| Popis: |
Viruses tend to be given a negative connotation due to their nature and the diseases they are known to carry. If you look past their malicious behavior, it is easy to see that nature has provided us a means to solve one of medicine’s most challenging aspects – effective, localized drug delivery. Therapeutics have made a great deal of headway since their first introduction, but sadly we still fall short when it comes to the two aforementioned issues. Virus-like particles such as the Qβ capsid allow us to tackle this issue head on by utilizing the non-infectious versions of a parent virus as a vessel we can load cancer drugs into and further functionalize to suit our intended goals. We seek to use the Qβ capsid, which upon self-assembly in its E.coli host system, encapsulates random tangles of mRNA. This allows us to use intercalating therapeutics such as Doxorubicin, which can be bound reversibly to the RNA inside through simple diffusion of the small molecule through any of the 32 pores on the capsid’s surface. In our lab, it has been demonstrated that the efficacy of Dox loading and that the capsid does not leak Dox over a 24-hour period. Further modification can be made to the drug carrier using the disulfide lined pores of Qβ – the addition of gold salts (HAuCl4) and sodium borohydride as a reducing agent allows for the templated growth of gold nanoparticles (AuNPs) on the capsid surface (Figure A). Molecular modeling performed by the Nielsen group (Figures B-D) indicates that the nanoparticle sits within the pore of the viral capsid such the surrounding proteins fold around the growing sphere to provide stability and confine the particle size. The hexameric pores of Qβ are predicted to cover 23% of the AuNP surface whilst the pentameric more only cover about 9% indicating that these particles may not be as tightly held to the VLP and may be more unstable than their hexameric counterparts. The addition of the AuNPs to the Qβ capsid was intended to give a mechanism of release for our drug delivery system, depicted in Figure E, which utilizes intercalated Doxorubicin (Dox) and improve the localization of this release. It is postulated that a single pulse from a laser centered at 532 nm – a value close to the absorbance λmax of our AuNPs – generates localized heating of the AuNPs causing small changes in the protein structure allowing the Dox to be released. This has been corroborated with through the use of circular dichroism to monitor changes in the capsid protein’s secondary structure as can be seen in Figure F by the decrease in the peak depth at 220 nm which indicates distortion of the β-sheets. It is also clear through cell studies with RAW 264.7 cells that laser irradiation releases the Doxorubicin from the capsid and additionally, when the laser beam is confined in size to about 1.3 mm, cells that do not receive stimulation do not release the drug. This can be visualized in Figure G where there is a clear line of demarcation where Dox release can be seen from the cells within the laser path. We even went one step further to demonstrate the selectivity of the system for green laser light. The experiment was repeated using a longer wavelength of light, 1064 nm, which is outside of the absorption of the nanoparticles and it yielded no Dox release under this wavelength. |
