September 5, 2016.
With rapid and significant development, progress and profit achieved in nanomaterials and nanotechnology, nanotoxicity has been put in the research agenda and a new subject termed nanotoxicology has emerged. Tremendous examples of nanomaterial toxicity have been found and demonstrated in the past decade. This toxicity effect should be carefully taken into account for nanomaterial-mediated gene delivery for therapy. When DNA is ligated and immobilized with nanomaterials, interaction occurs between them. Effect of this interaction on DNA must be clarified otherwise changed gene might be delivered and finally applied.
In our study, we directly made nanomaterials simply contact with naked DNA and cultured the mixture. Subsequently, the DNA was analyzed for their topological form change and also the nanomaterial-interacted DNA was transferred into bacterial cells to observe mutation. From screened mutants, DNA was extracted, isolated and analyzed by gel electrophoresis to observe DNA damage and sequenced as well to study DNA mutation types. To observe the effects from the simplest and then the most probable condition where nanomaterials meet with naked DNA without any additional activation, we only adopted the condition of a simple contact between the nanomaterials and naked DNA, but in two different environments, namely dry and wet (in solution). As DNA is inside the biological cell, a wet environment is natural for the nanomaterial-DNA contact. For an understanding of pure interaction between the nanomaterials and DNA, the dry environment was also tested.
Two different types of nanomaterials, carbon nanotubes (CNTs) and tungsten trioxide (WO3) nanoplates (Fig. 1), which were all synthesized in the laboratories of the Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, were used as testing materials. Plasmid DNA pGFP (containing the green fluorescence protein, GFP), a simple DNA molecule normally employed as a model DNA in many biological investigations, was used as the counterpart with the nanomaterials.
Fig. 1. SEM images of in-house synthesized (a) carbon nanotubes and (b) WO3 nanoplates.
Contacts between the nanomaterials and naked DNA were made by simple and direct mixing of the two counterparts in vitro in either dry or wet mixing environment for varied time periods at room temperature. DNA transfer into bacterial E. coli competent cells was operated using the standard electroporation. DNA extracted from mutated cells was sequenced. After simple and direct mixing of the naked DNA with the nanomaterials, DNA topological forms were analyzed using gel electrophoresis (Fig. 2). The DNA band light intensity was quantified for the amounts of the DNA forms. The original supercoiled form decreased while the single-strand break (SSB) represented relaxed form increases. It was indicated that the SSB occurrence had a high time rate and the wet contact was more offensive in breaking DNA strands than the dry contact.
Fig. 2. Gel electrophoresis of plasmid DNA mixed with 1 µg of CNT (the mass ratio was CNT:DNA = 1:100) and incubated for 3 [(a)], 6 [(b)], 12 [(c)], 24 [(c)] and 48 [(d)] hours. Lane No.: in all figures, 1 – marker (100 bp), 2 – control (without mixing with nanomaterials); (a) 3 and 4 – dry mixing for 3 hrs, 5 – wet mixing for 3 hrs; (b) 3 and 4 – dry mixing for 6 hrs, 5 – wet mixing for 6 hrs; (c) 3 – dry mixing for 12 hrs, 4 – wet mixing for 12 hrs, 5 – dry mixing for 24 hrs, 6 – wet mixing for 24 hrs; (d) 3 – dry mixing for 48 hrs, 4 – wet mixing for 48 hrs. S: supercoiled form. R: relaxed form.
Fig. 3. Quantified result from the gel electrophoresis in Fig. 2 and data fitting of the measured DNA form percentage to the exponential equation for the case of contacting with CNT in the mass ratio of CNT:DNA = 1:100.
To study the SSB occurrence time rate and quantitatively compare the time rates in the dry and wet contacting modes, the percentages of the supercoiled form and the relaxed form as a function of the contacting time were fitted to mathematic equations and the time rate was found to best follow an exponential style
Fs(t) = A exp(-αt) + B, or Fr(t) = 1 – Fs(t) = 1 – [A exp(-αt) + B].
Here, Fs(t) and Fr(t) are the percentages of the DNA supercoiled form and the relaxed form, respectively, A and B are two constants with B the stabilized supercoiled form percentage and thus A+B the initial percentage of the supercoiled form in the control, t is the contacting time period (in hour), and α is a positive coefficient, named as the time evolution coefficient, depending on the nanomaterial type and the contacting condition, provided that other DNA forms and fragments are neglected. Fig. 3 shows the fitting for the case of contacting with CNTs. The best fitting parameters are A = 37.5%, B = 36.5%, αdry = 1/8.7 = 0.115, αwet = 1/3.10 = 0.323. The time evolution coefficient α for the wet case is seen to be greater, nearly triply, than that for the dry case, representing a more rapid exponential change of the DNA form. It can be speculated that nanomaterials damaging DNA is a direct effect but also assisted by some indirect effects such as wet environment. The wet environment may provide a contacting medium between the two counterparts to link them by certain physical and/or chemical/biological factors such as charge, dielectric potential field and radicals, or even biological signals. It is also possible that wet contact may more easily involve oxidative reaction due to nanoparticle-induced water splitting, even without assistance of external energy such as light, heat or electricity. Some differences between the CNT and WO3 cases were noticed, indicating that CNTs were more toxic.
Fig. 4. Percentages of the survival and mutation of the bacteria E. coli transferred with WO3 (left) and (right) CNT nanomaterials-mixed DNA.
The plasmid DNA was transferred into bacterial cells of E. coli after mixing with the nanomaterials. Cell death and mutation were observed (Fig. 4). After the DNA transfer, in most cases, about 90% of the cells were dead and the survived bacteria were almost all mutants. The fact of nearly 100% mutation induction in the case of the wet contact which is more practical implies nanomaterials so toxic in inducing cancers or other potential diseases by changing DNA. The DNA sequencing result showed that DNA point mutations occurred after DNA contacting with the nanomaterials and transversion was the dominant mutation type.
The results provide clear evidence that one of the mechanisms involved in the nanomaterial toxicity is direct damaging to DNA by the nanomaterials and the damaged DNA can cause biological cell death and mutation. The induced mutation could be sources of diseases or cancers. Our result could give a warning to nanomaterial-mediated gene therapy that the gene delivered by nanomaterials might be changed and thus the gene would be no longer as original and the therapy might fail or even be wrong.
Assoc. Prof. Dr. Yu Liangdeng (L.D. Yu)
Thailand Center of Excellence in Physics
P.O. Box 70, Chiang Mai University, A. Muang, Chiang Mai-50202, Thailand
Email : firstname.lastname@example.org