Virus-like particles of Macrobrachium rosenbergii de man nodavirus for discovery of anti-virus peptides and delivery of a cancer drug
Macrobrachium rosenbergii nodavirus (MrNv) is the causative agent of white tail disease (WTD) in the giant freshwater prawn. The recombinant capsid protein can be expressed in Escherichia coli, and it self-assembles into virus-like particles (MrNVLP). Currently, there is no treatment for WTD and the...
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| Format: | Thesis |
| Language: | English |
| Published: |
2019
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/90664/1/FBSB%202020%209%20-%20IR.pdf |
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| Summary: | Macrobrachium rosenbergii nodavirus (MrNv) is the causative agent of white tail disease (WTD) in the giant freshwater prawn. The recombinant capsid protein can be expressed in Escherichia coli, and it self-assembles into virus-like particles (MrNVLP). Currently, there is no treatment for WTD and there is no thermally-responsive VLP for drug delivery. The main aims of this study were to characterise the MrNVLP, and applied the VLP for the screening of antivirus agents, as well as delivery and thermally-controlled release of a cancer drug. MrNVLP was compared with the native MrNv that was purified from naturally virus-infected post larvae of M. rosenbergii. Western blotting demonstrated that both virus particles were detectable using the rabbit anti-MrNv capsid protein serum. Transmission electron microscopy showed that MrNVLP was spherical in shape similar to that of native MrNv. Dynamic light scattering (DLS) showed the size of MrNVLP was stable for approximately 4 weeks, and MrNVLP was non-cytotoxic as determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, indicating the potential of MrNVLP for in vitro applications. The amount of RNA in MrNVLP was reduced using the RNase treatment to study the changes of the particles. Interestingly, the size of MrNVLP was inversely related with the amount of RNA packaged. Since MrNVLP was structurally similar with the capsid of native MrNv, MrNVLP was used as a target to screen for anti-MrNv agents in order to control WTD. Two dominant phages harbouring the amino acid sequences HTKQIPRHIYSA and VSRHQSWHPHDL were selected and they bound strongly to MrNVLP. Chemically synthesised peptides HTKQIPRHIYSA and VSRHQSWHPHDL inhibited the internalisation of MrNVLP in Spodoptera frugiperda (Sf9) cells. The peptides also inhibited infection of native MrNv as shown by higher cell viability. Real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) assay revealed that the highest MrNv inhibition was observed when both peptides were simultaneously applied. Owing to the nature of MrNVLP, which is non-infectious and non-cytotoxic, thus it has the potential to be developed as a nanocarrier in drug delivery. In this study, folic acid (FA) was covalently conjugated to lysine residues located on the surface of MrNVLP, while doxorubicin (Dox) was loaded inside the VLP using an infusion method. This nanoparticle, namely FA-MrNVLP-Dox, released Dox in a sustained manner, and the rate of drug release increased in response to a hyperthermia temperature at 43 °C. The FA-MrNVLP-Dox enhanced the delivery of Dox to HT29 cancer cells, which expressing higher level of folic acid receptor (FR) than CCD841CoN normal cells and HepG2 cancer cells. As a result, FA-MrNVLP-Dox increased the cytotoxicity of Dox on HT29 cells but demonstrated lower drug’s cytotoxicity on CCD841CoN and HepG2 cells. In conclusion, MrNVLP is molecularly and morphologically similar compared to the capsid of native MrNv. Two peptide inhibitors that blocked MrNv infection in vitro were identified. In addition, MrNVLP was also shown to function as a thermally-responsive nanocarrier for delivery of Dox to colorectal cancer cells. This study demonstrated the potentials of MrNVLP in identification of WTD inhibitors, and targeted delivery of cancer drugs. |
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