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The protein cage thermosome as versatile delivery platform

Nussbaumer, Martin G.. The protein cage thermosome as versatile delivery platform. 2015, PhD Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_11345

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Abstract

Protein cages represent a versatile platform for a variety of functions such as targeted delivery of drugs or imaging agents. These proteins protect their cargo from premature degradation and enhance uptake in desired tissue. The versatility results from the different modification sites of protein cages. Various cargoes can be packed in the interior of such protein cages. The exterior can be modified with cell-targeting and/or -penetrating moieties. Furthermore, protein engineering allows for the introduction of additional linking sites.
To this end, we use the thermosome (THS), a chaperonin from T. acidophilum. THS features two cavities that can accommodate guest molecules. Polyamidoamine (PAMAM) was covalently bound to the genetically introduced cystein residues in the interior. This cationic polymer is able to bind and stabilize small interfering RNA (siRNA) by electrostatic interactions. Oligonucleotides, such as siRNA, are normally rapidly degraded in serum. Our experiments showed the uptake of siRNA into the THS-PAMAM hybrid as well as the enhanced stability of siRNA against RNase. To achieve cell-uptake, THS was modified on its outer surface with different cell targeting and penetrating ligands. Beside the cell penetrating peptide TAT, also VEGF was able to increase the cellular uptake of THS into PC-3 and HUVEC cells, respectively. Interestingly, it turned out that unmodified THS is taken up by U87 cells, whereas other tested cell-lines did not show any uptake of THS. Silencing effect of siRNA delivered by THS-PAMAM to U87 cells and TAT modified THS-PAMAM to PC-3 cells was observed. To expand the concept of THS as a nano drug delivery system, we modified the THS’ cavity with a dye, which served as a drug model, through a reduction sensitive linker. In this study cell-internal triggered release of drugs from THS was examined.
THS showed also the capacity to be used in other contexts. By conjugating HRP into the cavity of THS and controlling the conformation of the THS’ pore with ATP analogues, we created an ATP-controllable enzyme nano-reactor, which could lead, in the long run, to enzyme replacement therapies. In a further study, we focused on the formation of gold-nanoparticles (AuNP) within the cavity of THS. Thereby, we benefited from the fact that PAMAM serves as scaffold for the formation of AuNP. Combining the ability for THS to transport cargo to the cell while containing AuNP cargo would make THS interesting in the field of photo thermal therapy.
In summary, we illustrate the versatility of THS as a nano drug delivery system. Thereby, the main objective was to show siRNA transfection by the protein-polymer conjugate THS-PAMAM and broaden its field of application by modifying it with cell targeting ligands. Furthermore, we show that THS can be modified in a way that THS can serve in other fields of nanomedicine, such as enzyme activity control, triggered drug release and photo thermal therapy.
Advisors:Meier, Wolfgang Peter
Committee Members:Weil, Tanja
Faculties and Departments:05 Faculty of Science > Departement Chemie > Chemie > Makromolekulare Chemie (Meier)
Item Type:Thesis
Thesis no:11345
Bibsysno:Link to catalogue
Number of Pages:121 S.
Language:English
Identification Number:
Last Modified:30 Jun 2016 10:58
Deposited On:10 Sep 2015 07:49

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