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Posted: Sep 10, 2012

Nanoparticle approach to simultaneously deliver proteins and DNA into plant cells

(Nanowerk Spotlight) Nanotechnology is gaining significant interest in plant sciences with research focusing on investigating plant genomics and gene function as well as improving crop species. The impact on agriculture could be dramatic.
Most of the work done with nanoparticles in plant sciences relies on a passive uptake of nanoparticles by plant cells – a process that cannot be controlled. Plant cells differ from animal cells in several aspects, a major one being that they possess a wall surrounding them to provide, among other things, mechanical and structural support. Researchers are commonly using surface-functionalized silica nanoparticles as nonviral nanocarriers for experimental drug and DNA delivery into animal cells but their use with plants so far was limited due to the barriers posed by the cell wall.
In a previous Nanowerk Spotlight ("Biolistic guns to shoot nanoparticle bullets into plant cells") in 2007, we reported on the use of mesoporous silica nanoparticles (MSNs) as a transmembrane delivery system. In this technique, the nanoparticles' mesopores are loaded with DNA and chemicals and then – in what is called the biolistic approach, biolistic being short for biological ballistics – the MSN with their payload is shot under pressure into the plant cells. This technique is much easier and faster to perform than the tedious task of micro-injection.
Once inside the cell, chemical uncapping of the nanoparticles releases their payload under controlled release conditions.
Following up on their 2007 work, a team from Iowa State University, led by Kan Wang, professor in the Department of Agronomy and director of the Center for Plant Transformation and and Brian G. Trewyn, adjunct professor in the Department of Chemistry, has made this process more efficient, and introduced a much more challenging biomolecule to plant cells – proteins.
Reporting their findings in a recent edition of Advanced Functional Materials ("Gold Functionalized Mesoporous Silica Nanoparticle Mediated Protein and DNA Codelivery to Plant Cells Via the Biolistic Method"), first-authored by Susana Martin-Ortigosa, a postdoctoral research associate in Wang's team, and Justin S. Valenstein, a graduate student at Trewyn's lab, the researchers show for the first time how gold functionalized mesoporous silica nanoparticles can be functionalized to be more efficient to overcome the plant cell wall as they are propelled using a gene gun.
Plant intracellular co-delivery of DNA and proteins loaded in gold plated mesoporous silica nanoparticles
Plant intracellular co-delivery of DNA and proteins loaded in gold plated mesoporous silica nanoparticles. Gold plated mesoporous silica nanoparticles were loaded with eGFP protein and coated with plasmid DNA. After bombardment of onion epidermis tissues with these nanoparticles, mCherry plasmid DNA expression and eGFP protein delivery could be observed simultaneously in the same cells, confirming intracellular delivery of both biomolecules. (Image: Dr. Susana Martin-Ortigosa, Iowa State University)
"This nanoparticle modification with the only purpose of making better nanoprojectiles allows the convergence between bionanotechnology and plant sciences," says Wang. She points out that these findings are important for two reasons:
"The first one is that for the first time we show a MSN functionalization, a gold plating technique, which increases the overall density of the nanoparticles without altering its porous nature. Since we deliver the nanoparticles through the biolistic method, using a gene-gun that propels the nanoparticles to the plant material, these new denser MSN materials are introduced efficiently to plant cells, overcoming the plant cell wall."
The second reason has to do with protein delivery. While DNA delivery into cells has become a routine procedure, delivering proteins to plant cells has proved more challenging. So far, there haven't been any nanoparticle-based methods for delivering proteins into plant cells.
"Delivery of proteins or codelivery of proteins and DNA to plant cells has great biological significance," Wang explains to Nanowerk. "In addition to the potential of enhancing genetic transformation and gene targeting in plants, researchers can assess the loss or gain of function of different post-translationally modified forms of a protein, and protein interactions with other biomolecules. Furthermore, direct delivery and release of proteins in plant cells could facilitate the understanding of cellular machinery or signal pathways more effectively."
The use of the gold-plated MSN now allows plant scientist to deliver not only nucleic acids but also proteins as large as bovine serum albumin into plant cells.
"This protein delivery or protein and DNA codelivery has a tremendous importance for plant scientist since it broadens the molecular tools that can be used to answer fundamental questions in plant biology and also for more practical biotechnological applications," adds Martin-Ortigosa.
The most immediate application of this work could be the delivery of enzymes designed for genome engineering. Adding DNA sequences to specific target locations in the genome, deletion of unwanted genes, or precise modification of genome sites are all techniques that scientists use to improve or modify agricultural plants.
These modifications are nowadays achieved through the delivery of gene encoding for enzymes like recombinases, or crosses between the plant to be modified and a plant harboring the recombinase. After the desired genome modifications have been achieved, at least one more generation of plants has to be analyzed in order to segregate this gene encoding the recombinase.
The direct delivery of the recombinase protein through MSN allows direct genome modification without DNA (carrying the recombinase gene) integration and further generation segregation and analysis – a simplified process that saves time and efforts.
One of the challenges that remain is the loading of bigger proteins into the pores. Right now, the pore size is 10 nm so any protein bigger than that is not suitable for loading and subsequent intracellular delivery. The synthesis of new materials to harbor bigger proteins is going to be one of the team's future efforts.
"While we hope that the direct application of our technology can help solve specific problems, we also would like to encourage plant scientist to start looking at nanotechnology as a way to obtain the tools necessary for conducting research," says Wang. "In the coming years, we expect to see more applications of nanoparticles designed only for plant sciences."
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