The development of nanotechnology is rapidly increasing, with an enormous number of applications in material science, consumer products, medical diagnosis and treatment, and drug delivery vehicles.1 Currently, there are more than 1600 nanotechnology products available for consumer use logged in the Nanotechnology Consumer Products Inventory (CPI).2 Research and commercial use necessitate disposal, thus, nanoparticles will inevitably be released into the environment. Questions arise as to the safety of such nanoparticles, especially concerning their effects on plant life consumed by humans.3 Further, even less is known in regards to the effects of functionalized nanoparticles; functionalization of nanoparticles for specific commercial uses or to reduce environmental impact may be induced by surface modification. Thus, this project aims to test the phytotoxicity of coated nanoparticles.
Metal oxide (Iron oxide, titanium oxide, etc.) nanoparticles have nearly unlimited potential for research applications.1 Applications of these nanoparticles range from solar cells to MRI contrast agents and drug delivery vehicles.3,4 Challenges present themselves in developing reliable methods to uniformly coat metal oxide nanoparticles; the coating offers the advantage of controlling the spacing between the solid particle and the pliable outside surface. Compounds with a tricarboxylic “head” group (Figure 1) can attach to iron oxide nanoparticles.5 In this manuscript we demonstrate that the same head group, when conjugated to a long-chain lipid (4-(2-Carboxyethyl)-4-(3-octadecyloxycarbonylamino)heptanedioic acid (3CCb18), Figure 1), can attach to titanium oxide nanoparticles (TiNPs). These TiNPs can be suspended in aqueous solution and subsequently used for further experimentation.
Due to their unique properties such as enhanced catalysis, lowered melting point, and increased conductivity, nanoparticles like TiNPs have potentially harmful effects to many organisms in the global ecosystem and ultimately pose a threat to humans.1,3 Preliminary studies on the impact of nanoparticles on plants have shown both positive and negative effects. In one study, nano-titanium oxide (TiO2) promoted the growth of spinach; however, the majority of phytotoxicity studies identified solid metal nanoparticles as toxic.6 Nano-aluminum oxide inhibits root elongation in corn, cucumber, soybean, cabbage, and carrots, while nanozinc oxide terminates the growth of various plant species.7,8
Although there have been many toxicity experiments conducted with uncoated nanoparticles, there has been little evaluation of functionalized or coated nanoparticles. Studies on the toxicity of nanoparticles and the effects of various coatings will elucidate the dangers of this technology and provide guidance for disposal and efficacious drug design. Phytotoxicity experiments aim to determine the effect of 3CCb18 coated (Figure 2) and uncoated TiNPs on the root development, seed germination, and overall germination index of Zea mays (corn). Z. mays was chosen based on availability, worldwide consumption, and previously conducted toxicity experiments.7-9 Toxicity was quantified using the germination index, a simple metric reported in nanotoxicology text that takes into account both total germination and early seedling development.9
2.1 Nanoparticle Coating
Optimum modification of the TiO2 surface was achieved by heating TiNPs (Sigma, 21 nm diameter) in a mixture of chloroform (CHCl3), toluene (PhMe), 3CCb18, and triethylamine. 3CCb18 (5 µM, previously synthesized in lab) was stirred in a 250-mL round bottom flask with PhMe/CHCl3 1:1 v:v (40 mL). Triethylamine (9 µL) was added to the mixture to neutralize the carboxyls. Next, a suspension of aqueous TiNPs (0.3 mL) was added dropwise, and the mixture was stirred at 80 ℃ for 24 h. The final mixture had a milky white color with noticeable precipitate at the bottom of the round bottom flask. Isolation of suspended nanoparticles was best achieved by rotary evaporation. The solvent was removed from the round bottom flask with rotary evaporation, and the nanoparticles were transferred to a small vial and dried under high vacuum for 24 h to give a cream-colored powder.
2.2 Thermogravimetric Analysis
The surface modification of the TiNPs was analyzed with thermogravimetric analysis (TGA) from 0 to 600 ℃ at 10 CHCl3/min. Samples of TiNP powder, 3CCb18 lipid, and 3CCb18-coated-TiNPs (5-10 mg) were analyzed with a TA Instruments TGA Q500.
To evaluate toxicity, the growth media of germinating Z. mays seeds was varied between three groups: coated and uncoated TiNPs as well as a DI water control. A modified procedure as published previously was followed.9 Prior to conducting experimentation, nanoparticle solutions were prepared by adding TiNPs (0.04 g) to deionized (DI) water (20 mL) and sonicating for 1 h. Afterwards, the solutions were diluted further to 100 µg/mL by adding DI water (380 mL) and stirring thoroughly. The viability of the Z. mays seeds (Hirt’s Gardens, Medina Ohio) was verified by suspending the seeds in DI water and selecting the seeds that settled to the bottom. The seeds were then soaked in 10% sodium hypochlorite solution, which acts as a surface sterilizing agent; after 10 min they were rinsed thoroughly in DI water. After rinsing, the seeds were sorted into three groups of thirty, which would be used for each nanoparticle solution. The three solutions evaluated were DI water (control), 3CCb18-coated-TiNPs, and uncoated TiNPs. Each group consisting of thirty seeds was then placed in solution and stirred for 2 h. Subsequently, nine petri dishes were prepared with Whatman filter and soaked with the corresponding nanoparticle suspension (5 mL) or DI water (5 mL). The seeds were drained, patted dry, and subsequently transferred to the petri dishes containing the filter paper, which were then sealed with parafilm and placed in a dark environment for 4 d. Root length and total germination measurements were taken, and germination index was calculated (Eq. 1).
2.4 Scanning Electron Microscopy
Following procedure 2.3, several samples were taken at random from each experimental group (DI water control, uncoated TiNP, coated TiNP) and analyzed with Scanning Electron Microscopy (SEM). Z. mays seeds were sliced open and sputter coated in Au/Pd 60:40. Samples were loaded into a LEO (Zeiss) 1550 field-emission SEM and electron micrographs were obtained. Visual analysis was used to identify TiNPs embedded in the Z. mays seedlings.
3. Results and Discussion
3.1 Coating and Thermogravimetric Analysis
The TGA of TiNPs and 3CCb18 lipid provide controls for comparison with coated TiNPs. TiNPs do not vaporize and lose zero weight percent (Figure 3a), whereas 3CCb18 lipid vaporizes completely at 350 CHCl3 and loses 100% of its weight by the end of the experiment (Figure 3b). With these control TGA experiments, it may be inferred that any lipid conjugated to a TiNP should vaporize by 350 CHCl3, leaving uncoated TiNPs for the remainder of the experiment. Further TGA experiments were then conducted on coated TiNPs that utilized 5 µM of 3CCb18 during synthesis (Figure 3c); the loss of 7% of the initial mass and retention of 93% of the input weight at 350 CHCl3 shows that lipid molecules successfully coated the TiNPs.
3.2 Phytotoxicity Assay
|Group||Seeds Germinated (of 30)||Average Root Length of Germinated Seeds (cm)||Germination Index|
|Control||27||2.3 ± 1.5||100|
|TiNPs||23||1.7 ± 1.5||63|
|Coated TiNPs||21||2.9 ± 1.1||96|
|Table 1: Germination rates, average root lengths of the seeds that germinated from each experimental group, and calculated germination index (Eq. 1) for each experimental group.|
p < 0.025 using paired single factor ANOVA analysis. Uncoated nanoparticles had a germination index (Eq. 1) of 63.3 compared to 100 for the DI water control; hence, uncoated TiNPs have detrimental effects on Z. mays during early development. According to the literature, concentrations of 100 µg/mL of various nanoparticles begin to show negative effects on root growth and overall germination on different varieties of seeds.8 The TiO2 data presents similar results: an uncoated nanoparticle concentration of 100 µg/ mL displayed prominent phytotoxicity. Both coated and uncoated nanoparticles showed a reduced percentage of seeds germinated compared to the control; yet, the coated nanoparticles’ germination index was very close to 100, comparable to the control’s growth rate. These results suggest that coated nanoparticles had little or no phytotoxic effects on seed germination, whereas uncoated nanoparticles exhibited prominent phytotoxicity. Additionally, average root growth was enhanced in coated nanoparticles (Figure 4); this positive effect may be explained by the lipid coating. This coating may have helped hydrophobic molecules absorb more effectively into corn, increasing nutrient uptake and providing for better growth. Alternatively, the lipid may have been hydrolyzed and removed from the coating on the TiNPs, creating a fatty alcohol that provided the Z. mays seeds with a source of energy and a component utilized in cell membranes. Experimental results are summarized in Table 1.
SEM micrographs show the presence of TiNPs in both coated and uncoated experimental groups, suggesting that their uptake contributed to experimental results (Figures 5a, 5b, and 5c). Elemental analysis was conducted in tandem to SEM spectroscopy to confirm the presence of titanium (data not shown)
The TGA data suggest that there is a strong interaction between the tricarboxylic acid head group and the TiO2 core. This shows that the coating procedure and tricarboxylic head group can be used to build novel uniformly coated metal oxide nanoparticles, and a tricarboxylic-acid head group will associate strongly with TiO2.
Phytotoxicity evaluation used Z. mays, a worldwide staple of food consumption. SEM showed that Z. mays uptakes nanoparticles (Figures 5b and 5c). Alarmingly, if the seeds carry these TiNPs to their gametes, they may pass into the human body if ingested. Uncoated TiNPs hindered root growth at a concentration of 100 µg/mL, while a solution of 3CCb18-coated-TiNPs at the same concentration promoted root growth when compare to a DI water control (Table 1 and Figure 4). Collectively, these results show that precautions must be taken to prevent the release of TiNPs into the environment. Further analysis of environmental retention must be conducted in order to better assess the direct implications of nanoparticles on the environmental health and human safety.
5. Future Work
This paper details initial synthesis and toxicity data for a lipid-coated-nanoparticle; more experiments are necessary to elucidate the long-term exposure effects on plant life and mechanism of toxicity. Additionally, we aim to use the knowledge gained from this study to ultimately create and optimize a minimally toxic nanoparticle for drug delivery applications.
5.1 Nanoparticle Synthesis
Surface modifications of nanoparticles enable unique properties for functionalization. We will synthesize and conjugate polymer linker molecules consisting of a polyethylene glycol spacer and a cholestanol anchor to the solid metal nanoparticle. Hydrophobic interactions with the cholestanol anchor will enable the spontaneous formation of a phospholipid bilayer around the nanoparticle, allowing the incorporation of both hydrophobic and hydrophilic compounds for drug delivery purposes. Further tests on the uniformity of coatings are also necessary before proceeding to the encapsulation of compounds.
5.2 Biological Evaluation
Ongoing biological evaluation is necessary to assess the environmental and human impacts of nanoscale devices. Several different coatings on many types of nanoparticles should be assessed with a variety of concentrations of nanoparticles. Further phytotoxicity and cytotoxicity studies using a broad array of plant and animal models for a statistically representative conclusion on nanoparticle toxicity. Further understanding of toxicity mechanisms will help in designing more effective nanoparticles for commercial use and in shaping public policy regarding nanoparticles utilized in consumer products.
This manuscript is based on results obtained through a honors biology project in conjunction with chemistry undergraduate research. The authors would like to express our gratitude to our advisor, Professor Richard D. Gandour, for his guidance and resources throughout the project. We thank Professor Amanda J. Morris and Mr. Andrew Haring for providing TiO2 nanoparticles. SEM work was conducted at the Nanoscale Characterization and Fabrication Lab (NCFL) at Virginia Tech; the authors would also like to acknowledge Mr. Stephen McCartney for the use of the SEM and his flexibility, and the department of biology for SEM funding.
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