Microbotics - nanoparticles hitching a ride on bacteria

Nanowerk Spotlight) Vaccination has resulted in the eradication of smallpox and control of measles, rubella, tetanus, diphtheria, and other infectious diseases in many areas of the world (at least where vaccines are available and affordable; providing vaccines to many parts of the developing world still is one of the basic medical needs that is far from being met). The basic idea of vaccination (the word comes from the Latin vacca - cow - because the first vaccine was derived from a virus affecting cows) is to inject weakened or killed forms of pathogens such as bacteria or viruses into the body in order for the immune system to develop antibodies against them; if the same types of microorganisms enter the body again, they will be destroyed by the antibodies. About 25 years ago, the basic idea of vaccination gave rise to bactofection - the technique of using bacteria as non-viral gene carriers into target cells. The DNA cargo is transported inside the bacteria and, once it arrives at the target location, the bacteria is broken up in order to release the therapeutic gene or protein. A novel technique takes advantage of the invasive properties of bacteria for delivery of nanoparticles into cells. Here, the gene or cargo is not carried inside the bacteria, but rather remains on the surface conjugated to nanoparticles. Consequently, this approach does not require bacterial disruption for delivery, or any genetic engineering of the bacteria for different cargo.

Although more than one gene can be delivered by means of bactofection, many more copies of a target cargo can be carried with one bacterium using our method. We also show that nucleic acid-based model drugs loaded on the nanoparticles can be released from the carriers and eventually find their way into the nucleus, with subsequent transcription and translation of their respective proteins, for both in vitro and in vivo conditions. Such bacteria, which we call microbots, can potentially be used to carry proteins, small molecules and even synthetic objects like sensors and therapeutic moieties into different types of cells.

Three steps were necessary to make our microbots says Bashir: First we treated the bacteria with a biotin-carrying antibody that acts against and will therefore attach to proteins on the bacterial surface called muraminidase. Next, we mixed the treated bacteria with nanoparticles, ranging from 40nm to 200 nm, coated with streptavidin, a protein that binds strongly to biotin. Finally, the nanoparticle-loaded bacteria were mixed with plasmid DNA carrying biotin, which binds to the free streptavidin sites on the surface of the nanoparticles.

Because the nanoparticles are linked to the bacteria by means of an antigen antibody interaction, the cargo and the bacteria can readily separate in the lower pH environment of the subcellular compartments. Rashid says that other factors, such as intracellular enzymatic processing or destabilization of antigen antibody binding or a reduction in the biotin streptavidin interactions can also be involved in the release mechanisms of the DNA, and all of these possibilities can potentially be used for endowing microbots with smart cargo release ability.

In their experiments, the researchers at Purdue found that microbots successfully delivered their cargos of nucleic acid-based model drugs, plasmid DNAs for firefly luciferase and SEAP enzymes into multiple organs of live mice, and the delivered genes also resulted in functional protein expression by three days post-treatment.

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