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Posted: Oct 02, 2008
Natural protein vaults for nanotechnology cargo applications
(Nanowerk Spotlight) Spheres can be found at all scales in both the inanimate and living world for the basic physical property of encapsulation. The protein shell of a virus (called a capsid), for example, encloses space by using the geometry of the icosahedron, thus exploiting the economy of this form in terms of both surface area-to-volume ratio and genetic efficiency of subunit-based symmetric assembly. Many viruses' capsids use icosahedral symmetry to form particles ranging from 20 to 200 nanometers in size. This makes viruses an attractive research subject for exploring their use as nano-containers for drug delivery applications.
Researchers have also begun to copy Nature's icosahedral-symmetry design principles for artificial molecular containers, which could solve the problem of designing and synthesizing stable molecular containers having very large interior (see: Artificial virus shells as practical nano containers).
There has been a fair amount of work by nanotechnology researchers on putting non-biological molecules or clusters into viruses or virus-like artificial nanocontainers. Although viruses are a type of protein cage, they are not a natural part of the cell. There is a class of biological protein nanocapsules though – called 'vaults' – that is part of cells – although their cellular functions and gating mechanism are not yet understood. Vault nanoparticles are already present in human cells in high numbers (approx. 10,000 per cell) and their hollow barrel-like structure with a large internal volume seems well suited for encapsulation purposes.
Transmission electron microscope images of vaults. Scale bar is 100nm (Image: Leonard Rome website)
"The biocompatibility of vaults and the ability to encapsulate and protect fluorescent materials inside provides a wide range of possibilities for vaults as inert markers for biological imaging" Dr. Sarah H. Tolbert tells Nanowerk. "Moreover, understanding how to encapsulate macromolecules inside vaults and developing strategies to seal them is an important first step toward the use of vault protein cages as vessels for drug delivery."
Tolbert, a Professor of Chemistry in the Department of Chemistry and Biochemistry at UCLA in Los Angeles, explains that scientists have been working for some time now to integrate biological systems with synthetic materials where the properties can be controlled using established chemical means. In this way, there is the potential for greatly increased functionality in bio-compatible systems.
"Our work is part of this larger goal" says Tolbert. "We took biological protein vaults and filled the interior space of the cage with a highly fluorescent semiconducting polymer. Vaults are found in high copy number in many higher eukaryotes (including humans) and our goal was to see if we could use them as biologically compatible nanocapsules. Because the semiconducting polymers are so highly fluorescent, we could use the polymer fluorescence to prove that the polymer was really encapsulated inside the protein cage."
The final materials could find applications in fluorescence labeling of cells. The more fundamental result, however, as described in a recent paper in Nano Letters ("Encapsulation of Semiconducting Polymers in Vault Protein Cages") is that foreign molecules can easily be put into these novel protein cages. That could eventually lead to the use of vault cages for nanomedicine applications like drug delivery.
Tolbert mentions that she personally got started with this work because her research group has been exploring ways to tune the optical properties of semiconducting polymers by putting them in confined nanoscale spaces for many years now. "The extension to a biological system seemed like an exciting new place to go" she says.
The other principal investigators in the work described in the Nano Letters paper are professors Harold Monbouquette and Leonard Rome, who have been working with vaults for a longer period of time. Rome was the original one to discover vault cages some 20 years ago. On his website is lots of information about these vaults.
The researchers describe protein vaults as composed of four components: "Vaults contain 96 copies of the 100-kDa major vault proteins (MVP), the 193-kDa vault poly(ADP ribose) polymerase (VPARP), the 290-kDa telomerase-associated protein 1 (TEP1), and untranslated vault RNA. The 96 copies of MVP account for ∼74% of the protein mass, and recombinantly synthesized MVP can self-assemble into a vault like structure without the remaining components.16 This hollow cylindrical capped barrel structure is measured to be 41 nm in diameter and 72.5 nm in length. Each vault contains two identical cup like halves made of 48 copies of MVP."
In their experiments, polymer/vault complexes were produced simply by incubating the polymer with the vaults at room temperature in an acid buffer solution. The team estimates that approximately 8 µg of polymer are captured by each 100 µg of vaults.
"Although we do not know the exact molecular interaction of the polymer with the vault complex, our studies clearly demonstrate that polymer can be reliably encapsulated at low polymer/vault ratios" says Tolbert. "Our results confirm that vaults are dynamic structures that allow facile encapsulation of macromolecules from their environment in the time scale of seconds to minutes."
These findings indicate that a polymeric polyanionic drug could also be encapsulated inside the vaults. By depolymerizing the particle using a pH change or external irradiation, the drug could then be released from the vault. According to Tolbert and her collaborators, one could imagine similar applications for gene therapy where large DNAs or RNAs carrying genetic information would constitute the vault cargo.
While this has been a proof-of-principle type of work, the real challenge is to go from the concept of drug delivery, to the real use of these protein cages. Tolbert says that the team is also working to use these cages to sequester other species that could be toxic to the cell. "That goal requires us to get things into the vault cages, but not to get them out again."