Magnetofection-- a novel tool for gene transfer
Summary:
Transfer of whole genes rather than protein products has become a useful and valuable technique for gene analysis as well as a promising tool for genetic therapy. Although many viral and non-viral systems for such transfection have been proposed finding an ideal system that can efficiently carry out a gene transfer has been difficult to come across. While viral systems have been found to be highly efficient in transfection, their oncogenic properties, immunogenecity and other such disadvantages limits their use. Non-viral techniques, on the other hand, while low in toxicity and immunogenecity, usually result in inefficient DNA uptake and expression.
In this paper, a series of experiments were conducted to investigate the use of magnetofection using iron oxide (Fe3O4) nanoparticles as a novel non-viral technique for gene transfer of LacZ, and enhanced green fluorescence protein (EGFP) into mice osteoblasts and He99 lung cancer cells respectively.
First, the effect of the nanoparticles on cell viability was tested using diphenyltetrazolium bromide assay and cytotoxicity was not found at any of the Fe3O4 concentrations tested.
Endocytosis of the gene mediated by the nanoparticles was then tested by adding liposome-enveloped LacZ/Fe3O4 to osteoblasts cultures. Half of the cultures were placed above 3599 gauss magnets for five minutes while the other half was not. Successful transfection and Lacz expression was detected after 10 days in both groups (showing that endocytosis has occurred) but the expression was visibly higher in the cells that were exposed to the magnetic field.
Transfection of He99 lung cancer cells with EGFP was then performed. The cells were transfected with one of the following preparations: homemade liposome-enveloped EGFP-DNA with iron oxide nanoparticles, homemade liposome-enveloped EGFP without the nanoparticles, lipofectamine 2000-enveloped EGFP-DNA (positive control), or EGFP-DNA gene alone. Half of the cells were exposed to magnetic field while the other half was not. Expression of EGFP was observed and analyzed. The group found that both commercial EGFP, and homemade liposome-enveloped EGFP/Fe3O4 were successful while the EGFP gene alone and the EGFP without the nanoparticles did not show any transfection. Though transfection occurred both with and without the presence of a magnetic field, like before, the rate was higher when exposed to magnetic field. This was taken to be evidence that due to gravity, just the presence of magnetic nanoparticles results in better transfection.
In this paper, a series of experiments were conducted to investigate the use of magnetofection using iron oxide (Fe3O4) nanoparticles as a novel non-viral technique for gene transfer of LacZ, and enhanced green fluorescence protein (EGFP) into mice osteoblasts and He99 lung cancer cells respectively.
First, the effect of the nanoparticles on cell viability was tested using diphenyltetrazolium bromide assay and cytotoxicity was not found at any of the Fe3O4 concentrations tested.
Endocytosis of the gene mediated by the nanoparticles was then tested by adding liposome-enveloped LacZ/Fe3O4 to osteoblasts cultures. Half of the cultures were placed above 3599 gauss magnets for five minutes while the other half was not. Successful transfection and Lacz expression was detected after 10 days in both groups (showing that endocytosis has occurred) but the expression was visibly higher in the cells that were exposed to the magnetic field.
Transfection of He99 lung cancer cells with EGFP was then performed. The cells were transfected with one of the following preparations: homemade liposome-enveloped EGFP-DNA with iron oxide nanoparticles, homemade liposome-enveloped EGFP without the nanoparticles, lipofectamine 2000-enveloped EGFP-DNA (positive control), or EGFP-DNA gene alone. Half of the cells were exposed to magnetic field while the other half was not. Expression of EGFP was observed and analyzed. The group found that both commercial EGFP, and homemade liposome-enveloped EGFP/Fe3O4 were successful while the EGFP gene alone and the EGFP without the nanoparticles did not show any transfection. Though transfection occurred both with and without the presence of a magnetic field, like before, the rate was higher when exposed to magnetic field. This was taken to be evidence that due to gravity, just the presence of magnetic nanoparticles results in better transfection.
Why this paper?
Magnetism and nanoparticles are topics that have traditionally fallen in the realm of physics and material science. This paper showcases magnetic nanoparticles under a new light and provides evidence for its effective use in molecular biology and tissue engineering. Nanoparticles are biodegradable, and the technique is very efficient making it ideal for use in tissue engineering, gene analysis, and gene therapy. I found the concept to be very interesting and the same time logical and fairly easy to understand. I expect that in coming years this innovative technique will be perfected and widely used in many fields of molecular biology and genetics.
3 comments:
Very interesting topic! I wonder if there is a possible limit to the size of genes/DNA that can be transformed using this method. In my opinion nanotechnology is the next stepping stone in the further of drug therapies since it would allow for proper target specificity. I read a paper once that discussed how nanoparticles of gold were coated with antibodies for particular cancer cells. In this way, the nano'drugs' were able to search out the tumor and attach to the outside surface. This allowed doctors to use radiation therapy to heat the tumor cells specifically without effecting the surrounding tissues.
I have heard the buzz about using magnetic nanoparticles as well and is the reason I opened this article, but it seemed that this particular use (gene transfer) wasn't quite what I was expecting with the use of magnets (most papers I've read/heard about use magnetic nanoparticles because of it's ability to be controlled with regards to location).
I thought overall the paper was well done, with numerous controls. I am glad the team decided to control for both homemade and commercial liposomes because they were only testing the Fe3O4 nanoparticles when formed with their homemade liposome.
As I was reading the paper I assumed the magnetic force was going to be used to force their way through the membrane (somewhat like particle guns), but they just used the magnets to localize the liposome-dna complex to the cells. If this was the case, and the problem is a diffusion-limited one (once the liposome-dna complex reaches the cell membrane it becomes endocytosed), I don't see why just letting non-magnetic liposome-dna complexes sit in solution a bit longer might not solve the problem. Looking at the graph, they go out to 600hours (roughly 20-30 days) and the magnetic liposome-dna complexes have significantly higher transfection rates, so I guess there is a part of the picture I am missing here.
I also thought the cell viability assay for this paper is not representative of the condition they were trying to test. They just had magnetic Fe3O4 particles sitting in solution with the cells so they probably never got endocytosed into the cell. The condition they should be testing for is the cell viability when Fe3O4 gets taken up by the cell, because I assume it will have much more of an effect inside the cell than out. I also found it weird that their cell viability tests showed that the cells cultured with Fe3O4 had a higher cell viability, almost double (if optical density is a direct measurement of cell viability) than the control. The absorbance is done with an ELISA based on the formation of formazan crystal so I don't think the Fe3O4 caused any abnormal absorbance readings, so I am wondering why the absorbance is 2x higher in the Fe3O4 treated cells.
Thanks for the paper!
I had read about hollistic treatment for cancers using Magnetic Therapy and the fact that it is, "safe, non-addictive, and there are no known harmful exposure levels". Clinical tests had also shown that magnets reduce pain by creating "a magnetic field that energizes, alkalizes, and oxygenates the blood, improving the immune system performance and the body's healing abilities".
Magnetofection for gene therapy along with the boost to the immune system..wow two for one :)
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