-alpha) rich in histidines served as model proteins. LK801 is a TNF-alpha analogue with double histidine mutation (Glu107HisGly108HisTNF-alpha) in the tip region of the bell-shaped molecule. Due to the symmetrical trimeric structure, histidine residues in the tip region form an almost planar cluster of six well accessible histidines resulting in strong binding to Immobilized Metal Affinity Chromatography (IMAC), which was used for efficient single step purification. IMAC was also used for preparation of His10-TNF, a TNF-alpha analogue with His10 tag and amino acid sequence responsible for enterokinase cleavage added to the native N-terminus and H7dN6TNF analogue, which has a tag comprised of seven histidines on N-terminus. Two types of inorganic nanoparticles containing metal ions were prepared, zinc phosphate nanoparticles by precipitation, and zinc modified silica by adsorption of zinc ions on commercially available silica nanoparticles. For the proof of concept, in preliminary experiments bovine serum albumin (BSA) was used for binding. BSA contains naturally surface exposed histidines and was also expected to coordinatively bind to nanoparticles with metal ions. When such particles were exposed to low pH buffers or buffers containing imidazole, release of BSA was confirmed by SDS-PAGE analysis, thus proving the reversibility of metal-specific binding. In the next steps, histidine rich TNF analogues were used for binding to Zn-phosphate nanoparticles and release studies were performed under different conditions. We also measured biological activity of TNF-alpha analogues prior to binding and later after the release from inorganic nanoparticles. The controlled formation of TNF-alpha analogues nanoparticles was tried by protein self-assembly using metal ions (Zn2+). First experiments were performed with only the addition of zinc ions and later we performed experiments using Zn2+ and different biocompatible chelates (phytic acid and 1,4,8,11-Tetraazacyclotetradecane-1,4,8,11-tetraacetic acid). Upon administration of above mentioned protein aggregates to the testing animals, an increased immune response is anticipated. In the case of TNF-alpha analogues, an enhanced formation of antibodies against TNF-alpha would be advantageous serving as a basis for developing new drugs for chronic diseases associated with pathogenically elevated TNF-alpha levels (rheumatoid arthritis, Crohn disease, psoriasis, etc.). His10-TNF analogue appears especially interesting, since it exhibits very low in vitro cytotoxic activity. Upon formation of nanostructures, a significantly diminished number of accessible receptor binding sites and consequently even more reduced cytotoxicity is expected, leading to safe formation of anti-TNF-alpha antibodies.
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