Design and analysis of peptide based nanoparticles (2008)

Viruses are naturally formed bionanoparticles (BNPs), ranging in size from 22-150 nm. Remarkably, small viruses are composed of one single protein chain folding into a capsid structure with icosahedral symmetry. The icosahedron is built up from 60 asymmetric units and is the largest closed shell in which every subunit is in an identical environment. It is characterized by 2-fold, 3-fold and 5-fold rotational symmetry axes. By superposition of different protein oligomerization domains onto the symmetry axes of the icosahedron, a nanoparticle with icosahedral symmetry can be designed. To test our concept, we have designed a peptide comprising a slightly modified form of a pentameric coiled-coil domain of cartilage oligomeric matrix protein (COMP) linked to a de novo designed trimeric coiled-coil domain as a single chain. These two different oligomeric domains were linked by two glycine residues to provide flexibility between them and were fixed in their relative orientation with an intramolecular disulfide bridge. Computer modeling predicted that such a design would result in an icosahedral peptide based nanoparticle with a diameter of 17 nm. We chemically synthesized the above designed peptide and performed refolding studies and biophysical characterization using analytical ultra centrifugation (AUC) and electron microscopy (EM), thereby showing the formation of icosahedral nanoparticles with a diameter of ~ 17 nm. Subsequently, we switched to recombinant expression of the designed peptide. Again, we performed refolding studies with the expressed peptide and biophysical characterization (AUC and EM), thereby showing the formation of icosahedral nanoparticles with a diameter of ~ 17 nm. In addition, we showed that during refolding, parameters such as ionic strength, pH of the refolding buffer and presence of glycerol influence the formation of icosahedral nanoparticles. Moreover, we observed icosahedral nanoparticles in conditions in which the formation of intramolecular disulfide bridges is not possible. This showed that even in the absence of intramolecular disulfide bridge the helices of the two different oligomeric domains like to be arranged close to each other, as this will favor the formation of icosahedral nanoparticles.\nIn parallel, we also modified the designed peptide to include charged residues at the\ninterface between the two oligomeric domains. The idea was to fix the relative orientation\nbetween the two oligomeric domains through ionic interactions. The subsequent\nexpression, refolding studies and biophysical characterization showed the formation of\nnanoparticles, but they were lacking icosahedral symmetry. This result showed that the\ndesign has to be optimized further to obtain icosahedral nanoparticles.\nMoreover, we wanted to study whether in our design principles oligomerization motifs\nother than coiled-coils can be used to form icosahedral nanoparticles. Accordingly, we\nused globular foldon domain as the trimerization domain and the COMP as the\npentamerization domain. The results showed the formation of nanoparticles, but they\nwere lacking icosahedral symmetry. In addition, in the above design we studied the effect\nof linker region by increasing the liker region to four and six residues, but it did not help\nin the formation of icosahedral nanoparticles. However, when the foldon domain\nextended with the trimeric coiled-coil domain as a single trimerization domain and with\nCOMP as the penatmerization domain, we observed icosahedral nanoparticles.\nViruses are known for their induction of strong antibody (B-cell) mediated immune\nresponse in the host even against the self-antigens. This property is conferred by the\nrepetitive arrangement of the antigens on their surface. Peptide based nanoparticles have\nsimilar properties to viruses, such as self-assembly and most importantly the repetitive\narrangement of subunits (peptide based nanoparticles are composed of 60 identical\nmonomeric chains). Therefore, we wanted to use our system as a ‘repetitive antigen\ndisplay carrier’ in vaccination, as an alternative to viral and viral based platforms such as\nvirus like particles (VLPs). To this end, and in order to understand the effect of our\nnanoparticle size on immune response, we built different constructs displaying the pilin\nepitope of Pseudomonas pathogen at their C-terminus. Computer modeling indicated that\nthese constructs would form icosahedral nanoparticles of three sizes: small (18 nm),\nmedium (23 nm) and large (29 nm). From the refolding studies, we observed mostly\naggregation and precipitation of the nanoparticles.\nThis effect presumably due to interparticle cross-linking, by the cysteine residues of\npilin epitope which are present at the periphery of nanoparticles, as we observed\naggregation of small size icosahedral nanoparticles upon changing from reducing to\noxidizing condition. Immunization results, because of the aggregation and precipitation\nbehavior of nanoparticles, showed poor immune response. However, the immunization\nresults showed that our nanoparticles present the attached epitope in their native form, as\nwe observed binding against native pilin protein. Moreover, immunization results from\nour laboratory using medium size nanoparticles displaying Salmonella epitope D2\nshowed promising results, as we got antibody titer values which were well comparable to\nthe values obtained with VLPs. This places our system alongside with VLPs, which are in\nclinical trials as a carrier in vaccination.