Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

Summary

Here we present a protocol to synthesize novel, high-aspect ratio biocomposites under biological conditions and in liquid media. The biocomposites scale from nanometers to micrometers in diameter and length, respectively. Copper nanoparticles (CNPs) and copper sulfate combined with cystine are the key components for the synthesis.

Abstract

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper and cystine, with either copper nanoparticles (CNPs) or copper sulfate contributing the metallic component. Synthesis is carried out in liquid under biological conditions (37 °C) and the self-assembled composites form after 24 hr. Once formed, these composites are highly stable in both liquid media and in a dried form. The composites scale from the nano- to micro- range in length, and from a few microns to 25 nm in diameter. Field emission scanning electron microscopy with energy dispersive X-ray spectroscopy (EDX) demonstrated that sulfur was present in the NP-derived linear structures, while it was absent from the starting CNP material, thus confirming cystine as the source of sulfur in the final nanocomposites. During synthesis of these linear nano- and micro-composites, a diverse range of lengths of structures is formed in the synthesis vessel. Sonication of the liquid mixture after synthesis was demonstrated to assist in controlling average size of the structures by diminishing the average length with increased time of sonication. Since the formed structures are highly stable, do not agglomerate, and are formed in liquid phase, centrifugation may also be used to assist in concentrating and segregating formed composites.

Introduction

Copper is a highly reactive metal that in the biological world is essential in some enzyme functions ^1,2, but in higher concentrations is potently toxic including in the nanoparticulate form ^3,4. Concern over copper toxicity has become more relevant as CNPs and other copper-based nanomaterials are utilized, due to the increased surface area/mass for nanostructures. Thus, even a small mass of copper, in nanoparticle form, could cause local toxicity due to its ability to penetrate the cell and be broken down into reactive forms. Some biological species can complex with and chelate metal ions, and even incorporate them into biological structures as has been described in marine mussels ⁵. In studying the potential toxic effects of nanomaterials ⁴, it was discovered that over time, and under biological conditions used for typical cell culturing (37 °C and 5% CO₂), stable copper biocomposites could be formed with a high-aspect ratio (linear) structure.

By a process of elimination, the initial discovery of these linear biocomposites, which occurred in complete cell culture media, was simplified to a defined protocol of essential elements needed for the biocomposites to self-assemble. Self-assembly of two types of highly linear biocomposites was discovered to be possible with two starting metal components: 1) CNPs and 2) copper sulfate, with the common biological component being cystine. Although more complex, so called “urchin” and “nanoflower” type copper-containing structures with nanoscale and microscale features have been previously reported, these were produced under non-biological conditions, such as temperatures of 100 °C or greater ^6-8. To our knowledge, synthesis of individual, linear copper-containing nanostructures that are scalable in liquid phase under biological conditions has not been previously described.

One of the starting materials utilized for synthesis of nanocomposites, namely CNPs, has been reported previously to be very toxic to cells ⁴. It has recently been reported that after the nanocomposites are formed, these structures are less toxic on a per mass basis than the starting NPs ⁹. Thus, the synthesis described here may be derived from a biological and biochemical reaction that has utility in stabilizing reactive copper species, both in the sense of transforming the NP form into larger structures and in producing composites less toxic to cells.

In contrast to many other nanomaterial forms which are known to aggregate or clump upon interaction with biological liquid media ^10,11, once formed, the highly linear composites described here avoid aggregation, possibly due to a redistribution of charge which has been previously reported ⁹. As detailed in the current work, this avoidance of aggregation is convenient for the purposes of working with the structures once formed for at least 3 reasons: 1) composite structures once formed may be concentrated using centrifugation and then easily dispersed again using vortex mixing; 2) formed structures can be decreased in average size by sonication for different periods of time; and 3) the formed linear structures may provide an additional tool for avoiding the recently described “coffee ring effect” ¹² and thus provide a dopant for creating more evenly distributed coatings of materials, especially those containing spherical particulates.

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Protocol

1. Planning of Experiments

1. Determine the volume of copper nanocomposites needed for synthesis. On that basis, choose a number of small volume flasks (25 cm²), or larger flasks as indicated below in preparation of materials.
2. For this synthesis, use a 37 °C incubator with 5% CO₂ and at least 40% humidity. Ensure that such an incubator is available and that it will not be repeatedly disturbed over the period of synthesis (approximately 24 hr).
   CAUTION: Repeated opening and closing of the incubator will certainly cause temperature fluctuations which may result in altered synthesis of the nanocomposite structures.

2. Preparation of Materials

1. Prepare all materials fresh before the start of an experiment, by adding solid materials to solvents right before synthesis is to begin. Keeping stock solutions of cystine and copper starting materials in liquid for long times before the experiment is not recommended and may lead to variable results. Once opened from the vendor, keep starting materials dry by wrapping the top of the container with Parafilm.
   Note: The following protocol is used as an example for reaction in a 25 cm² cell culture flask using 7 µl of cystine, 6,643 µl of sterile water, and 350 µl of CNPs.
2. Prepare a 2 mg/ml solution of copper nanoparticles by weighing out at least 2 mg of CNPs. Wear disposable gloves during this step to prevent possible contact of CNPs with skin. Place the nanoparticles in an empty sterile 16 ml glass vial.
   1. To the vial containing CNPs, add sterile deionized water in the appropriate volume to make a 2 mg/ml solution and vortex the solution for 20 sec to provide dispersion of the nanoparticles before synthesis starts (at least 1 ml total volume is recommended). Do not fill the vial more than half way with water as this will inhibit mixing by vortexing. CNPs will quickly settle to the bottom of the vial and will appear dark in color (grey to black).
   2. Sonicate the CNP solution for 17 min at RT to provide maximal dispersion of CNPs before start of synthesis. Periodically check to make sure that CNPs are mixing due to sonication. After a successful sonication, CNPs remain suspended in solution for at least 30 min and the solution will be dark in color.
3. Weigh out sufficient mass of cystine to make a 72.9 mg/ml solution for the synthesis. Since cystine is not directly soluble in water, place the weighed cystine in an antistatic weighing vessel.
   1. To the weighing vessel containing cystine, add sufficient volume of sterile, 1 M NaOH, so that the cystine completely dissolves. For example, dissolve 7.29 mg of cystine completely in 100 µl of 1 M NaOH, to make a 72.9 mg/ml solution.
   2. To maintain sterile conditions, carry out this step in a sterile flow tissue culture hood.
      CAUTION: NaOH at 1 M concentration is caustic, so wear disposable gloves during this step to prevent contact of concentrated NaOH with skin
4. Working in a sterile tissue culture hood, add 7 µl of cystine with 6,643 µl of sterile water to the sterile synthesis flask first, and let incubate for 30 min in the incubator at 37 °C with the flask cap vented (loose) to provide effective mixing. Resuspend the 2 mg/ml CNP solution by vortexing for 30 sec, since CNPs will have settled after the sonication step.
   1. Add sufficient CNP solution to the synthesis flask (using sterile technique) to maintain the following component ratios: combine 1 parts cystine, 50 parts CNPs, and 949 parts sterile water in a 25 cm² cell culture flask to start the synthesis. For example, for a 7 ml synthesis volume, combine 7 µl of cystine stock solution, 350 µl of CNPs, and 6,643 µl of sterile water. Replace the cap on the flask and tighten so that it is secure.
   2. After combining all components for the synthesis, gently mix in the flask by swirling 4-5 times. Place flask in the CO₂ incubator and vent the flask by loosening the cap so that there will be gas exchange in and out of the flask during synthesis.
5. Allow synthesis to run in the incubator for approximately 24 hr. During synthesis, one can observe, with microscopy and by eye, formation of highly linear composites.
   Note: The process of formation of the structures may happen suddenly in the sense that structures are initially hard to detect, then appearance proceeds quickly to an increasing density. Formation may therefore occur before 24 hr. The process can also be observed by eye once structures become larger and their density increases. While generation of the structures can be observed over time under the microscope and by eye at later time points, continuously interrupting the synthesis conditions and temperature will lead to poor synthesis results.
6. Terminate synthesis of biocomposites by tightly capping the synthesis flask and storing the vessel in a refrigerator (4 °C). Structures, once generated, remain stable in this form for at least a year. Label the flask with synthesis conditions, including components utilized, date of the synthesis, and incubation time of the synthesis before termination.

3. Synthesis Using Copper Sulfate

1. Carry out self-assembly synthesis by replacing CNPs with copper sulfate salt. Using sterile technique, dissolve at least 2 mg of copper sulfate in sufficient volume of sterile deionized water to make a 2 mg/ml solution. The copper sulfate crystals easily go into solution at this concentration, but vortex the vial if needed, and inspect by eye to ensure all crystals are dissolved.
2. After preparation of the copper sulfate, carry out synthesis as described previously, but replacing CNPs with the copper sulfate.
   Note: Self-assembled nanocomposites using copper sulfate as a starting material were found to be much more consistent in final shape than for structures synthesized from CNPs.
3. Terminate the synthesis of copper sulfate biocomposites as for CNP composites (step 2.6) and store them long-term at 4 °C.

4. Characterization and Handling of Biocomposites Post-synthesis

1. Characterize biocomposites derived from CNPs and from copper sulfate by white light microscopy⁹ and by electron microscopy⁹.
   1. For characterization and inspection of biocomposites post-synthesis by white light microscopy, use an inverted microscope as composites will settle to the bottom surface of the flask within a few minutes of laying the flask flat, and can then be brought into focus. Use the bright field setting on the microscope to maximize contrast between biocomposites and the liquid medium. Composites derived from CNPs and copper sulfate will both appear clear to opaque in color, but unreacted CNP aggregates will appear very dark in color.
      1. Use a digital camera connected to the microscope to capture images of the composites. A range of lengths for the individual structures will be observed.
   2. For characterization and inspection of biocomposites post-synthesis and after storage at 4 °C, allow flasks to come to RT for at least 15 min as flasks will form condensation initially upon removal from refrigerator, which will obscure effective focusing while carrying out microscopy imaging. After allowing equilibration to RT, wipe the top and bottom surfaces of the flask with a clean paper towel to maximize microscopy imaging quality.
   3. When working with or imaging composites that have been stored long-term, vortex the flask for 30 sec to dissociate clumps of composites that form while in the refrigerator. After vortexing, inspect the structures with an inverted microscope to ensure that aggregates have dissociated, and repeat vortexing as necessary.
   4. Use inverted white light microscopy to assess the efficacy of the synthesis for a given experiment using CNPs. For example, document the presence or absence of unreacted CNPs in synthesis flasks used for CNP-derived biocomposites from flasks with different parameters such as time of synthesis.
      Note: Individual CNPs are too small to observe with a light microscope, but unreacted CNP aggregates will appear as round-shape and dark objects, in contrast to the successfully synthesized CNP-composites which will have a high-aspect ratio, linear form, and will have a range of different lengths. Avoid carrying out synthesis for too long of a period of time before termination, as this will result in highly branched “urchin” type structures, which are difficult to disperse into individual structures once formed.
   5. Use inverted white light microscopy to assess the efficacy of the synthesis for a given experiment using copper sulfate. Since copper sulfate goes fully into solution using this protocol, the solution will appear less dark than the solution from synthesis using CNPs. Document the size and extent of copper sulfate composites by comparing flasks with different synthesis conditions such as time of synthesis before termination.
      Note: Successfully synthesized composites will show a range of different lengths. Avoid carrying out synthesis for too long of a period of time before termination, as this will result in highly branched aggregates of composites, some of which will be “urchin-like” in structure, and which are difficult to disperse into individual structures once formed.
2. To concentrate biocomposites post-synthesis, centrifuge solutions of composites in a centrifuge tube. Add 6 ml of either CNP-derived structures or copper sulfate-derived structures to a 15 ml centrifuge tube. Centrifuge for 10 min at 500 x g at RT to form a pellet. For smaller volumes, add 500 µl of structures in solution to 0.6 ml sized tubes. Centrifuge at 2,000 x g at RT for at least 10 min to form a pellet.
   1. After centrifuging for sufficient time (at least 10 min for microfuges), save the observable pellet at the bottom of the tube where the structures are concentrated by carefully removing the supernatant liquid above the pellet. Biocomposite structures derived from copper sulfate appear blue in color and structures derived from CNPs are darker (grey to black).
   2. Add more composites to this tube and repeat the process in the same tube to concentrate structures if desired. To disperse the concentrated pellets, add the desired volume of solution to the tube, and vortex for 10-30 sec.
3. Sonicate structures once formed, to move the average population size (lengths) of the structures to lower values. Place structures in sterile deionized water and sonicate for at least 10 min. Using this process, over time, structures become fragmented and smaller in average length (see Figure 6 of the text). Document changes in composite sizes with different sonication times using an inverted white light microscope and digital camera.

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Results

Figure 1 shows a flow-chart schematic of the synthesis steps to form the linear biocomposites described in this work. CNPs or copper sulfate as starting materials are combined with sterile water to form a 2 mg/ml solution, this solution is mixed and sonicated to provide an even mixture, and this copper solution is then mixed in the following ratio for synthesis: 949 parts sterile water: 50 parts copper mixture: 1 part cystine stock solution. The actual volumes may be increased or decreased according to t...

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Discussion

While evaluating potential toxic effects of nanomaterials including CNPs, it was observed that over the long-term, CNPs were transformed from an initially more dispersed particulate distribution to a larger, aggregated form (Figure 2). In some cases, these highly aggregated formations which were produced in the cell culture dish, under biological conditions, formed highly linear projections from the central aggregate, reminiscent of previously described copper- containing “urchins” ⁶

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Materials

Name	Company	Catalog Number	Comments
Mini Vortexer	VWR (https://us.vwr.com)	58816-121
CO2 Incubator Model # 2425-2	VWR (https://us.vwr.com)	Contact vendor	Current model calalog # 98000-360
Eppendorf Centrifuge (Refrigerated Microcentrifuge)	Labnet (http://labnetinternational.com/)	C2500-R	Model Prism R
Cell Culture Centrifuge Model Z323K	Labnet (http://labnetinternational.com/)	Contact vendor	Current model Z206A catalog # C0206-A
Sonicator (Ultrasonic Cleaner)	Branson Ultrasonics Corporation (http://www.bransonic.com/)	1510R-MTH
Balance	Sartorius (http://dataweigh.com)		Model CP225D similar model CPA225D
Olympus IX51 Inverted Light Microscope	Olympus (http://olympusamerica.com	Contact vendor
Olympus DP71 microscope digital camera	Olympus (http://olympusamerica.com	Contact vendor
external power supply unit - white light for Olympus microscope	Olympus (http://olympusamerica.com	TH4-100
10X, 20X, and 40X microscope objectives	Olympus (http://olympusamerica.com	Contact vendor
Scanning Electron Microscope	Hitachi (http://hitachi-hitec.com/global/em/sem/sem_index.html)	model S-4800
Transmission Electron Microscope	Zeiss (http://zeiss.com/microscopy/en_de/products.html)	model Libra 120
Table Top Work Station Unidirectional Flow Clean Bench	Envirco (http://envirco-hvac.com)	model PNG62675	Used for sterile cell culture technique