David Dickinson and Dr. William Pitt, Chemical Engineering
Cancer is one of the leading causes of death worldwide. Although surgeries and chemotherapy have had limited success, these techniques are largely unable to solve the problem and may cause significant damage to a patient’s entire body. Some patients are killed by the chemotherapy. Since many cancers have been found to be caused by the lack of a protein due to mutated DNA, a plasmid DNA carrying a replacement for this gene may be able to solve the problem. P52 is one of the more famous missing proteins, but others have and will continue to be discovered. If DNA could be effectively transported to and then transfected into only tumor cells, then these people could be relieved of their disease and have the freedom to live a healthy life.
Previously DNA transportation systems have been made using lipid-coated gas bubbles filled with perfluorohexane. These bubble carriers combined with DNA and ultrasound have proven to be much more effective than plasmid DNA alone in delivering DNA to the site of focused ultrasound, but it can only be delivered to endothelial cells of the circulatory system and perhaps one cell layer beyond. Penetration beyond the capillaries is limited due to the large size of the gas bubbles. The gas bubble has an average micelle diameter between 2 and 3 microns. Dreher et al. have found that an ideal size for such carriers to penetrate through tumor capillaries is much smaller. Since tumor cells have leaky capillaries (there is a gap between endothelial cells where there should be a tight junction in other cells), an ideal size will be small enough to accumulate in the tumor but large enough to not be quickly cleared by the lymphatic system. Tumor capillary gaps are generally between 50 nm and 2 µm. Though these gas bubbles are able to transfect a little bit, a smaller sized carrier could provide for a much better transport system and provide a greater accumulation of carrier into the tumor.
In order to improve the technology currently available, I have created nanoemulsions with an average size about an order of magnitude smaller (about 200 nm). Tumors with leaky capillaries should accumulate a larger amount of carrier with this smaller size1. A smaller transport system is the key to safely reaching and treating tumors. By creating a positively charged micelle, there is a coulombic attraction to hold the DNA to the carrier. The method of release will be as follows: Initially the micelles will be filled with perfluorohexane and DNA will be attracted to the outside. When ultrasound is applied to the carrier, the perfluorohexane will become a gas and the bubbles will begin to vibrate. They will then be able to shear the cell walls a little through their oscillation and will push the plasmid DNA into the cells. Thus one can see the importance of having the carrier next to tumor cells rather than just endothelial cells.
At this point, I have synthesized an amide quaternary amine to be used as the carrier. The amide quaternary amine was made utilizing perfluoro octanoyl chloride and N,N-dimethyl ethylene diamine. The compound becomes a quaternary amine at neutral pH because the terminal nitrogen has a pKa around 10. NMR analysis has proven that the compound synthesized is distinct from either of the two reagents used and indicates that the compound synthesized is indeed an amide. The perfluorocarbon portion of the molecule will make this surfactant capable of stabilizing an emulsion of the perfluorohexane which will be inside of the carrier.
James Lattin, one of Dr. Pitt’s graduate students, is doing graduate work with the stability of emulsions. He has been using this molecule to test how well it can form stable emulsions. Currently emulsions have been made but have not been tested on cells to determine in vitro uptake levels.
A concern which has arisen since beginning this project is the toxicity of cationic molecules in the body. Studies have shown that cationic lipids interrupt cell membranes and are likely to trigger apoptosis inside of cells. Although cationic molecules in literature are found to have relatively high transfection rates, they are also found to have high cell mortality rates. The number of cells that are killed by cationic emulsions will likely be similar to those of cationic lipids. This unfortunately makes the carrier which has been developed less useful than originally hoped for.
Currently further studies are being performed into developing a carrier with disulfide bonds. Disulfide bonds may be effective as a shell to get genes into the cell. Once inside the cell, lysosomes will try to digest the molecule. The acidic environment will then break the shell into many pieces which will cause a very high osmotic pressure within the lysosome. This high osmotic pressure will burst the lysosome allowing the genes to escape and stand a better chance at transfecting a larger number of cells without killing them.
Though this simple project has been unable to cure cancer, it has given us new insights into methods that can be used to help target cancer cells with gene therapy. Though initially cationic surfactants were appealing due to the coulombic attraction which would exist with DNA, the toxicity seems to make it an improbable choice for in vivo usage. The disulfide carrier is currently promising, but only further studies will show if the cell mortality will be minimized while the cancer destruction will be maximized.
References
Dreher, Matthew R., Wenge Liu, Charles R. Michelich, Mark W. Dewhirst, Fan Yuan, Ashutosh Chilkoti. 2006. Tumor vascular permeability, accumulation, and penetration of macromolecular drug carriers. Journal of the National Cancer Institute 98:335-344