21 February 2017 Photoswitchable dye-nanoparticle probes with photothermal switching of light-dark states and colors (Withdrawal Notice)
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Publisher's Note: This paper, originally published on 21, February, 2017, was withdrawn per author request.
Harrington, Haji, Galanzha, Nedosekin, Nima, Watanabe, Ghosh, Biris, and Zharov: Photoswitchable dye-nanoparticle probes with photothermal switching of light-dark states and colors




Photoswitchable Moieties

Photoswitchable compounds, compounds in which certain properties such as their absorption can be changed (i.e. “switched”) in response to remote light, have garnered a growing interest in various fields of engineering, optics and medicine in the past decade.1-9 Photoswitching has been used to improve the resolution7 of microscopy and create micro/nano valves or photo-switches10,11, utilizing a compound’s ability to be influenced remotely by light. Photoswitchable proteins, in particular, have been used for intra- and inter-cellular tracking of organelles and even whole cells.1,2. The ability to remotely switch the optical properties of these proteins has allowed for more precise, inter-cellular tracking of a few, or even single, circulating tumor cells (CTCs) when combined with In vivo Photoacoustic Fluorescent Flow Cytometry (PAFFC).12,13 Using PAFFC to monitor the blood stream in vivo, photoswitchable proteins give off photoacoustic signals when they pass through a laser focused on a blood vessel based on their original absorption. Once these signals are detected in a semi-regular fashion, a “switching” laser can be used to change the absorption of only a few single cells containing photoswitchable proteins, and thus allow for the tracking of single circulating cells.14

However, one of the major dilemmas that face photoswitchable compounds for use in biomedical applications is the need for high-energy, ultra-violet (UV) light to switch the compound. UV light has the potential to be quite damaging to skin and other organs, and is greatly attenuated by bio-tissue which limits the use of these compounds to superficial monitoring.15-17 These limitations must be surpassed to achieve the full potential of photoswitchable clinical applications.


The Near-Infrared Window and Thermochromism

One approach to address the issues that photoswitchable compounds typically have is to engineer a way in which the photoswitchable compounds can be switched via the use of light in the near-infrared range (NIR). NIR light is not particularly dangerous to body tissue and bio-tissue is relatively transparent to NIR light.17 However, the mechanism of switching for most photoswitchable compounds is inherent in the molecule or protein itself, making it difficult to directly engineer the compound to switch at an alternative wavelength. Here, we describe a novel method of creating a system that indirectly engineers photoswitchable that are responsive to NIR light. To do this, we use thermochromic dye (TCD), which changes color through the mechanism of heating (thermochromism), combined with magnetic nanoparticles that absorb across the spectrum, including the NIR range. Thus, the hybrid probes, referred to as TCD-NP probes throughout, will absorb NIR light via the magnetic nanoparticles, heating the system and causing the thermochromic dye to change color (“switch”).




TCD-NP probe concept

To create TCD-NP probes, two different thermochromic dyes were selected from LCRHallcrest®18, having two different transition temperature at which they changed color (absorbance). These were leuco dyes composed of three components, a solvent (such as Bisphenol A), dye and developer, micro-encapsulated in melanine formaldehyde.19 As the temperature of inside each microcapsule (referred to as “bead” throughout) surpassed the transition temperature (melting temperature of the solvent), the solvent would liquefy and the dye and developer would disassociate, allowing the chemistry of the dye to change in a way in which its absorbance was lost.19 The two dye we selected had different properties: 1) we selected a green dye that came in a powder form, which was constituted in a non-polar solvent (DMSO), and transitioned from green to transparent at a transition temperature of 31±3°C; 2) we selected an orange dye that came in a slurry (aqueous dispersion), which was diluted in water, and transitions from orange to yellow at a transition temperature of 47±3°C. Iron (II, III) oxide magnetic nanoparticles (MNPs) were added to the green and red dyes to create the TCD-NP probes. Each probe was tested on the macro scale in bulk solution to observe any color (absorbance) changes due to laser illumination in bulk according to the shown schemes in Figure 1. Then the orange TCD-NP probe was chosen (for reasons discussed below) to observe on the microscopic/single bead level.

Figure 1

TCD-NP probe concept. a) Schematic showing the use of MNP and laser energy to switch green TCD beads from colored to colorless. b) Schematic showing the use of MNP to switch orange (red + yellow beads) TCD from orange (red + yellow) to yellow (colorless + yellow).



Macro/bulk results

Before any conclusions could be drawn in reference to the color change of the TCD-NP probes, it was necessary to show that any color change observed indeed came from the TCDs and not the nanoparticles themselves. The green TCD, orange TCD and a solution of MNPs were heated in a water bath separately and any resulting color changes were observed. Figure 2a shows that only the green and orange TCDs change color, whereas the MNPs remain constant. After this was confirmed, the TCDs were mixed with MNPs for 1-2 hours. The TCD was then placed in a cuvette in an Ultrospec 3300 pro UV/Visible spectrophotometer and an 808 nm Diomed CW laser was positioned ~10 cm above the cuvette to provide illumination and heating. Temperature was monitored by IR FLIR camera. A baseline absorbance was taken for each TCD-NP probe, then the probes were heated with the 808 nm laser past the transition temperature, and periodic spectra were recorded as the probes cooled. As can be seen in Figure 2c, the absorbance for each probe was lower as the temperature of the solution was higher due to the heating of the MNPs via laser. It can also be seen in Figure 2d that the absorbance remains fairly constant above and below the transition temperature, which is estimated by the apparent inflection point of each curve. Water and DMSO were tested as vehicle controls, and no significant absorbance change was observed.

Figure 2

TCD color changes. a) Preliminary study showing the color change (top before heating, bottom after heating) of orange (left) TCD, green (center) TCD and MNPs (right) alone after being heated in a water bath. b) Microscope image of orange TCD beads before (left) and after (right) heating with a heat gun, showing that only the red TCD beads change color. c) Spectroscopy for green (left) and orange (right) TCD bulk solutions at different temperatures. d) Absorbance/temperature profile for green (left) and orange (right) TCDs.



Micro/single bead level results

Once it was confirmed that color change could be achieved in bulk solution via laser heating, the orange TCD was selected to carry out work on the microscopic scale to observe color change at the single bead level. The orange TCD was selected because it was dispersed in water and had a transition temperature that was much more advantageous for use at biological temperatures. When the orange TCD was observed on the microscopic level, it was noted that it was composed of two separate types of beads: yellow static beads and red thermochromic beads. When the solution is heated, the yellow beads remain yellow, and the red beads transition from red to transparent (Fig. 2b), following the typical leuco dye scheme. The red beads were separated from the yellow beads via centrifugation to remove any static noise that the yellow beads caused, and red TCD-NP probes were created by using the red beads alone mixed with MNPs in a 1:4 ratio. These red TCD-NP probes will be the focus of the remainder of the paper, unless otherwise noted.

At the single bead level, the change in color was much more apparent than on the macro level (Figure 3a,c). The red TCD-NP probes rapidly responded to NIR laser illumination (805 nm, ~0.25W), losing their absorbance within seconds of being hit with the laser. This color change was highly reversible, as the color returned to the probes even faster (milliseconds), and repeatable over several cycles (Fig. 3b). We also wanted to show that a shift in absorbance is possible, not just a decrease as is shown for the red TCD-NP probes. To achieve this goal, we use the original orange TCD-NP probes (without the removal of the yellow static beads) on the microscopic level. Figure 3d shows a slight shift in absorbance as the color of the probes changes from orange (red + yellow beads) to yellow (transparent + yellow beads), though it is not as dramatic as the decrease in absorbance observed via laser heating of the red TCD-NP probes.

Figure 3

Single bead level. a) Spectral difference before and after heating (805nm Diomed laser). b) Laser heating and cooling cycles showing repeatability (time is in arbitrary units corresponding to the order of seconds) c) Transmission image of red TCD before, during and after laser irradiation. d) Orange TCD at the single bead level showing a slight shift in absorbance maxima before and during heating, with corresponding transmission images.



In vivo preliminary work

Though these are proof-of-concept results and much work is needed to optimize these probes if they are to be used in biological contexts, we wanted to first determine if it was even possible to visualize these probes inside cells. To do this, MDA cells were incubated with red TCD alone and the red TCD-NP probes overnight, and photoacoustic imaging was used in conjunction with dark field (scattering, see methods) imaging to see the probes inside of cells. Figure 4 shows that cells can uptake the probes and these probes can be visualized. Using two color photoacoustic imagining, we were able to separate the TCD signals from the MNP signals via 532 nm and 820 nm lasers (see methods). Figure 4a shows the co-localization of signals in a single cell. Further, when EAHY cells were incubated with red TCD alone, we showed that the photoacoustic signals given by the TCD beads taken up by the EAHY cells subsequently disappear when the medium was heated with a heat gun, indicating the possibility of switching inside cells (Fig. 4b).

Figure 4

In vivo work. a) EA.hy926 cells (left) and MDA cells (right) incubated with nothing (control), TCD alone or TCD-NP probes (TCD/IONP). Bright field, photoacoustic and merged images are shown. b) Bright field, photoacoustic and merged images of EA.hy926 cells incubated with red TCD before (top) and after (bottom) heating with a heat gun.





Choice of NPs and dye

The goal of this project was to create a photoswitchable probe that was responsive to NIR light. We chose two TCDs that had transition temperatures that were around the normal body temperature and focused on the red TCD which had a transition temperature that was slightly above the normal body temperature. This transition temperature (47±3°C) would allow the probes to remain un-switched under normal body conditions. MNPs were chosen as the avenue of NIR absorption for multiple reasons: 1) MNPs absorb across a broad spectrum, and thus can absorb a variety of laser wavelengths; 2) MNPs absorb energy via other methods such as exposure to a magnetic field, allowing for alternative methods of switching20; 3) MNPs are currently used in clinical settings and have low toxicity21-23; 4) MNPs can function as multimodal agents, providing contrast for magnetic resonance imagining; and 5) MNPs have the potential to be used as photothermal agents in the hypothermic treatment of cancer via heating. Combining these MNPs with the chosen TCDs, we were able to create novel photoswitchable probes that were fast, reversible and that would lay the proof-of-concept ground work as a foundation to create more sophisticated and stable probes in the future.


Advantages and limitations

What makes these new photoswitchable probes advantageous over photoswitchable compounds that are currently available is their ability to respond to NIR light. These probes repeatedly showed the ability to change color when illuminated with an 805 nm laser. These probes switched very rapidly, and remained stable over several cycles. The color change was seen even more drastically on the single bead level, likely owning to the different mechanisms of heating (i.e. heating individual beads directly at the single bead level as opposed to heating the medium of the solution at the macroscopic level). Another advantage that these probes have are the ability to change out different TCDs for ones with a desired transition temperature or color change. We showed that not only could laser switching cause the TCD to switch from a colored state to a colorless state, but using a different color scheme, we could switch from one color (orange) to another (yellow). This switch, though it shows the proof-of-concept, was not a dramatic as the switch from color to colorless, probably due to the relatively small spectral difference between orange and yellow and the fact that the orange TCD was made up of static yellow beads and red thermochromic beads rather than being truly orange.

Another advantage to our system is that we were able to show the proof-of-principle that cells could uptake our probes in vivo. Though this is in the early stages of development, we wanted to observe if the new probes showed any promise of being used for in vivo work in the future. The TCD and MNPs were shown to co-localize in MDA cells. We also showed evidence that the TCD could be switched in vivo in EAHY cells using a heat gun. Much more work needs to be done in this area if these probes are to be routinely used in in vivo work, however we have shown the first steps.

As much of this work is at the proof-of-concept stage, there are some limitations. Cytotoxicity work needs to be done to verify if the probes can be safely used in biological settings. Further, there is no covalent attachment from the TCD beads to the MNPs that would enhance the probes switching ability. Finally, other NPs should be explored, such as gold nanorods, which have better absorption in the NIR and narrower bands of absorption maxima for specific applications.


Future directions

Future directions in this project would address many of the limitations listed above. A covalent linker should be used to attach TCD beads and MNPs, making the probes more stable for routine use. We would also explore other NP options such as gold NPs or gold nanorods that have better absorption in the NIR for deeper light penetration, and thus better detection. These probes lay the proof-of-principle work to make probes that can be switched remotely using NIR light. Future applications could include cell tracking in the bloodstream, the building blocks for a micro/nano-thermometer and the contrast agents that would provide laser tracking in vivo though indication by laser switching. It is the ultimate goal of this project to be able to label and track circulating bacteria cells in the bloodstream using these or similar photoswitchable probes, opening up opportunities to study the dynamics of bacterial dissemination throughout the body during infection.




Dye and nanoparticles

The dye and the nanoparticles used in this project were obtained from two commercial companies. The TCDs were thermochromic leuco dyes obtained from LCRHallcrest (Glenview, IL).18 These leuco dyes consist of a dye, a color developer (Bisphenol A) and a solvent encapsulated in melamine formaldehyde. Below the transition temperature (i.e., the melting point of the solvent), the dye and the developer are held in close contact with one another, and the bead (i.e., the microcapsule) is colored. Once the transition temperature is surpassed, the solvent liquefies and the developer separates from the dye, causing the bead to lose color.19 One of the dyes chosen had a green color in the un-switched state and lost all color after it passed its transition temperature (31±3°C). The second dye appeared orange below its transition temperature (47±3°C) and switched to a yellow color above this temperature. Upon further inspection of the orange dye, it was observed that the orange color was engineered using a mixture of two types of beads: 1) a yellow, non-switchable bead and 2) a red thermochromic bead. When the transition temperature is surpassed, the red beads lose their color, and the yellow beads remain constant. Thus, the orange (red bead + yellow bead mixture) TCD changes to a yellow (transparent bead + yellow bead mixture) color after heating. For experiments on the microscopic scale, the red beads were separated from the yellow beads via centrifugation and used alone to study only the thermochromic nature of the system, unless otherwise stated.

The MNPs were purchased from Sigma-Aldrich (St. Louis, MO) and came in a 5 mg/mL dispersion of 20 nm (average) iron oxide (II, II) nanoparticles. MNPs were physically mixed with TCDs in a 4:1 ratio in an Eppendorf tube, creating the TCD-NP probes. These probes were then tested for photoswitchable properties on both the macro and micro level.


Heating and spectroscopy

The TCD beads and MNPs were tested for inherent color change using UV-VIS spectroscopy (UV-3600, Shimadzu) and a heated water bath. Samples of different concentrations of green and orange TCD alone in Eppendorf tubes were placed in a water bath and observed below and above the transition temperature. Samples of MNPs were similarly tested in the same manner to ensure that color change was from the TCDs alone.

To test the photoswitching abilities of the TCD-NP probes in bulk solution (i.e., on the macro scale), samples of each TCD-NP probe were placed in a cuvette located under a NIR Diomed laser (Dotmed World) with a laser wavelength of 805 nm. The laser was positioned 10 mm above the sample and set to deliver a continuous wave output of 15 W. An Ultrospec 3300 pro UV/Visible spectrometer (Amersham Biosciences) was used to measure the absorption of the samples as they were heated and cooled using the Diomed laser. Both and IR thermometer (PTM0.1) and an IR FLIR camera (FLIR systems, Thermovision A40M) were used to record the temperature of the sample during the cooling periods. Each sample was irradiated, and thereby heated, with the laser for three minutes and allowed to cool for two to three minutes. IR images were taken every ten seconds during this period. Kinetics were obtained by heating the sample via laser for 85 seconds (intervals of one second of laser illumination, one second laser off) and recording absorption spectra at various temperatures as the sample cooled.

To test photoswitching at the single bead level (i.e., microscopic level), TCD-NP probes were placed on microscope slides for use on an Olympus microscope (IX81), also used for bright-field imaging. Absorption spectra was recorded using an Ocean Optics USB4000 spectrometer, which measured absorption spectra in a specific area of the field of view given by the microscope. Samples were illuminated with the same 805 nm Diomed laser as in bulk experiments (beam fed through the microscope optics) at various intensities ranging from ~0.25W to ~2W.


Cell culture and imaging

MDA-MB-231 (ATCC HTB-26) adenocarcinoma cells and EA.hy926 (ATCC CRL-2922) epithelial cells were cultured in DMEM and tested to demonstrate that live cells could take up the TCD. Each cell line was cultured in Nunc Lab-TEK II Chamber Slide Systems, so that cells would grow on the slides that would later be used for bright-field and photoacoustic imaging. Cells were allowed to grow until confluency, and then incubated with TCD, MNPs or TCD-NP probes overnight. After incubation, the slides were washed 3-5 times and filled with PBS to prepare for imaging. Optical images were obtained using a scattering (dark-field) technique where light was shown at an angle, not directly hitting the detector, so that all light detected was scattered light.


Photoacoustic imaging

Photoacoustic imaging of the cells incubated with TCD, MNPs or TCD-NP probes was carried out in vivo using a custom laser scanning photoacoustic microscope based on an Olympus IX81 inverted microscope platform. Galvo mirrors (6215H, Cambridge Technologies, Lexington, MA) steered 532 nm and 820 nm laser beams (10 kHz pulse repetition rate) coupled to the microscope using single mode optical fibers. The imaging area was limited (150 μm) due to thefocal area of the transducer (V316, 20 MHz, 12 m focal distance, Olympus-NDT Inc.). Signals from transducer were amplified (5662B, Panametrics) and recorded using a high-speed digitizer (PCI-5124, 12-bit card, 128 MB of memory, National Instruments, Austin, TX). A digital waveform generator (DG4062, Rigol, Beijun, China) gave control over the mirrors and synchronization of the system. A custom LabView program was then used to translate signals generated from this scanning into a photoacoustic image.



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© (2017) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.
Walter Harrington, Mwafaq R. Haji, Ekaterina I. Galanzha, Dmitry A. Nedosekin, Zeid A. Nima, Fumiya Watanabe, Anindya Ghosh, Alexandru S. Biris, Vladimir P. Zharov, "Photoswitchable dye-nanoparticle probes with photothermal switching of light-dark states and colors (Withdrawal Notice)", Proc. SPIE 10079, Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications IX, 100790D (21 February 2017); doi: 10.1117/12.2254991; https://doi.org/10.1117/12.2254991

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