Abstract
Phase change nanoemulsions have been proposed as theranostic agents, using light to induce vaporization into bubbles (also called optical droplet vaporization). The current work uses perfluorohexane nanoemulsions (PFH-NEs) stabilized by a highly biocompatible and optically absorbing fluorosurfactant shell. Once vaporized, the bubbles can be used for contrast enhanced ultrasound (CEUS) imaging but also to enhance photoacoustic (PA) signals due to the presence of bubbles and optical absorbing shell material. The formation and expansion of these gas filled bubbles leads to increasing photoacoustic signals for imaging. Compared to other contrast agents which may not give stable signals due to photo-degradation, these contrast agents are shown to be stable up to 24 hours. The source of PA signal enhancement is through the presence of long lasting perfluorohexane (PFH) bubbles resulting from the optical vaporization. These bubbles generated from the PFH-NEs directly generate photoacoustic signals due to the optical absorption from the fluorosurfactant shell, but also secondary signals from the subsequent scattering of the photoacoustic waves from the PFH bubbles. In addition, the pressures generated from vaporization of NEs and ability to load chemotherapeutic agents enable these nanoparticles to also be used for cancer therapy by contributing to drug delivery and transport.
© 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Perfluorocarbon (PFC) nanoemulsions (NEs) have been extensively used in the biomedical field for therapy and imaging. These type of nanoparticles have been used in various imaging modalities such as MRI, photoacoustic imaging and ultrasound. For example in MRI, PFC NEs have been used for tracking, detecting and quantifying cells for determining severity of inflammation [1]. Because of the extremely low concentration of naturally occurring fluorine atoms in the body, 19F MRI imaging shows high specificity to labelled cells only (due to negligible background signal from tissue). In ultrasound imaging, PFC nanodroplets are used for extravascular imaging of cancer [2] through ultrasound mediated vaporization into PFC bubbles. For photoacoustic (PA) imaging, PFC nanodroplets have been synthesized and loaded with organic semiconductors and photosensitizers to both trigger vaporization for enhancing the PA signal and deliver cytotoxic oxygen for therapy [3].
PFC NEs are also important therapeutic carriers for the treatment of disease. For example, the ability of perfluorocarbons to carry oxygen makes PFC nanodroplets important oxygen carriers for treatment of hypoxic tumors [4]. The PFC NEs can also carry therapeutic agents such as antigens for targeting dendritic cells to improve immune response after vaccination [5], as well as chemotherapeutics for both tumor growth regression and imaging (i.e., theranostics) [6]. However, taking into consideration the major advantages of using PFC nanoemulsions, there still remain several challenges in creating very stable imaging agents required for providing sufficient contrast for long term imaging of tumor growth/regression during therapy. In addition, common therapeutic and imaging agents rely on the use of toxic components (e.g. organic and inorganic components making quantum dots, titanium oxide in nanoparticles) for enhancing contrast and/or therapeutic outcome in vitro and in vivo [7]. Therefore, there remains a need for highly biocompatible and physically stable contrast agents for effective theranostics.
We have previously shown that these agents form stable bubbles that can be imaged with contrast enhanced ultrasound. Here we show how they can be used to amplify the signals generated during photoacoustic imaging and imaged with cells giving significant signals in tissue mimicking phantoms which mimick the optical and acoustic properties of tissue. The current work presents perfluorohexane nanoemulsions (PFH-NEs) that are highly biocompatible, with the optically absorbing fluorosurfactant shell used to generate photoacoustic (PA) signals through vaporization and the formation of stable bubbles. The PA signal enhancement is through the presence of long lasting perfluorohexane (PFH) bubbles which generate strong scatterers that scatter the photoacoustic signals generated from the light absorption of the PFH-NEs and PFH bubbles. The PFH bubbles generated in vitro can last at least for 24 hours at physiological conditions and are one of the most stable contrast agents [8]. In addition to being stable imaging agents, these PFH-NEs can be used for therapy of cancer cells through light induced vaporization into PFH bubbles. Cell death can be achieved either through the mechanical disruption resulting from the vaporization of PFH-NEs, or through the loading of PFH-NEs with chemotherapeutic agents (i.e., doxorubicin).
2. Materials and methods
2.1. Synthesis of fluorosurfactant coated PFH-NEs
Perfluorohexane emulsions of nanometer size (PFH-NEs) were synthesized by first making PFH microemulsions. Volumes of 600 µL perfluorohexane (PFH) (1100-2-07, Synquest Laboratories), 150 µL of Zonyl FSP fluorosurfactant (with anionic phosphate group) (09988, Sigma-Aldrich) and 4250 µL of Milli-Q water were mixed for 1 minute under vortexing at 2,700 revolutions per minute (rpm). To make PFH-NEs, ultrasonication at 20 kHz was used (11 Watts, 2 minutes total time, 10 seconds on/ 20 seconds off cycles on ice). Previous experiments confirmed the size of PFH-NEs to be monodisperse and below 100 nm using dynamic light scattering (DLS) with size reported from the mean ± standard deviation from the Gaussian fitted curve of the size distribution from three replicates.
2.2. Absorption spectroscopy and transmission electron microscopy
The absorption coefficients (in units of cm−1) of PFH-NEs were determined using a Shimadzu UV-3600 UV-VIS-NIR spectrophotometer with medium scan speed, single scan mode, 8 nm slit width and 1 nm sampling interval. The NEs (10 mg/mL) were placed in a 2 mm quartz cuvette (Starna Cells Inc., USA) (45 mm × 4.5 mm × 12.5 mm) and placed in an integrating sphere sample port. The double beam integrating sphere technique was used to determine the total transmittance and diffuse reflectance of sample. Using the transmittance and reflectance of samples the optical absorption was determined using the inverse adding doubling software program (developed by Prahl, 2011). The absorption coefficients of a well-known biological absorber (mainly red blood cells in whole blood) were measured for comparison with the PFH-NEs. The use of blood for experiments was approved for research use from the Canadian Blood Services. To characterize the PFH-NEs a JEOL JEM-1200 electron microscope (JEOL Ltd., Tokyo, Japan) was used at a beam energy of 80 kV. The PFH-NEs were imaged after placing a very dilute suspension of NEs on a carbon grid.
2.3. PA and US imaging
PFH-NEs were imaged after vaporization in rectangular inclusions (channels) using a Vevo LAZR (FUJIFILM VisualSonics Inc.) commercial imaging system. Tissue mimicking phantoms were made by using 2% v/v formaldehyde (252549, Sigma-Aldrich) and 10% w/v gelatin (Type A, 250 Bloom, G2500, Sigma-Aldrich) to represent the mechanical properties of soft tissue [9]. Channels for placing nanoparticles were made using ∼ 1 mm thick metal rods placed through the gelatin phantoms and removed after cooling and solidification of the gelatin.
To image MCF-7 breast cancer cells with PFH-NEs at non-cytotoxic concentrations (viability > 90% after 48 hours cell incubation using trypan blue viability test), cells were first grown for 24 hours at an initial concentration of 125,000 cells/mL in Dulbecco’s Modified Eagle Media (DMEM) (comprising 4500 mg glucose/L, L-glutamine, NaHCO3, and sodium pyruvate with 10% fetal bovine serum) before being incubated next day with 10 mg/mL PFH-NEs for an additional 4, 24 or 48 hours. All cells used for experiments were grown in an incubator at 37 °C and 5% CO2. Cells were then trypsinized using 0.05% Trypsin-EDTA after washing cells three times (1 mL each time) with phosphate buffered saline (PBS) and mixed with 10% w/v gelatin with 2% v/v formaldehyde. Inclusions were then made by placing drops of the mixture on top of a base layer with the same concentration of gelatin and formaldehyde. To make tissue mimicking layers with optical and acoustic properties of tissue [10–13], 0.1 g/mL of type A gelatin (G2500, Sigma-Aldrich), 0.67 mg/mL human hemoglobin (H7379, Sigma-Aldrich) and 7 mg/mL from 20% intralipid (I141, Sigma-Aldrich) were mixed and placed on inclusions (after 24 hours incubation of nanoparticles with cells at a non-cytotoxic concentration of 125 mg/mL with viability of 90.2 ± 2.3% using trypan blue viability test). Inclusions were then imaged at 37°C using Vevo LAZR commercial imaging system, using a Nd:YAG laser with a laser fluence of 20 mJ/cm2 at 700 nm, repetition rate of 20 Hz and pulse duration of 4–6 ns. To acquire photoacoustic (PA) and ultrasound (US) signals from inclusions, a 21 MHz transducer with both laser excitation and ultrasound emission capabilities was used. A gain of 70 dB for photoacoustic imaging and 2D gain of 55 and 35 dB were used for ultrasound imaging of PFH-NEs alone and PFH-NEs with MCF-7 cells (to avoid signal saturation), respectively. As a control, MCF-7 cells were incubated with exact experimental procedure in gelatin inclusions as above but without PFH-NEs.
To determine the long term stability of PFH-NEs and PFH bubbles, the same procedure was used as for making inclusions involving cells. The PFH-NEs at a concentration of 20 mg/mL were imaged in gelatin inclusions at day 0 (which is the initial day inclusions were made) and day 1 or 24 hours after incubation of inclusions at physiological temperature (37°C) in a water bath. Each inclusion with particles were excited for 10 seconds at 700 nm (using the PA imaging sequence) at each day using the Vevo LAZR commercial system. The stability of PA and US signals were determined by acquiring signals using the same US and PA acquisition settings for comparison of signals between day 0 and 1. The averages reported for PA and US signals for all experiments are the mean ± standard deviation from three independent replicates. These averages were calculated from the gray scale values enclosed within a 3 mm x 1 mm region within the center of the inclusions.
To compare the stability of photoacoustic signals from PFH-NEs and PFH bubbles in cells to a reference, DiR (1,1’-Dioctadecyl-3,3,3’,3’-Tetramethylindotricarbocyanine Iodide) (D12731, Thermo Fisher Scientific), an optical absorber was used to label MCF-7 cells using 2.5 µg/mL of solution (using protocol by Invitrogen). Cells were labelled for 10 minutes prior to washing and removing free dye using phosphate buffered saline (PBS). The labelled cells were then used to make inclusions as mentioned above and excited at 700 nm wavelength using the Vevo LAZR using the same image acquisition settings for comparison. Signals for all experiments were analyzed from images directly after laser excitation at frame 1 for US and PA images for better comparison.
2.4. Optical imaging
A light microscope was used to determine vaporization of PFH-NEs into PFH bubbles after laser excitation at 700 nm using the Vevo LAZR imaging system. To determine the viability of MCF-7 cells after vaporization of PFH-NEs into bubbles, 100 µL of a 100 mg/mL solution of PFH-NEs was mixed with cancer cells (926,000 MCF-7 cells) prior to vaporization at 700 nm for 10 minutes. This was followed with another 100 µL volume of the same concentration of PFH-NEs with cells and another 10 min interval of 700 nm excitation (using Vevo LAZR) after mixing the cells with nanoparticles. MCF-7 cells were then mixed with propidium iodide (P3566, Thermo Fisher Scientific) and imaged using fluorescence microscopy (ZOE fluorescent imager) with an excitation of 556/20 nm and emission filter of 615/61 nm to determine non-viable cancer cells.
PFH-NEs were loaded with doxorubicin (DOX) (LC Laboratories, D-4000) by mixing an aqueous solution of the fluorescent drug with 150 µL Zonyl FSP followed by adding required amount of PFH and Milli-Q water (using the exact synthesis method mentioned in section “Synthesis of fluorosurfactant coated PFH-NEs”). The drug loaded emulsions were then sonicated at the above mentioned settings. The resulting nanoparticle solution was then washed to remove free DOX using 15 mL 10 kDa centrifugal filters (UFC901024, Millipore, Sigma-Aldrich) (1,000xg, 5 minutes each for 8 spins) before incubation with MCF-7 cells for 24 hours (at a doxorubicin concentration of 30 µM). Percent encapsulation for DOX was greater than 95% by determining absorption of known concentrations of DOX drug and comparing with initial amount of DOX used for loading. A ZOE fluorescent imager (Bio-Rad) was used to determine localization of DOX loaded NEs with MCF-7 cells using excitation of 480/17 nm and emission filter of 517/23 nm.
2.5. Cell viability
Trypan blue viability dye was used to quantify the amount of cell death after vaporization and treatment with DOX loaded PFH-NEs using the Vi-Cell XR Cell Viability Analyzer (Beckman Coulter). Viability results reported are those from treatment of MCF-7 cells with PFH-NEs and 700 nm light (using Vevo LAZR) for two intervals of 10 minutes each (at final concentration of 10 mg/mL of PFH-NEs, see above section 2.4. Optical imaging for details) or from treatment of cells with 7 µM DOX loaded in PFH-NEs after 24 hours incubation. For controls, viability from cells with PFH-NEs only and cells treated with laser only (700 nm using Vevo LAZR for two 10 minute intervals) were determined. The amount of MCF-7 cells initially used for each type of treatment was 926,640 cells for determining therapeutic outcome.
3. Results and discussion
3.1. Characterization of PFH-NEs and bubbles
PFH-NEs were synthesized to have an average size below 100 nm using a concentration of 12% v/v PFH and 3% Zonyl FSP (anionic fluorosurfactant). Sonication of PFH emulsions resulted in a monodisperse size distribution size, with average size of 61 ± 6 nm for nanoparticles determined using dynamic light scattering (DLS) [14]. Transmission electron microscopy (TEM) images of PFH-NEs showed that nanoemulsions had a spherical morphology with particles clearly distinguishable due the high electron dense perfluorohexane core of particles (Fig. 1(a)).
To determine the potential of PFH-NEs as photoacoustic contrast agents the absorption extinction coefficients of PFH-NEs were determined in the ultraviolet-near-infrared (UV-NIR) region (Fig. 1(b)). PFH-NEs show strong absorption in the NIR region, which can be advantageous in vivo to give higher signals (relative to background tissue signals) for tumor monitoring compared to the very weak NIR endogenous absorption from chromophores (i.e., lipids, collagen, proteins) and cells [15]. Because perfluorohexane shows very weak absorption in the UV-Vis-NIR region measured for NEs (Fig. 8 in the Appendix), the likely source of absorption is from the Zonyl FSP fluorosurfactant shell surrounding particles. The absorption in the NIR region from PFH-NEs are similar to other photoacoustic (PA) contrast agents (i.e., titanium particles) used in vivo [16]. Compared to a well know endogenous chromophore (i.e., red blood cells in whole blood) the absorption coefficient from PFH-NEs (using 10 mg/mL solution of nanoparticles) is ∼1.5 times greater when comparing the peak absorptions between the two optical agents in the NIR region (700–850 nm) (Fig. 1(b) and 1(c)). This suggests that PFH-NEs can be used within the 700–850 nm NIR optical window to enhance the signals from tumor targeted NEs relative to the weaker endogenous chromophores present in background tissue (i.e., from cells and connective tissue) for effective tumor monitoring during treatment. Thus absorption based imaging techniques such as photoacoustic imaging can be used to take advantage of the highly absorbing PFH-NEs, which is a modality which provides greater sensitivity and deeper penetration in tissue for imaging compared to other modalities (i.e., optical coherence tomography (OCT), optical imaging) [17].
Because the core of NEs is made of volatile perfluorohexane, the PFH-NEs can convert into PFH bubbles (Fig. 1(d) and 1(e)) upon laser excitation [18,19]. After laser excitation at 700 nm using the Vevo LAZR, microbubbles (Fig. 1(e)) as well as nanobubbles were detected with average sizes of 6 ± 2 µm and 236 ± 83 nm, respectively. While the average size of microbubbles was determined using optical microscopy, nanobubbles were sized using Archimedes particle metrology system which is able to distinguish between positively buoyant bubbles from negatively buoyant nanoparticles [14,20]. The laser induced expansion of PFH-NEs into PFH bubbles not only increases the PA signals from volume expansion of particles but also has the potential to increase cancer cell death from the local stress generated from vaporization.
3.2. PA and US imaging after vaporization of PFH-NEs
Strong PA and US signals were detected after vaporization of PFH-NEs (Fig. 2(a) and (b)) at 700 nm using the Vevo LAZR imaging system. Ultrasound and photoacoustic signals both increased with concentration of PFH-NEs (Fig. 2(c) and 2(d)) due to the increase in the number of PFH bubbles formed from vaporization (with no significant signals seen when PFH-NEs are not present, Fig. 9 in the Appendix). To determine the stability of PFH-NEs at physiological conditions (i.e., at 37°C) PFH-NEs were incubated for 4, 24 and 48 hours with cancer cells prior to being imaged in inclusions (Fig. 3(a) and 3(b)). Both US and PA signals from vaporized PFH-NEs were stable for up to 48 hours with cells suggesting high stability of NEs at 37°C and physiological saline conditions. US signals did not significantly decrease with time, with average signals of 152.98 ± 4.85, 171.27 ± 15.67, 153.07 ± 8.65 after 4, 24 and 48 hours, respectively (Fig. 3(c)). PA signals on the other hand increased with average signals of 34.73 ± 10.44, 41.56 ± 4.40, 43.16 ± 6.33 after 4, 24 and 48 hours, respectively (Fig. 3(d)). These results show the potential use of these PA contrast agents in vivo for cancer imaging, giving enhanced US and PA signals compared to cancer cells only (Fig. 10 in the Appendix). Compared to MCF-7 cells only, PA signals from PFH-NEs were more than 50 times greater with US signals being more than 2.5 times greater from the vaporized NEs (supplementary Fig. 10(c)–10(f)).
Because signals in tissue can be dominated by endogenous absorbers (i.e., red blood cells, proteins, melanin) [15], US and PA signals were measured using MCF-7 cells loaded with PFH-NEs relative to background signals from tissue-mimicking phantoms. PA signals from vaporized PFH-NEs were more than 4 times stronger compared to background signals from tissue-mimicking layer (Fig. 4(c) and 4(e)) suggesting the potential of PFH-NEs to give greater signals and contrast for effective tumor monitoring during therapy. In addition to the PFH-NEs being stable, the ability of the resulting vaporized PFH bubbles to be used to enhance the photoacoustic signals was studied. As can be seen in Fig. 5, PA signals are enhanced after 24 hours due to the presence of stable PFH bubbles which scatter the PA signals and can be used as “acoustic amplifiers” to increase photoacoustic signal strengths. Due to the acoustic scattering properties of gas filled bubbles, even weak PA signals can be enhanced by the larger acoustic scattering cross sections of the generated PFH bubbles. Since the US signals do not significantly change after 24 hours (Fig. 5(a), 5(c) and 5(e)), even a small increase in the amount of PFH bubbles can be used to enhance PA signals more than 10 times (Fig. 5(b), 5(d) and 5(f)) (see Fig. 6 for how PFH bubbles were used for acoustic signal amplification). These results highlight the potential ability of PFH-NEs to be used for long term tumor monitoring, compared to other biomedical optical contrast agents (i.e., DiR) [21–23] which have reduced PA signals within few seconds after laser excitation (Appendix Fig. 11) possibly due to photobleaching. Moreover, since it is hypothesized that most of the bubble vaporization will occur in tumor tissues rather than within blood vessels (due to NP accumulation via the Enhanced Permeability and Retention (EPR) effect), the bubbles will be locally entrapped in the tumor cells or extracellular matrix.
3.3. PFH-NEs as cancer therapeutic agents
To determine the potential of PFH-NEs for cancer therapy, MCF-7 cells were mixed with PFH-NEs before vaporization at 700 nm using the Vevo LAZR system. As can be seen from Fig. 7(a) and 7(b), vaporization of NEs led to significant damage to most of the cancer cells. The non-viable cancer cells have irregular morphology perhaps due to severe damage to cell membranes from pressures generated from the expansion of nanoscale NEs into much bigger microbubbles. Viability of MCF-7 cells after treatment with PFH-NEs and laser was 75 ± 7% compared to viability of 95.0 ± 0.6% and 96.4 ± 0.2% for cells treated with PFH-NEs only and with laser only [14]. It is possible that such severe damage from vaporization of NEs can prevent cancer cells from replicating. To determine the ability of PFH-NEs to deliver conventional chemotherapeutic agents, doxorubicin was loaded in particles. After 24 hours incubation of DOX loaded NEs with cancer cells, the NEs have completely localized with cells (Fig. 6(c) and 6(d)). Viability of MCF-7 cells after 24 hours treatment with 7 µM DOX loaded in PFH-NEs was 52 ± 1% (compared to 99 ± 1% when treated with blank PFH-NEs) [20]. This might be due to enhanced ability of these nanoparticles to internalize in cancer cells, serving as a promising therapeutic agents for cancer therapy.
4. Conclusions
Perfluorohexane nanoemulsions (PFH-NEs) serve as promising imaging and therapeutic agents. These contrast agents are able to be stable at physiological conditions for much longer times. Compared to common endogenous absorbers (i.e., blood), photoacoustic signals from PFH-NEs can be comparable, if not greater, in the near-infrared region, which can be used for giving greater contrast relative to background signals from tissue in vivo even in the absence of the use of specialized spectroscopic techniques for chromophore identification. The ability of stable PFH bubbles formed from the vaporization of NEs to enhance photoacoustic signals is unique with the addition of being able to damage cancer cells through vaporization and potentially drug delivery. Future work will involve the use of these theranostic agents in vivo in animal models to answer questions such as whether nanoemulsion optical droplet vaporization can be achieved in vivo and what is the lifetime of the formed microbubbles in vivo.
5. Author contributions
D.A.F. designed and carried out experiments as well as analyzed data and wrote the paper. M.C.K. supervised the work. All authors discussed the results and contributed to the final manuscript.
Appendix
Supplementary Figures
Funding
Canadian Institutes of Health Research; Canada Foundation for Innovation.
Acknowledgements
This work was supported by grants from Canadian Institutes of Health Research (CIHR) and CFI (Canada Foundation for Innovation) to M.C.K. TEM experiments were performed at the Nanoscale Biomedical Imaging Facility, user facility operated by SickKids.
Disclosures
The authors declare no conflicts of interest.
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