Team:Tec-Chihuahua/Project
Introduction
Nanotechnology is the study and application of extremely small things and can be used across all the other science fields, such as chemistry, biology, physics, materials science, and engineering. One of the nanostructures which will revolutionize the future nanotechnological devices are the Carbon nanotubes (CNTs), which can be imagined as rolled up graphite sheets held together by van der Waal’s bonds. These CNTs can be functionalized to improve their solubility in physiological solutions and selective binding to biotargets. That is why they can be used as a new DNA delivery system including enhancement of cell membrane interactions due to electrostatic forces, increased cellular uptake by endocytosis, an improved trafficking to the nucleus, and competing against traditional methods for greater efficiency of transfection and transformation in different biological systems.
Background
Nanotechnology
Nanotechnology is science, engineering, and technology conducted at the nanoscale, which is about 1 to 100 nanometers.
Nanoscience and nanotechnology are the study and application of extremely small things and can be used across all the other science fields, such as chemistry, biology, physics, materials science, and engineering.
It’s hard to imagine just how small nanotechnology is. One nanometer is a billionth of a meter, or 10-9 of a meter. Here are a few illustrative examples:
- There are 25,400,000 nanometers in an inch
- A sheet of newspaper is about 100,000 nanometers thick
- On a comparative scale, if a marble were a nanometer, then one meter would be the size of the Earth
Although modern nanoscience and nanotechnology are quite new, nanoscale materials were used for centuries. Alternate-sized gold and silver particles created colors in the stained glass windows of medieval churches hundreds of years ago. The artists back then just didn’t know that the process they used to create these beautiful works of art actually led to changes in the composition of the materials they were working with.
Today's scientists and engineers are finding a wide variety of ways to deliberately make materials at the nanoscale to take advantage of their enhanced properties such as higher strength, lighter weight, increased control of light spectrum, and greater chemical reactivity than their larger-scale counterparts (United States National Nanotechnology Initiative,2015)
Carbon nanotubes
Carbon nanotubes (CNTs) are a new allotrope of carbon originated from fullerene family, which will revolutionize the future nanotechnological devices. CNTs can be imagined as rolled up graphite sheets held together by van der Waal’s bonds. They have long cylinder made of hexagonal honey comb of lattice of carbon, bound by two pieces of fullerenes at the ends. There are two types of CNTs: MWCNT and SWCNT. The multi walled CNTs (MWCNT) consists of two or more concentric cylindrical shells of graphene sheets coaxially arranged around a central hollow area with a spacing between the layers which is close to that of the interlayer separation as in graphite. In contrast, single shell or single walled nanotubes (SWCNT) are made of single graphene (one layer of graphite) cylinders and have a very narrow size distribution (Sobhi, et. al, 2007).
The nanodimension of the CNTs provide very large surface area. The surface area of the SWCNTs were found to be one magnitude higher than that of graphite but smaller compared to the activated porous carbon (Sobhi, et. al, 2007).
Functionalization/grafting of CNTs
The surface functionalization will aid the carbon nanotube materials in becoming biocompatible, improving their solubility in physiological solutions and selective binding to biotargets. The functionalization of CNTs may be separated into two categories a noncovalent wrapping or adsorption and the covalent tethering. The best stability, accessibility and selectivity will be achieved through covalent bonding because of its capability to control the location of the biomolecule, improved stability, accessibility, selectivity and reduced leaching. The chemicals that can form covalent or irreversible van der Waal’s bonds with the nanotube could alter the sp2 hybridization of CNTs to sp3 hybridization, as a result of functionalization (Liu et. al, 2005).
In order to attach the molecules to the nanotubes covalently, the first requirement is the formation of functional groups on the CNTs. The carboxylic acid group is often the best choice because it can undergo a variety of reactions and is easily formed on CNTs via oxidizing treatment. Functionalized CNTs have received much interest for dispersion enhancement in processing or chemical modification. Shortening of CNTs by ultrasonication with oxidizing acid mixtures is frequently used to functionalize CNTs (Liu et. al, 2005).
The sidewall of CNTs is chemically stable, making the functionalization of CNTs with sensing molecules, a challenge. Also nanotubes have distinct inner and outer surfaces, which can be functionalized either chemically or biochemically. Thus, the development of simple and cost effective chemical methods for covalent functionalization of carbon nanotube materials is becoming an area of growing fundamental and industrial importance (Sobhi, et. al, 2007).
Technique of functionalization of CNTs by EDAC
One of the universal methods for connecting biomolecules to CNTs is diimide activated amidation by direct coupling of carboxylic acid to proteins using N-ethyl-N-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) or N,N dicyclohexyl carbodiimide (DCC) as a coupling agent (Jiang et. al, 2004)
DNA is an important and promising molecule with all the basic properties necessary for the assembly of nanoscale electronic devices. The combination of CNT with DNA has attracted the attention of several research groups recently. DNA chains have been used to create various functional structures and devices through the sequence-specific pairing interactions (Sobhi, et. al, 2007).
DNA-CNTs
CNTs can be used as a delivery system, but they have to be efficient for successful gene therapy. This will allow the transfer and expression of the therapeutic gene in the target organ or tissue. To this end, both viral and nonviral vectors are currently in use.
Although viral gene delivery achieves high levels of gene expression, it has several disadvantages that make it problematic for human use. In particular, viral vectors can be immunogenic, or induce inflammation that render transgene expression transient, or can have oncogenic effects (Liu et. al, 1995). Nonviral vectors might be more desirable since they can overcome some of these concerns. In addition, because these vectors are typically assembled in cell-free systems from well-defined components, they can have significant manufacturing and safety advantages over viral vectors. However, improvements of nonviral vectors to achieve therapeutically relevant levels of gene expression are still needed.
The use of cationic molecules such as various synthetic lipids, polylysine, protamine sulfate, and cationic dendrimers to condense DNA and form complexes able to enhance the efficiency of gene transfer in vitro and in vivo is well documented (Xiao et. al, 2005) (Varcarcel et. al,2005). Such DNA condensates are commonly of a spherical morphology, while the molecular interactions between DNA and the cationic component greatly determine a number of biological processes responsible for efficient gene expression.
These include enhancement of cell membrane interactions due to electrostatic forces, increased cellular uptake by endocytosis, and improved trafficking to the nucleus. To optimize f-CNTs as gene delivery vehicles, it is essential to characterize their interactions with DNA.
DNA-PEI-CNTs
The functionalization of carbon nanotubes (CNTs) has been carried out in various ways for numerous applications in biotechnology, including for the preparation of sensors, as scaffolds for cell growth, imaging reagents, and transporters for drug delivery. One way is to immobilize DNA onto the surface of CNTs through noncovalent interactions or covalent bonds. Covalent-bond approaches might compromise and even spoil the functions of DNA owing to chemical reactions and the difficulty in releasing DNA. Nevertheless, noncovalent approaches developed to date may only provide metastable immobilization of DNA onto the surface of CNTs. It was reported that the migration of DNA linked covalently to CNTs was retarded in gel electrophoresis but noncovalent interactions between DNA and CNTs did not completely prevent migration (Dwyer et.al, 2002).
Polyethylenimine (PEI) is a type of polymer with a high density of amines, thus DNA may be immobilized securely onto the surface of multiwalled carbon nanotubes (MWNTs) that have been functionalized with PEI through strong electrostatic interactions arising from these amines.
Hence, we have adopted a grafting-from approach to prepare polyethylenimine-graft multiwalled carbon nanotubes (PEIg-MWNTs).
Genetic transformation and transfection
Genetic transformation is the introduction of foreign genetic material in prokaryotes (bacteria) and non animal eukaryotic cells such as fungi, algae or plants and transfection when introduced into animal cells. Some techniques used in the laboratory for transforming and transfecting cells are based on the production of competent cells by chemical and physical treatments; however these treatments have low efficiencies. Other methods use bacterial and / or viral vectors capable of infecting the cell of interest and introduce exogenous genetic material. Despite being effective they have some disadvantages as not all species are susceptible to being infected by these microorganisms, and it is not accessible technique for laboratories with little microbiology experience.
Efficiencies of transformation and transfection with traditional methods
Direct microinjection-Mammalian cells
Specific genes can be introduced into cultures mammalian cells by chromosome-mediated gene transfer and by purified DNA- mediated gene transfer. DNA of interest can be injected in the pronucleus of an embryo in the two cells state. This technique is well known and generally used in many laboratories, but it has not have a considerable efficiency progress, which is 0.1-5% depending on the considered specie (Fernández, 2008)
Electroporation technique- E.coli
Electroporation is a technique in which bio-membranes are permeabilized by pulsed electric fields of several kV cm amplitude and submicrosecond duration. Thereby membrane pores occur temporarily and solvated molecules, like DNA or drugs, can be transferred into living cells. However, existing electroporation technology is limited in its ability to treat large quantities of cell material and DNA.
Additionally, the application of high electric field pulses can lead to irreversible electroporation and, consequently, cell lysis.
Inoue in 1990 reported that the efficiency for plasmid transformation was 1-3 x 109 cfu/µg of DNA in at least three E.coli strains (DH5α, JM109 and HB101).
Biobalistics-Nicotiana tabacum callus
For genetic transformation of any plant species, it is essential to have an optimized protocol for regeneration to give birth previously transformed tissue, so it is important to properly adjust the driving conditions in vitro according to each species and variety.
In the literature the percentage of stably transformed clones obtained per cell surviving DNA delivery and continuing normal development is generally referred to as the stable transformation efficiency of microinjection. A study made under optimal conditions by Kost in 1995 described, on average, 23% of the successfully injected cells developed into microcalli and 12.2% gave rise to paromomycin-resistant stably transformed clones. The average efficiency of stable transformation can therefore be calculated to be 53%.
CNTs in our biological systems
Novel developments in the area of nanotechnology have provided advanced knowledge and technological platforms with applications in a variety of scientific areas, ranging from medicine, aerospace, electronics, and sensing to defense industries. Lately, given the need to understand the interaction between engineered nanomaterials and various biological systems, a significant research interest has developed around the use of nanotechnology-based approaches for agricultural and food systems.
The unique properties of nanosized materials (small size, high biochemical reactivity, ability to penetrate cells, and swift distribution inside organisms) make them an attractive tool for growing management techniques.
CNTs- Bos taurus embryos in early development
So far, no studies have been reported on this interaction, however it has found as an area of opportunity for research thanks to the features that make the CNTs unique in its field.
CNTs- E.coli
As we mentioned before the electroporation is a technique with many limitations, for that reason, miniaturized protocols for electroporation by localized electropermeabilization have been developed. These new techniques include, among others, electrolyte filled capillaries, micropipettes and chip structures.
Compared with bulk electroporation, these techniques not only reduce the voltage applied (a few volts versus kilovolts), but also the amount of cell material and agents, considerably, and make it possible to electroporate single cells.
Though these techniques are well suited for treating single mammalian cells, they are still limited for targeting bacteria and microorganisms because of the microscopic scale of the electroporative devices. Moreover, these methods seem to be difficult to use in areas where the cell surface is hard, such as plant cells, cell walls of Gram positives or capsules. Further miniaturization of the electroporation systems to the nanoscale will allow selective targeting and electroporation of cell organelles in eukaryotic cells and prokaryotic microorganisms.
Rojas-Chapana et. al in 2005 revolutionized the electroporation by the use of CNTs as physical ‘‘electroporation vectors’’. The technique differs fundamentally from other methods used today because electrodes are not needed. Water dispersible CNTs having an anionic surface charge attach to the surface of Gram negative bacteria. 6
This arises mainly due to an electrostatic interaction between the CNTs and the likewise charged bacterial surface. On the other hand, the effect of a microwave electromagnetic field pulse on the interaction of CNTs with the cells leads to electropermeabilization through individual CNTs. Using this technique, they demonstrated a reversible electroporation, in which cell growth and cell morphology were not affected.
CNTs-Nicotiana tabacum callus
Specific types of nanoparticles in low doses have not been found harmful to plants but instead are capable of activating specific physiological processes. Nevertheless, future perspectives on nano-biotechnological approaches for the regulation of plant productivity will depend on a thorough understanding of the molecular mechanisms responsible for the activation of seed germination and plant growth in the presence of complex engineered nanomaterials.
Carbon nanotubes have shown promise as regulators of seed germination and plant growth. Khodakovskaya et. al in 2012, demonstrated that multiwalled carbon nanotubes (MWCNTs) have the ability to enhance the growth of tobacco cell culture (55-64% increase over control) in a wide range of concentrations (5-500 μg/mL). Activated carbon (AC) stimulated cell growth (16% increase) only at low concentrations (5 μg/mL) while dramatically inhibited the cellular growth at higher concentrations (100-500 μg/mL). They found a correlation between the activation of cells growth exposed to MWCNTs and the upregulation of genes involved in cell division/cell wall formation and water transport.
Justification
Some laboratory techniques used to transform and transfect prokaryotic cells are based on the production of competent cells by chemical and physical treatments, which originate micropores in their wall that allow the entry of exogenous DNA. These techniques have some disadvantages as not all species are susceptible to being infected by these microorganisms or systems. The efficiencies of these traditional methods have been studied and reported by previous investigations which show low rates of transformation efficiencies.
Nowadays there are several studies in where researchers are looking for new methodologies to infect cells which are more efficient and less complicated and expensive. The nanotechnology has the potential to create new structures with the ability to cross cell membranes and increase solubility, stability and bioavailability of biomolecules, improving efficiency.
Carbon Nanotubes, a nanomaterial, can be used as a delivery system and offer many advantages when is functionalized with DNA. The combination of CNTs with DNA has recently attracted the attention of several research groups, because they work as a vessel for DNA to create various functional structures and devices through the sequence-specific pairing interactions.
General objective
To evaluate the efficiency of DNA-CNTs transformation and transfection in different cell systems.
Specific objectives
- To synthetize, purify and characterize CNTs with low toxicity.
- To evaluate the internalization capacity of fluorescent functionalized CNTs in E.coli, Nicotiana tabacum callus and embryos in early development of Bos taurus.
- To synthesize our part of DNA with GFP gene and CMV promoter.
- To functionalize CNTs by ionic double bond between the polyethilenamine and our part of DNA, and to characterize them.
- To transform and transfect the different cell systems with pDNA-CNTs system delivery and with traditional protocols.
- To compare efficiencies of the different transformation and transfection methodologies (DNA-CNTs vs. traditional protocols)
Hypothesis
The pDNA - CNTs (part-CNTs) system will have greater efficiency in the transformation and transfection in different cell systems than traditional methods
Results
Synthesis and characterization of NP-NCTs and P-NTCs
The size and elemental composition of NP-CNT and P-CNT were characterized by SEM, and the structural quality of CNTs were determined by Raman spectroscopy. The dimensions obtained for NP-NCTs varied from 40 to 75 nm in diameter and 37.79 µm in length (Figure 5, A and B respectively). Similarly P-CNTs presented the same diameters distribution but the length decreased to 1.8 µm (Figure 6: A and B respectively). Semiquantitative analysis of elemental composition of NP-CNTs and P-CNTs (Table 1) showed a decrease of Fe from 2.53% to 1.43%, in P-CNTs as well an increase of O content from 14.29 to 16.30 %, in purified samples. On the other hand P-CNTs showed a 14.41% of S content. These results indicates an effective removal of Fe form surface of UP-CNTs. This Fe comes from the synthesis of CNTs because we used ferrocene as catalyzer, and is important removed it due the toxic effect in cell cultures that this produces. The increase of O content indicate an addition of oxidized groups (COOH, OH, CO) on the surface of the CNTs (Saito et. al., 2002; ) due to the strong interaction of concentrated acid allowing open-end formations and promoting the oxidation of exposed carbon atoms (Zhang et. al., 2003). This suggestion is confirmed by decreasing of the length of P-CNTs. The S content probably comes from purification process with H2SO4/HNO3 concentrated acid. Because of this more washes were performed on P-CNTs. Oxidized groups are very important for functionalization process which will be discussed later.
According to Montes-Fonseca et. al., 2012 the purification process by sonication with concentrated acid produce more open-end on P-CNTs as can see in Figure 6. Therefore these results are consistent with previously published (Montes-Fonseca et. al., 2012).
The raman spectra for UP-CNTs and P-CNTs showed in Figure 7. Each spectra consist of two characteristic bands, namely D-band at 1338 cm-1 and G-band 1600 cm-1. The G-band is a characteristic feature of the graphitic layer and corresponds to the tangential vibration of the carbon atoms, while de D-band is a typical sign for defective graphitic structures on CNTs. The comparison of the ratios of these two peaks intensities (IG/ID) gives a measure of the quality of the samples. If bands have similar intensity this indicates a high quantity of structural defects (Keszler et. al., 2004; Datsyuk et. al., 2008). The ratio IG/ID of the P-CNT and UP-CNT was 1.2526 and 0.9748 respectively. These results suggest an increase of the structural quality of CNTs due purification process.
Fuctionalization of P-CNTs with fluorescein diacetate
For internalization studies P-CNTs were functionalized with fluorescein diacetateby carbodiimide activate amidation (see methodology). To confirm that functionalization process was successful, the CNTs functionalized with fluorescein diacetate (FD-CNTs) were observed in Epifluorescent in inverted microscope (Axio Vert.A1) where we could see little fluorescents dots on the accumulation of FD-CNTs (Data not shown).
On the other hand for corroborate fluorescent functionalization, FD-CNTs where analyzed by Raman spectroscopy. The raman spectra for these samples (Figure 8) indicate the specific band G at 1338 cm-1 for CNTs layers, however the spectra is altered due to the interference of the fluorescence, therefore, its an indicator that the functionalization was effective.
Internalization studies
In the image 9.A we can observe the oocyte nucleus due to the staining with 4,6-diamidino-2-Phenylindole (DAPI) which stains nucleic acids. The oocyte in the Figure 9.B was put into interaction with the FD-NTC and little fluorescent green dots can be seen inside the nucleus’ area. In Figure 9.C the same protocol was done and similar fluorescent green dots can be viewed inside the nucleus’ area and the cytoplasm. We can infer that the FD-NTCs passed successfully through the pellucide zone, since there is fluorochrome inside the nucleus’ cell and the cytoplasm.
Parts
After transforming the cells with the GFP and the CMV plasmids, we miniprepped them to carry on with the digestion protocol. We quantified them via Nanodrop 2000 but the graphs seemed odd.
Not happy with this, we ran an electrophoresis, which explained the oddity:
In this gel we can see a general RNA contamination, and in the wells 2, 3 and 4 are bands of like-weight DNA, which correspond to GFP plasmids.
In this gel we can also see RNA contamination, as well as like-weight DNA in wells 1, 2 and 5, which also corresponds to GFP plasmids. No CMV whatsoeverL.
With this, we decided not to make the new part, because we wouldn’t have time to transform new cells with CMV, miniprep, digest, ligate, transform, miniprep and lyophilize all over again.
Despite this, we decided to submit our part to the registry because we expect to work on it in the subsequent months.
Future applications
Transformation and biological transfection through nanotubes would be used as an effective tool for the creation of drugs, treat diseases and meet the demand for food products by means of transgenesis. Carbon nanotubes are used as carriers of biomolecules, in the case of our project its transport capacity applies to the creation of a system of genetic modification within cells eukaryotes and prokaryotes. This capacity holds 4 fundamental advantages which could replace the current systems of genetic modification; carbon nanotubes are effective in the transformation of a simple or difficult to change cell system, its toxicity is void due to the established purification process, the use of the technique is not exclusive to equipped laboratories and trained personnel.
In the case of genetic transformation by means of carbon nanotubes:
- Serves as an efficient and simple system of modification.
- For the creation of production of metabolites in high-scale systems.
- The transformation of specific bacteriological lines for the production of drugs can be achieved.
This new technology can also be used in eukaryotic cells applied above and promptly in the case of our project in bovine embryos. This can help to improve the techniques of gene transfection for treating diseases. For example: using RNA's mufflers anchored in nanotubes.
References
A. M. Keszler, L. Nemes, S. R. Ahmad, and X. Fang, “Characterisation of carbon nanotube materials by Raman spectroscopy and microscopy - A case study of multiwalled and singlewalled samples,” Journal of Optoelectronics and Advanced Materials, vol. 6, no. 4, pp. 1269–1274, 2004. [26] V. Datsyuk, M. Kalyva, K. Papagelis et al., “Chemical oxidation of multiwalled carbon nanotubes,” Carbon, vol. 46, no. 6, pp. 833–840, 2008.
C. Dwyer, M. Guthold, M. Falvo, S. Washburn, R. Superfine, D. Erie (2002), Nanotechnology 13, 601.
Fernández Narbón, Patricia (2008). Microinyección pronuclear. TRANSFERENCIA GÉNICA EN ANIMALES. Recovered from UNED: http://www.uned.es/experto-biotecnologia-alimentos/TrabajosSelecc/PatriciaNarbon.pdf
Hersam, Mark C. (2009) Material science: nanotubes sorted using DNA. Nature 460, 186-187
Inohue H, Nojima H, Okayama H (1990): High efficiency transformation of Escherichia coli with plasmids. Gene 96:23-28.
J. Liu, A.G. Rinzler, J. Dai, J.H. Hafner, R.K. Bradley, P.J. Boul, A. Lu, T. Inverson, K. Shelimov, C.B. Huffman, F. Rodriguez Maeias, Y.S. Shon, T.R. Lee, D.T. Colbert, R.E. (1995) Smalley, Fullerene pipes, Science 280 1253–1256.
J. Zhang, H. Zou, Q. Qing et al., “Effect of chemical oxidation on the structure of single-walled carbon nanotubes,” Journal of Physical Chemistry B, vol. 107, no. 16, pp. 3712–3718, 2003.
Khodakovskaya Mariya V., De Silva Kanishka, Biris Alexandru S., Dervishi Enkeleda, and Villagarcia Hector (2012). Carbon nanotubes induce growth enhancement of tobacco cells. ACS nano, Vol. 6, No. 3, 2128-2135
K. Jiang, L.S. Schadler, R.W. Siegel, X. Zhang, H. Zhang, M. Terrones (2004), Protein immobilization on carbon nanotubes via a two-step process of diimide-activated amidation, J. Mater. Chem. 14, 37–39.
Kost, Benedikt, Galli, Alessandro, Potrykus, Ingo and Neuhaus, Gunther (1995). High efficency transient and stable transformation by optimized DNA microinjection into Nicotiana tabacum protoplast. Journal of Experimental Botany, Vol. 46, No. 290, pp. 1157-1167, September 1999
L. Montes Fonseca et. al., “Citotoxicidad de Nanotubos de Carbono Funcionalizados con Proteínas de Superficie de Entamoeba histolytica”, Centro de Investigación en Materiales Avanzados (CIMAV), 2012.
M. Valcarcel, B.M. Simonet, S. Cardenas, B. Suarez (2005), Present and future applications of carbon nanotubes to analytical science, Anal. Bioanal. Chem. 38 1783–1790.
Rojas-Chapana, José, Troszczynska, Julia, Firkowska, Izabela, Morsczek, Christian and Gierig (2005). Multi-walled carbon nanotubes for plasmid delivery into Escherichia coli cells. Lab Chip, 5, 536-539
S. Chen, W. Shen, G. Wu, D. Chen, M. Jiang (2005), A new approach to the functionalization of single-walled carbon nanotubes with both alkyl and carboxyl groups, Chem. Phys. Lett. 402, 312–317.
S.F. Xiao, Z.H. Wang, G.A. Luo (2005), The progress in functionalization of carbon nanotube, Chin. J. Anal. Chem. 3 261–266.
Sobhi, Daniel et.al (2007). A review of DNA funtionalized/grafted carbón nanotubes and their characterization. ScienceDirect. Sensors and Actuators B 122 (2007) 672-682
T. Saito, K. Matsushige, and K. Tanaka, “Chemical treatment and modification of multi-walled carbon nanotubes,” PhysicaB, vol. 323, no. 1-4, pp. 280–283, 2002.
United States National Nanotechnology Initiative (2015). What is Nanotechnology? Recovered from: http://www.nano.gov/nanotech-101/what/definition
Veena Choudhary and Anju Gupta (2011). Polymer/Carbon nanotube nanocomposite. INTECH. ISBN 978-953-307-498-6