Mohamed H. Lahiani

Impact of Carbon Nanotube Exposure to Seeds of Valuable Crops

Multiwalled carbon nanotubes (MWCNTs) affected seed germination, growth, and the development of three important crops (barley, soybean, corn). Early seed germination and activation of growth in exposed seedlings was observed when MWCNTs were added to sterile agar medium. Similarly, seed germination was activated for all tested crop species when MWCNTs were deposited on seed surfaces. The ability of MWCNTs to penetrate the seed coats of corn, barley, and soybean was proven by detection of nanotube agglomerates inside MWCNT-exposed seeds using Raman spectroscopy and transmission electron microscopy (TEM). Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that the expression of genes encoding several types of water channel proteins was increased in soybean, corn, and barley seeds coated with MWCNTs compared with uncoated control seeds. Our results indicate that the positive effect of MWCNTs on the germination and growth of seedlings is reproducible between crop species and can be observed for different methods of delivering carbon nanotubes. Such studies could prove the significant potential of carbon nanotubes as regulators of germination and plant growth.

Comparative study of plant responses to carbon-based nanomaterials with different morphologies.

The relationship between the morphology of carbon-based nanomaterials (CBNs) and the specific response of plants exposed to CBNs has not been studied systematically. Here, we prove that CBNs with different morphologies can activate cell growth, germination, and plant growth. A tobacco cell culture growth was found to increase by 22%-46% when CBNs such as helical multi-wall carbon nanotubes (MWCNTs), few-layered graphene, long MWCNTs, and short MWCNTs were added to the growth medium at a concentration of 50 μg ml(-1). The germination of exposed tomato seeds, as well as the growth of exposed tomato seedlings, were significantly enhanced by the addition of all tested CBNs. The presence of CBNs inside exposed seeds was confirmed by transmission electron microscopy and Raman spectroscopy. The effects of helical MWCNTs on gene expression in tomato seeds and seedlings were investigated by microarray technology and real time-PCR. Helical MWCNTs affected a number of genes involved in cellular and metabolic processes and response to stress factors. It was shown that the expression of the tomato water channel gene in tomato seeds exposed to helical MWCNTs was upregulated. These established findings demonstrate that CBNs with different morphologies can cause the same biological effects and share similar mechanisms in planta.

Plasmonically active nanorods for delivery of bio-active agents and high-sensitivity SERS detection in planta

Multifunctional, plasmonically-active nanorods based on gold cores and silver shells (AuNR/Ag), after exposure to tomato plants, were found to be taken up in rather short times, being detected by SERS in both roots and stems, while exhibiting lack of phytotoxicity in the concentrations used during this study. Additionally, we report that AuNR/Ag have the ability to deliver auxin growth regulator 2,4-D, which resulted in a significant impact on the regulation of tobacco cell culture growth. The unique morphology of these nanorods allowed their sensitive SERS detection, resulting in a technological approach that combines both high-sensitivity detection and delivery of biologically active molecules to plant tissues or cells. Here, we demonstrate that the multiplex combination of plasmonically active agents, laser spectroscopy, and plant biology could represent a new direction in bio-nano-engineering that integrates the use of spectroscopically-active systems for delivery of active compounds and ultra-sensitive detection in plants.

Graphene and carbon nanotubes activate different cell surface receptors on macrophages before and after deactivation of endotoxins

Nanomaterial synthesis and handling in a non‐sterile environment can result in the final product becoming contaminated with bacterial endotoxin or lipopolysaccharides (LPB). During toxicological testing, the effects caused by endotoxin‐contaminated nanomaterials can be misinterpreted in the end‐point analysis (such as cytotoxicity and immune responses) and could result in erroneous conclusions. The objective of this study was twofold: (i) to test different carbon‐based nanomaterials (CBNs) [pristine graphene and multi‐wall carbon nanotubes (MWCNTs)] for the presence of endotoxin and develop strategies for depyrogenation, and (ii) to compare the immune response exhibited by macrophages after exposure to native CBNs versus depyrogenated CBNs. The gel‐clot limulus amebocyte lysate (LAL) and chromogenic‐based LAL assays were used to detect endotoxins. Results revealed that the CBNs contained greater amounts of endotoxin than are approved by major regulatory agencies (0.5 EU ml−1). Three repeated cycles of autoclaving reduced the endotoxin in the test materials. Macrophages were incubated with pyrogenated and depyrogenated pristine graphene and MWCNTs to test differences in phagocytosis, cytotoxicity, and expression of genes involved in macrophage activation. The uptake of depyrogenated CBNs was significantly reduced as compared with pyrogenated CBNs. Exposure of macrophages to depyrogenated CBNs resulted in a distinct pattern of gene expression for TLR signaling, NOD‐like receptor signaling, and downstream signal transduction molecules. Furthermore, macrophages exposed to both types of CBNs showed the downregulation of TLR5 and NLRC4 inflammasomes. The results of this study reaffirm that assessment of endotoxin and other bacterial contamination is critical when evaluating the cellular toxicity of nanomaterials. Published 2017. This article has been contributed to by US Government employees and their work is in the public domain in the USA. Published 2017. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Carbon nanotubes as carriers of Panax ginseng metabolites and enhancers of ginsenosides Rb1 and Rg1 anti-cancer activity.

A major benefit to nanomaterial based-medicine is the ability to provide nanosized vehicles for sporadic metabolites. Here, we describe how the conjugation of valuable ginseng secondary metabolites (ginsenoside Rb1 or Rg1) with carbon nanotubes (CNT) can enhance their anti-proliferative and anti-cancer effects. Ginsenoside-CNT conjugate (Rb-CNT or Rg-CNT) permitted the ginsenosides to be used at a low dose, yet achieve a higher incidence of cancer killing. We were able to demonstrate that the ginsenoside-CNT conjugate can decrease cell viability up to 62% in breast cancer cells (MCF-7) and enhance antiproliferation of drug-resistant pancreatic cancer cells (PANC-1) by 61%. The interaction of the ginsenoside-CNT conjugate with breast cancer cells was studied using Raman Spectroscopy mapping. Total transcriptome profiling (Affymetrix platform) of MCF-7 cells treated with the ginsenoside-CNT conjugate shows that a number of cellular, apoptotic and response to stimulus processes were affected. Therefore, our data confirmed the potential use of CNT as a drug delivery system.

Genetic reduction of inositol triphosphate (InsP3) increases tolerance of tomato plants to oxidative stress

AbstractMain conclusionWe demonstrate here that the reduction of InsP3, the key component of the phosphoinositol pathway, results in changes in ROS-scavenging machinery and, subsequently, increases the tolerance of tomato plants to light stress. Different plant stress signaling pathways share similar elements and, therefore, ‘cross-talk’ between the various pathways can exist. Links between the phosphoinositol signaling pathway and light signaling were recently found. Tomato plants expressing InsP 5-ptase and exhibiting reduction in the level of inositol 1,4,5-triphosphate (InsP3) demonstrated enhanced tolerance to stress caused by continuous light exposure. To understand the molecular basis of observed stress tolerance in tomato lines with decreased amount of InsP3, we monitored the expression of enzymatic antioxidants as well as important factors in light signaling associated with non-enzymatic antioxidants (secondary metabolites). Here, we demonstrated that InsP 5-ptase transgenic plants accumulate less hydroxide peroxide and maintain higher chlorophyll content during stress caused by continuous light exposure. This observation can be explained by documented activation of multiple enzymatic antioxidants (LeAPX1, SICAT2, LeSOD) at levels of gene expression and enzymatic activities during continuous light exposure. In addition, we noticed the up-regulation of photoreceptors LePHYB and LeCHS1, key enzymes in flavonoid biosynthesis pathway, transcription factors LeHY5, SIMYB12, and early light-inducible protein (LeELIP) genes in transgenic tomato seedlings exposed to blue or red light. Our study confirmed the existence of a correlation between phosphoinositol signaling pathway modification, increased tolerance to stress caused by continuous light exposure, activation of ROS-scavenging enzymes, and up-regulation of molecular activators of non-enzymatic antioxidants in InsP 5-ptase expressing tomato lines.

Polyphenolic extract of InsP 5-ptase expressing tomato plants reduce the proliferation of MCF-7 breast cancer cells

In recent years, by extensive achievements in understanding the mechanisms and the pathways affected by cancer, the focus of cancer research is shifting from developing new chemotherapy methods to using natural compounds with therapeutic properties to reduce the adverse effects of synthetic drugs on human health. We used fruit extracts from previously generated human type I InsP 5-ptase gene expressing transgenic tomato plants for assessment of the anti-cancer activity of established genetically modified tomato lines. Cellular assays (MTT, Fluorescent microscopy, Flow Cytometry analysis) were used to confirm that InsP 5-ptase fruit extract was more effective for reducing the proliferation of breast cancer cells compared to wild-type tomato fruit extract. Metabolome analysis of InsP 5-ptase expressing tomato fruits performed by LC-MS identified tomato metabolites that may play a key role in the increased anti-cancer activity observed for the transgenic fruits. Total transcriptome analysis of cancer cells (MCF-7 line) exposed to an extract of transgenic fruits revealed a number of differently regulated genes in the cells treated with transgenic extract compared to untreated cells or cells treated with wild-type tomato extract. Together, this data demonstrate the potential role of the plant derived metabolites in suppressing cell viability of cancer cells and further prove the potential application of plant genetic engineering in the cancer research and drug discovery.

Multiwalled Carbon Nanotubes Dramatically Affect the Fruit Metabolome of Exposed Tomato Plants

Here, we reported that multiwalled carbon nanotubes (MWCNT) added to hydroponics system can enhance fruit production of exposed tomato plants. We quantified the exact amount of MWCNT accumulated inside of fruits collected by MWCNT-exposed plants using an advanced microwave induced heating technique (MIH). We found that absorption of MWCNT by tomato fruits significantly affected total fruit metabolome as was confirmed by LC-MS. Our data highlight the importance of comprehensive toxicological risk assessment of plants contaminated with carbon nanomaterials.

Role of Nanoparticles for Delivery of Genetic Material

Use of nano-sized materials as systems for delivery of genetic material into living cells is new and promising approach. Recent data showed that carbon-based, metal-based, composite nanoparticles and polymer nanoparticles have a potential to carry nucleic acids into plant cells. The unique ability of nanomaterials to penetrate plant cell wall and move inside the cell in fast manner can open ways for improvement of a number of transformation techniques including particle bombardment. However, experimental attempts to use nanomaterials as carriers of DNA/RNA in planta are rare. Here, we summarize the reports on successful delivery and integration of genetic material inside plants by using different classes of nanomaterials as delivery systems.

Effects of carbon-based nanomaterials on seed germination, biomass accumulation and salt stress response of bioenergy crops

Bioenergy crops are an attractive option for use in energy production. A good plant candidate for bioenergy applications should produce a high amount of biomass and resist harsh environmental conditions. Carbon-based nanomaterials (CBNs) have been described as promising seed germination and plant growth regulators. In this paper, we tested the impact of two CBNs: graphene and multi-walled carbon nanotubes (CNTs) on germination and biomass production of two major bioenergy crops (sorghum and switchgrass). The application of graphene and CNTs increased the germination rate of switchgrass seeds and led to an early germination of sorghum seeds. The exposure of switchgrass to graphene (200 mg/l) resulted in a 28% increase of total biomass produced compared to untreated plants. We tested the impact of CBNs on bioenergy crops under salt stress conditions and discovered that CBNs can significantly reduce symptoms of salt stress imposed by the addition of NaCl into the growth medium. Using an ion selective electrode, we demonstrated that the concentration of Na+ ions in NaCl solution can be significantly decreased by the addition of CNTs to the salt solution. Our data confirmed the potential of CBNs as plant growth regulators for non-food crops and demonstrated the role of CBNs in the protection of plants against salt stress by desalination of saline growth medium.