(Nanowerk Spotlight) Scientists involved in cancer research are showing a lot of interest in carbon nanotubes (CNTs) to be used in basically three cancer-fighting areas. CNTs are being developed as targeted delivery vehicles for anticancer drugs right into cancer cells - think of really, really tiny injection needles. They are also used as the therapeutic agent itself; there is research that shows that CNTs can act as nanoscale bombs that literally blow apart a cancer cell. A third area of application is using CNTs as imaging agents. Particularly single-walled CNTs (SWCNTs) are under active development for various biomedical applications. One of the issues in using CNTs for therapeutic applications is the question of how to get them to the desired place within the organism, say a tumor cell. Another significant problem in applying CNTs for biological applications is that the nanotubes do not stay suspended as discrete nanotubes in aqueous solutions. Coupling the CNT with biomolecules, such as proteins, is a good method for targeting specific sites but has the disadvantage of either reducing protein activity or CNT absorption or both. A novel method demonstrates that it is possible to achieve complete retention of enzymatic activity of adsorbed proteins as well as retention of a substantial fraction of the near-infrared (NIR) absorption of SWCNTs.
"The problem we are seeking to solve is how to couple a protein to the SWCNTs and keep the SWCNTs completely suspended and the protein biologically active" Dr. Roger G Harrison tells Nanowerk. "We know we can't covalently couple the protein to the SWCNTs because previous studies have shown that this eliminates the strong optical absorption of the SNWTs, which is a property that must be retained in our application for cancer treatment. We therefore developed a sodium cholate suspension–dialysis method that gives us a stable SWCNT–protein suspension with complete retention of enzymatic activity of adsorbed horseradish peroxidase (HRP) and also retention of a substantial fraction of the NIR absorption at 980 nm of the SWCNTs."
Comparison of SWCNT suspensions. Left: no HRP protein was added and the suspension of SWCNTs in sodium cholate was dialyzed for two days using a 10 kDa membrane. Right: SWCNTs suspended in sodium cholate with HRP added were dialyzed, first with a 10 kDa membrane and then with a 100 kDa membrane. The SWCNT–protein suspension was held at 4°C for ten days. (Reprinted with permission from IOP Publishing)
Harrison says that they wanted to design a SWCNT/protein complex that binds to tumors, so that the tumor can be selectively destroyed after exposure to near-infrared (NIR) light. "We were motivated to do this research by a desire to target SWCNTs for the treatment of cancer" he says. He points out that SWCNTs have very strong optical absorbance at certain wavelengths in the NIR region. However, biological systems have very low levels of absorption of NIR light.
Previous research already attempted to adsorb different proteins on single-walled carbon nanotubes. In one ("Structure and Function of Enzymes Adsorbed onto Single-Walled Carbon Nanotubes"), a solvent displacement method was used to adsorb two proteins. For one protein (soybean peroxidase), only 28% of the native enzyme activity was retained. For the other protein (alpha-chymotrypsin), no greater than 1% of the enzyme activity was retained. In another ("Selectivity of water-soluble proteins in single-walled carbon nanotube dispersions"), nanotubes and protein were sonicated in aqueous solution, which resulted in temperatures up to 60-70°C. The nanotubes could not be dispersed for two of the proteins (papain and pepsin). For the other two proteins (lysozyme and albumin), the nanotubes could be dispersed, but there was a significant change in the structure of both proteins after adsorption, as measured by circular dichroism.
"For our method we have first completely suspended the nanotubes in an aqueous solution of sodium cholate, a bile salt which acts as a surfactant" Harrison explains. "Then, we added the enzyme to be adsorbed, in this case HRP, to the suspended nanotubes and remove the sodium cholate by a method called dialysis, in which the sodium cholate leaves the solution through a membrane but the enzyme is retained and is adsorbed on the nanotubes. Sodium cholate is generally compatible with proteins, and in this case it did not lead to denaturation and a change in the structure of the enzyme."
With this method Harrison and his colleagues have demonstrated that the sodium cholate suspension-dialysis method for adsorption of the enzyme horseradish peroxidase on single-walled carbon nanotubes gives complete retention of the biological activity of the enzyme after adsorption. This indicates that adsorption of this enzyme on the carbon nanotubes has not perturbed the biological function of this enzyme.
The researchers also found that the loading of protein on the SWCNTs is high and the overall yield of preparing the SWCNT–protein suspension is also high. This process, therefore, is promising for the preparation of SWCNT–protein suspensions for biological applications where maintaining protein activity and SWCNT absorption are important.
Besides the applications for the treatment of cancer, there are other possible applications of this work: Imaging of cancer tumors, using carbon nanotubes or other types of nanoparticles useful for imaging; Any other type of medical treatment or imaging in which a nanoparticle needs to be complexed with a protein and the biological function of the protein must be retained; Biosensors in which an enzyme or other protein is complexed with SWCNTs (retaining the enzyme’s activity or the protein’s biological activity is important for the biosensor to function.)
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