Nanofibers and nanoparticles from the insect-capturing adhesive of the Sundew (Drosera) for cell attachment
© Zhang et al. 2010
Received: 4 June 2010
Accepted: 18 August 2010
Published: 18 August 2010
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© Zhang et al. 2010
Received: 4 June 2010
Accepted: 18 August 2010
Published: 18 August 2010
The search for naturally occurring nanocomposites with diverse properties for tissue engineering has been a major interest for biomaterial research. In this study, we investigated a nanofiber and nanoparticle based nanocomposite secreted from an insect-capturing plant, the Sundew, for cell attachment. The adhesive nanocomposite has demonstrated high biocompatibility and is ready to be used with minimal preparation.
Atomic force microscopy (AFM) conducted on the adhesive from three species of Sundew found that a network of nanofibers and nanoparticles with various sizes existed independent of the coated surface. AFM and light microscopy confirmed that the pattern of nanofibers corresponded to Alcian Blue staining for polysaccharide. Transmission electron microscopy identified a low abundance of nanoparticles in different pattern form AFM observations. In addition, energy-dispersive X-ray spectroscopy revealed the presence of Ca, Mg, and Cl, common components of biological salts. Study of the material properties of the adhesive yielded high viscoelasticity from the liquid adhesive, with reduced elasticity observed in the dried adhesive. The ability of PC12 neuron-like cells to attach and grow on the network of nanofibers created from the dried adhesive demonstrated the potential of this network to be used in tissue engineering, and other biomedical applications.
This discovery demonstrates how a naturally occurring nanofiber and nanoparticle based nanocomposite from the adhesive of Sundew can be used for tissue engineering, and opens the possibility for further examination of natural plant adhesives for biomedical applications.
One of the unique properties of the Sundew adhesive is its highly elastic nature that allows it to be drawn into threads up to one meter in length . Early studies confirmed that the chemical structure of the adhesive was an acid polysaccharide containing various concentrations of sugars and acids, depending on the species [11, 12]. Isolation of D. capensis adhesive through gel filtration, cellulose acetate filtration, ion-exchange chromatography, and ultracentrifugation yielded one macromolecule with a molecular weight of 2 × 106 Daltons . It was discovered that the adhesive was formed by xylose, mannose, galactose, glucuronic acid, and ester sulfate in the ratio of 1:6:6:6:1 . In other species, the acid polysaccharide was found to have different ratios of chemicals. D. binata was reported to contain arabinose, xylose, galactose, mannose, and glucuronic acid in a ratio of 8:1:10:18:17 . Further analysis also found that these polysaccharides consisted of an abundance of metal cations, including 22 mM Ca++, 19 mM Mg++, 0.9 mM K+, and 0.2 mM Na+ in D. capensis. The D. capensis adhesive was composed of water (96%) and acid polysaccharide (4%) . The ratio of polysaccharide to water has proven to be crucial in the formation of the unique elastic properties of the adhesive, as seen with other polymers [13–17]. Due to the difference in chemical composition, varying material properties were expected for different Sundew species. Environmental factors and prey availability could have imparted selection pressure that influenced the development of the adhesives over the course of evolution.
In addition to chemical composition, nanoscale morphology also contributes to the physical properties of materials. Preliminary studies on structural properties of polysaccharide-based adhesives have been conducted [18, 19]. However, the relationship of the nanoscale morphology to the physical properties of adhesives remains largely unexplored. We report here our recent discovery of a nanofiber and nanoparticle-based network from the Sundew adhesive, and explore the potential of using this network for cell attachment.
The Sundew species (D. binata, D. capensis, and D. spatulata) were purchased from the Carnivorous Plant Nursery, Derwood, MD, USA and maintained in mineral depleted soil with distilled water. The Sundew are sensitive to high concentrations of minerals, and thus it was necessary to ensure that tap water was not given to the plants. The plants were exposed to direct sunlight for 12 hour periods, and maintained at a constant temperature of 21°C. After a period of one week, all plants began to produce adhesive on the tentacle heads. It should be noted that there is no variation in the chemical composition of the adhesive from tentacle to tentacle within a species [11, 12].
As shown in Figure 1, a small amount of adhesive forms on the head of each tentacle on the leaf surface. To coat a surface with this adhesive, the sample (silicon wafer, glass coverslip, and mica) was held with sterile forceps and gently brushed against the tentacle heads, allowing the adhesive to be transferred to the sample. Using this method, a different pattern of coating was achieved with each treatment. Due to the non-uniformity of the coating method, over six replicates for each species and substrate were examined. After applying the adhesive to the substrate, the samples were allowed to dry for 24 hours under a bio-safety cabinet.
Due to the large surface area of the 25 mm2 coverslips, for cell attachment studies, the coverslips were cut to 5 mm2 with a diamond etched pen. These smaller coverslips were then cleaned by sonication in acetone, ethanol and deionized water. Using these smaller coverslips, it was possible to more easily coat the entire surface area. To ensure that the coating covered the entire surface, an Alcian Blue pH 2.5 Periodic Acid Schiff Stain (Chromaview®) was applied to all coated samples per the manufacturer's instructions. With this staining procedure, acid polysaccharide stains bright blue and neutral mucosubstances stain pink. Upon completion of staining, the samples were imaged using an Olympus Fluoview 1000 microscope to visualize the stained adhesive.
In addition to the stained experimental samples, control samples were prepared for the cell attachment experiments using uncoated coverslips, and 0.1% poly-L-lysine (Electron Microscopy Sciences®) coated coverslips. PC12 and primary nerve cells have been shown to strongly attach to poly-L-lysine coated surfaces, but not to bare glass, so these uncoated and poly-L-lysine coated samples served as positive and negative controls. After the coverslips were coated with the adhesive, the samples were UV sterilized while submerged in Hank's Balanced Salt Solution, Formula III (Electron Microscopy Sciences®) for 15 minutes in a biosafety cabinet. Upon sterilization, the samples were seeded with PC12 cells in F12-K medium supplemented with 15% horse serum and 2.5% fetal bovine serum at a density of 5 × 104 cells/cm2. The cells were then incubated on the samples for 24 hours in a 37°C incubator with 5% CO2 to allow for attachment. After 24 hours, the samples were gently washed with sterile Milonig's Phosphate Buffer (Electron Microscopy Sciences®) warmed to 37°C. This prevented detachment due to temperature induced stress. Cells were then stained for 30 minutes with a live/dead viability dye containing calcein AM and ethidium homodimer-1 from Invitrogen (catalog number #L3224), live cells stained green and dead cells stained red. The samples were then washed and visualized using the fluorescent microscopy. Four fields of view under a 10× objective (0.0391 mm2) were used to determine the number of attached cells on each sample. The number of viable cells was determined by counting 100 cells at random and scoring as either alive or dead using the viability dye.
AFM imaging was conducted using both an Agilent 5500 AFM and an Agilent 6000 ILM/AFM. The purpose of using both systems was to control for potential artifacts, and to allow for microscopic imaging of the samples to determine the targeted scanning areas. In addition, all samples were examined by two independent investigators who prepared their samples separately to further eliminate the possibility of artifactual data. All imaging for both systems was conducted in air in AC mode. Both systems were equipped with intermittent contact mode tips, Budget Sensors® Tap150AL-G, with aluminum reflex coating. The tips had a resonant frequency of 150 kHz and a force constant of 5 N/m. Due to tip variation, manual sweeps were conducted on all tips prior to scanning to determine the actual frequency of the tip. Prior to scanning, a calibration grid was used to assure that the distance measurements of the Picoview® software were accurate. Publication quality scans were conducted at a scan speed of less than 1 ln/s and a resolution of 1024 × 1024 pixels.
Transmission electron microscopy (TEM) imaging and energy-dispersive X-ray spectroscopy (EDS) were conducted using a JEOL 2200 FS TEM with attached EDS at the Advanced Microscopy Center of Michigan State University. Copper grids were coated with ultra thin carbon films. By using the thin film copper grids, the sample could be deposited on the film, instead of falling through the mesh of the grid. Grids were then coated with the Sundew adhesive in the same manner using the technique described earlier. Briefly, the copper grids were grasped using sharp electron microscopic forceps and gently brushed against the tentacles of the Sundew. After coating with the adhesive, the samples were dried overnight for subsequent analysis.
The first stage of this study focused on determining the nanoscale structure of the dried adhesive on a variety of substrates. By determining the nanostructure of the adhesive, we could evaluate the potential uses for this material. Three Sundew species, D. binata, D. capensis, and D. spatulata, were chosen for this study. Adhesive from the tentacles from the three species were streaked onto silicon wafers, mica, and glass coverslips. After the samples were allowed to dry overnight in a biosafety cabinet, the samples were scanned using AFM.
To investigate whether the elastic properties were maintained from the liquid to the dried adhesive, force versus distance curves were generated on the dried adhesive. Since the adhesive was completely dried before conducting the AFM studies, there was no adhesive force observed from the network of nanofibers when compared to the bare silicon surface. However, as seen in Figures 7C-D, there was a significant increase in extension length. The extension from the dried adhesive was 320.6 nm, while the extension from the bare silicon surface was less than 49.2 nm. Similarly, the adhesive showed significant deformation compared to the bare silicon wafer. It is important to point out that the AFM experiments indicated that the dried adhesive adhered to the silicon wafer, and could not be removed using sharp probes in contact mode with fast scanning speeds (> 3 ln/s) and a negative setpoint. It is believed that a curing process takes place during drying that forms a strong bond between the adhesive and the substrate surface. This phenomenon is common for many epoxies, glues, and adhesives, where drying or chemical modification of a liquid adhesive often leads to the formation of tight bonding between the dried adhesive and the contact surface [26–30]. The stability of the dried adhesive on the surface, combined with the non-toxic components of the adhesive (salts, polysaccharide, and organic nanoparticles), and the porous network structure of the nanofibers, led to the hypothesis that the network could be used for applications in tissue engineering and wound healing.
To validate this hypothesis, it was essential to demonstrate that the Sundew network was capable of supporting cell growth. To test this ability, PC12 cells were chosen as a model system for nerve cell growth. PC12 cells were derived from a pheochromocytoma of the rat adrenal medulla , and are typically used as a model system for nerve cell growth and differentiation [32–34]. Three treatments were tested to determine if the network of nanofibers was capable of supporting cell attachment. Since PC12 cells do not attach to bare glass, this sample was used as a negative control. A positive poly-L-lysine coated control was used to determine the maximum number of cells that could attach on an ideal substrate. The third sample was a Sundew adhesive coated glass coverslip, stained with Alcian Blue to visualize the pattern of staining. Viability was determined by using a calcein/ethidium bromide live/dead assay and all samples were imaged using an Olympus Fluoview 1000 confocal microscope.
Through this study, we have systematically examined the nanoscale structure of the adhesive generated from the Sundew, and evaluated the potential of this material to be used for tissue engineering. It was determined that the adhesive is a nanocomposite composed of water, nanoparticles, polysaccharide, and salts. This nanocomposite was observed in three species of Sundew, and was shown to form a network of nanofibers independent of the surface. When dried, this adhesive serves as a suitable substrate to promote the attachment of PC12 neuron-like cells, and may be used for a variety of other cell types. Further study into the role of the nanoparticles within the nanocomposite will lead to a better understanding of how nanoparticles can be used in adhesives. Experimentally, nanoparticles have been shown to help increase adhesion of epoxy adhesives . The presence of nanoparticles in the Sundew adhesive may increase surface contact and generate larger force for initial binding to insects. Another possibility is that the nanoparticles may provide a mechanical support that allows the liquid polysaccharide to stretch beyond what has previously been observed. This could explain the high elasticity observed in the liquid adhesive. Moreover, the potential uses of composite materials from biological organisms show promises for a wide variety of applications . A Sundew adhesive inspired biomaterial can be proposed for a wide range of biomedical applications. In addition to tissue engineering, it may be used for biological treatment of wounds, regenerative medicine, or helping enhance synthetic adhesives. Further studies will focus on extending the results obtained from this study to evaluate the additional potential for this material to be used in biomedical applications.
We would like to thank partial support for this study by the UTK-ORNL Science Alliance Award#3318039.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.