Synthesis of Stable Multifunctional Perfluorocarbon Nanoemulsions

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SYNTHESIS OF STABLE MULTIFUNCTIONAL PERFLUOROCARBON NANOEMULSIONS FOR CANCER THERAPY AND IMAGING Donald Anthony Fernandes, Dennis Denzil Fernandes, Yuchong Li, Yan Wang, Zhenfu Zhang, Dérick Rousseau, Claudiu C Gradinaru, and Michael C. Kolios Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b01867 • Publication Date (Web): 26 Aug 2016 Downloaded from http://pubs.acs.org on September 7, 2016

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Scheme 1. Synthesis of PFH nanoemulsions. Schematic of three step process used to synthesize PFH nanoemulsions with diameter below 100 nm. 42x16mm (300 x 300 DPI)

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Scheme 2. Schematic of PFH-NE constructs. Illustrations of synthesized (a) unlabelled PFH-NEs, (b) PFH-NEs with inserted biotin-linker and encapsulated DiI-18C, (c) BODIPY500/510 labelled PFH-NEs. Repeating units of Zonyl-FSP not shown for simplicity. 58x27mm (300 x 300 DPI)


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Scheme 3. Functionalization of silica-coated gold nanoparticles (scAuNPs). Schematic of the step by step functionalization of silica-coated AuNPs with hydroxyl termination (grey-colour coded coating) with (3Mercaptopropyl)-trimethoxysilane (3-MPTMS), 1H,1H,2H,2H-perfluorodecyltriethoxysilane (PFDTES) and Texas Red (TR) maleimide. 67x26mm (300 x 300 DPI)


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Figure 1. Size and stability from PFH nanoemulsion synthesis. (a) Averaged number-weighted DLS distributions of nanoemulsions (three replicates each) at day 1 made of 2% PFH/1% FSP (blue), 12% PFH/3%FSP (black), and 9% PFH/1%FSP (red, bimodal size distribution). (b) 12% PFH/3% FSP nanoemulsions in 0% (blue) and 0.9% w/v (150 mM) NaCl (green) both at day 30 (three replicates each) . (c) Zeta potential distribution at day zero and (d) over the course of 225 days for 12% PFH/3% FSP nanoemulsions. Yellow star over 180 days in (d) indicates the day an aliquot of PFH and Zonyl FSP were used to make nanoemulsions labelled with DiI-18C for TIRF imaging and Fluorescence Correlation Spectroscopy (FCS). Error bars represent standard deviation between three replicate measurements. Results are summarized in Table 1. 69x56mm (300 x 300 DPI)


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Figure 2. PFH nanoemulsions as colloidally stable biological theranostic agents. (a) TEM image of nanoemulsions made from 12% v/v Perfluorohexane (PFH), 3% v/v Zonyl FSP fluorosurfactant (FSP) (0% NaCl, day 1) (scale bar: 200 nm). (b) Multicomponent Gaussian fitting of the size distribution of PFH nanoemulsions measured by TEM. (c) TIRF image (scale bar: 5 µm) of nanoemulsions with the PFH core containing the fluorescent dye DiI-18C. (d) FCS data of PFH nanoemulsions encapsulating DiI-18C (red circles) along with the hydrodynamic diameter (DH = 54 ± 4 nm) calculated from equation (1) by fitting the data to Supplementary equation (S2) (black line). Fitting parameters of TEM and FCS data are given in Table 2. 85x77mm (300 x 300 DPI)


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Figure 3. Co-localization & co-diffusion of PFH-NEs and scAuNPs. TEM images of PFH (12% v/v) nanoemulsions with unfluorinated (a) and fluorinated (c) scAuNPs (scale bar: 200 nm). Dual-color FCCS data (symbols) and fitting according to Supplementary equation (S4) (black lines) for unfluorinated (b) and fluorinated (d) scAuNP-PFH-NE tandems. Inserts in (a) and (c) are zoomed-in regions of interest, with scale bars of 200 nm. Parameters of FCCS fitted curves are given in Table 3. 85x76mm (300 x 300 DPI)


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Figure 4. Photoacoustic and ultrasound imaging of PFH-NEs and scAuNPs. (a) Schematic of VEVO LAZR system setup for photoacoustic and ultrasound imaging of scAuNPs and PFH-NEs. (b) Photoacoustic and (d) ultrasound images of scAuNP-PFH-NEs after excitation with 680 nm radiation and 21 MHz ultrasound frequency, respectively. Color-coded intensity bars in (b, d) are in units of decibels (dB). Signal amplitudes from simultaneous (c) photoacoustic and (e) ultrasound imaging as function of depth within the ~1mm microchannel inclusion loaded with scAuNP-PFH-NEs as indicated by the white arrows in (b, d). Signals for photoacoustic and ultrasound imaging in (c, e) are from a representative single RF line, whereas reported means are from at least 25 RF lines per replicate.

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Figure 5. Vaporization of the PFH-NE-scAuNP tandem. Representative time series images (optical phase contrast microscopy) of vaporisation of PFH nanoemulsions made from 12% Perfluorohexane (v/v) and 3% Zonyl FSP (v/v) with 5 nm (a) unfluorinated and (b) fluorinated scAuNPs, upon exposure to 532 nm radiation. Scale bar is 10 µm and elapsed time in (a) and (b) is ~30 sec. (c) Representative image showing cluster formation and expansion of microbubbles after exposure to 532 nm radiation with 330 picosecond (ps) pulse width and 4 kHz repetition rate, scale bar 15 µm (See Supplementary Video 1). (d) Shows the distribution of droplet expansion into microbubbles before imploding, fit to the Generalized Extreme Value (GEV) model according to Supplementary equation (S10). (e) Optical images (brightfield) of MCF-7 cells with internalised PFH-NEs, (f) unfluorinated PFH-scAuNP-NEs, and (g) fluorinated PFH-scAuNP-NEs after 6 hours incubation at 37oC in the absence of pulsed-laser radiation. White arrows in (f, g) show PFH microbubbles. 170x91mm (300 x 300 DPI)


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Figure 6. Cellular internalisation of PFH-NE-scAuNP tandem in MCF-7 cells. TIRF images of BODIPY labelled PFH-NEs (a, d), Texas-red labelled scAuNPs (b, e), and overlays from unfluorinated (c) and fluorinated (f) samples after 2 hours incubation. White arrows in (a, b) indicate the formation of PFH- microbubbles. Fluorescence confocal images of BODIPY labelled PFH-NEs (g, k), Texas-red labelled scAuNPs (h, l), DiRmembrane-labelled MCF-7 cells (i, m) and corresponding overlays for unfluorinated (j) and fluorinated (n) samples after 6-hour incubation (See Supplementary Video 2). Scale bar: 10 µm. 85x82mm (300 x 300 DPI)


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Graphical Table of Contents. Mechanism for scAuNP-PFH-NEs drug-delivery via cellular internalization through the endothelial lining of carcinogenic tissue (endocytosis) and exposure to non-ionizing radiation. 39x24mm (300 x 300 DPI)


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Figure S4. Control samples for FCCS experiments. (a) Negative control: an equimolar mixture (10 nM each) of Texas Red and BODIPY; data (symbols) and fit according to Supplementary equation S4 (black lines); (b) Positive control: 40 base pairs (bp) dsDNA labelled at opposite ends with Texas Red and Fluorescein, which is spectrally similar to BODIPY. 113x51mm (300 x 300 DPI)


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Figure S5. Photoacoustic and ultrasound imaging of fluorinated PFH-NEs and scAuNPs. Simultaneous (a) photoacoustic and (c) ultrasound control images of the 1 mm microchannel filled with Milli-Q water after exposure to 680 nm radiation and 21 MHz ultrasound frequency, respectively. Signal amplitudes from (b) photoacoustic and (d) ultrasound images as function of depth. Simultaneous (e) photoacoustic and (g) ultrasound images of fluorinated scAuNP-PFH-NEs after exposure to 680 nm radiation and 21 MHz ultrasound frequency, respectively. Signal amplitudes from (f) photoacoustic and (h) ultrasound images as function of depth within the ~1mm microchannel loaded with fluorinated scAuNP-PFH-NEs as shown by the white arrows in (e, g). Color-coded intensity bars in (a, c, e, g) are in units of decibels (dB). Signals for photoacoustic and ultrasound imaging in (b, d, f, h) are from a single representative RF line whereas reported means are from at least 25 RF lines per replicate. 175x77mm (300 x 300 DPI)


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Figure S8. MCF-7 cellular internalisation control images. Confocal images of unlabelled MCF-7 cells using the same excitation wavelengths and emission filters as indicated in the main text for the (a) BODIPY channel, (b) Texas-red channel and (c) DiR channel. 114x38mm (300 x 300 DPI)


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TITLE: SYNTHESIS OF STABLE MULTIFUNCTIONAL PERFLUOROCARBON NANOEMULSIONS FOR CANCER THERAPY AND IMAGING

AUTHORS: Donald A. Fernandes , Dennis D. Fernandes2, 3, ‡, Yuchong Li2, 3, Yan Wang4, Zhenfu Zhang2, 3, Dérick Rousseau1, Claudiu C. Gradinaru2, 3,*, and Michael C. Kolios1,4, * 1, ‡

AUTHOR AFFILIATIONS: Department of Chemistry & Biology, Ryerson University, 2 Department of Physics, University of Toronto, 3 Department of Chemical and Physical Sciences, University of Toronto Mississauga, 4Department of Physics, Ryerson University. *Authors to whom correspondence may be addressed. ‡ These authors made equal contributions to this work. 1

CORRESPONDENCE: Michael C. Kolios Address: Department of Physics, Ryerson University, Toronto, 350 Victoria Street Toronto, Ontario M5B 2K3, Ontario, Canada E-mail: [email protected] • Phone: +1 (416) 979-5000 x3157 • Fax: +1 (416) 979-5343 Claudiu C. Gradinaru Address: Department of Chemical Physical Sciences, University of Toronto, Mississauga, 3359 Mississauga Road North, Mississauga, L5L 1C6, Ontario Canada. E-mail: [email protected]. • Phone: +1 (905) 828-3833 • Fax: (905) 828-5425.

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ABSTRACT Nanotechnology provides a promising platform for drug-delivery in medicine. Nanostructured materials can be designed with desired superparamagnetic or fluorescent properties in conjunction with biochemically functionalized moieties (i.e., antibodies, peptides and small molecules) to actively bind to target sites. These multifunctional properties make them suitable agents for multimodal imaging, diagnosis and therapy. Perfluorohexane nanoemulsions (PFH-NEs) are novel drug-delivery vehicles and contrast agents for ultrasound and photoacoustic imaging of cancer in vivo, offering higher spatial resolution and deeper penetration of tissue when compared to conventional optical techniques. Compared to other theranostic agents, our PFH-NEs are one the smallest of their kind (< 100 nm), exhibit minimal aggregation, long-term stability at physiological conditions, and provide a non-invasive cancer imaging and therapy alternative for patients. Here, we show using high-resolution imaging and correlative techniques that our PFH-NEs when in tandem with silica-coated gold nanoparticles (scAuNPs), can be used as a drug-loaded therapeutic via endocytosis, and as a multimodal imaging agent for photoacoustic, ultrasound and fluorescence imaging of tumour growth. KEY WORDS: Perfluorocarbon, perfluorohexane, nanoemulsions, photoacoustic, ultrasound, nanocarriers, theranostic and silica-coated gold nanoparticles.


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INTRODUCTION Many chemotherapeutic drugs, especially those that are hydrophobic and non-polar are inefficient due to their toxicity, poor solubility and permeability in blood vessels. Most of these drugs rely on passive targeting which has the drawback of low intratumoural drug concentrations and circulation times, yet high systemic toxicity. Recent advances in nanotechnology however, have led to the development of nanoparticles that have the ability to both passively and actively target tumours1,2,3. Their high surface-to-volume ratio enable encapsulation, adsorption or surface attachment of both hydrophilic and lipophilic drugs4,5. Nanoparticles have improved bioavailability, permeability, and retention through the porous angiogenic vessels, enhancing ligand binding efficacy to receptors expressed on cancer cells6,7. Perfluorocarbon (PFC) nanoemulsions are a class of theranostic agents that can be used for both imaging and treating tumours8,9,10,11,12. These nanoemulsions consist of an amphiphilic shell and a hydrophobic PFC core enabling water insoluble drugs to be delivered to tumour sites. PFCs have long been used in patients as oxygen carriers because they are biochemically inert and easily expelled through the circulatory system13,14. Due to their volatility, when given sufficient energy these PFC nanoemulsions can undergo a phase transition from a liquid nanodroplet to a gaseous microbubble state. The phase transition can be triggered by high pressure ultrasound waves (Acoustic Droplet Vaporization, ADV)15,16, or laser light when optical absorbers are introduced in the proximity of the PFC nanoemulsions (Optical Droplet Vaporization, ODV)17,18. These microbubbles can oscillate, grow and/or collapse in a process called cavitation causing the encapsulated or bound drug to be released19. Photoacoustic microscopy (PAM) is an imaging technique that acoustically detects optical contrast via the photoacoustic effect20. PAM has a greater imaging penetration depth (a few millimeters) as it usually uses both near infrared light excitation and ultrasound detection, compared to conventional optical microscopic techniques (i.e. confocal, multiphoton microscopy) which typically use shorter wavelengths which lead to greater tissue scattering 21,22,23. In ODV, the nanoemulsion may be used as a photoacoustic contrast agent during and after vaporization24,25. Upon phase transition, the initial droplet expansion generates a large pressure signal 3 ACS Paragon Plus Environment

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that can be used for imaging tumours18,26. The subsequent microbubbles create sufficient echo contrast for ultrasound imaging. Due to the high boiling point of perfluorocarbons and high clinical use of these contrast agents, PFC nanoemulsions can be more easily vaporized using silica-coated gold nanoparticles (scAuNPs). These nanoparticles can reduce the ultrasound pressure threshold in ADV, or contribute to optical absorption in ODV. Here, we report on a synthesis technique which is able to produce monodisperse perfluorohexane nanoemulsions (PFH-NEs), stabilized by biocompatible zonyl-FSP (fluorosurfactant) shells27. Due to their small size, these PFH-NEs can target a variety of tumour types compared to other nanoparticles which may be restricted by the pore size of the tumour vasculature for targeting tumours. Furthermore, these nanoemulsions can carry and/or encapsulate both soluble and insoluble drugs on their polar zonyl-FSP and non-polar PFH core with the potential of having a strong therapeutic effect because of their ability to target a variety of receptors on tumour cells. Another important characteristic of these PFH-NEs is their ability to vaporize at low laser fluence levels, with minor biological effects in the intervening tissue. For these reasons, PFH-NEs and scAuNPs were synthesized and characterized in terms of size, stability and ability for vaporization, which can both enhance contrast for imaging and serve as a potent form of treatment to infected regions within the tissue.

RESULTS & DISCUSSION Size and stability of PFH nanoemulsions. PFH nanoemulsions (PFH-NEs) were synthesized with Zonyl FSP to create anionic particles using ultrasonication. PFH/Zonyl FSP nanodroplet sizes were controlled by varying the respective PFH to FSP concentration ratio (Scheme 1), and were characterized using Dynamic Light Scattering (DLS). To achieve a unimodal distribution below 100 nm, three PFH to FSP concentration (%v/v) ratios were tested, 2:1, 9:1, and 12:3 (Scheme 1, Table 1). At a fixed FSP concentration of 1 % (v/v), the average droplet size decreased as the PFH concentration was reduced from 9 to 2 (%v/v) (Figure 1a), shifting from a bi-modal distribution (red) to a more mono-disperse size (blue). The concentration of 12 % (v/v) PFH and 3% (v/v) FSP was optimal for creating a unimodal size below 100 nm (Figure 1a, black, Table 1) and for 4 ACS Paragon Plus Environment

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increasing the number of vaporization events during contrast imaging while remaining well below the maximum laser fluency experimentally available (i.e., 250-500 mJ/cm2). This was the PFH and FSP concentration used in all subsequent experiments. The average size (diameter) of these particles was 50 ± 5 nm (Scheme 2a, & Table 2). The size of these droplets did not significantly change after 30 days, although the mean diameter increased slightly to 64 ± 9 nm and the size distribution became broader (Figure 1b, blue). At physiological saline (0.9 % w/v or 150 mM NaCl) the nanoemulsions remain stable and exhibit an average diameter of 53 ± 5 nm possibly due to the strong negative charge of PFH-NEs (Figure 1b, green). Transmission electron microscopy (TEM) shows that in 0.9 % w/v NaCl, some of these nanoemulsions seem to cluster in some regions (Supplementary Figure S1) but do not coalesce into larger droplets bigger than the average cut-off size in the tumour vasculature for targeting tumour cells (i.e., ~400 nm)3. Their mean zeta potential, which is related to the surface charge, was -72 ± 5 mV (Figure 1c) and was found to be very stable for more than 6 months (Figure 1d). The strong negative charge reduces flocculation and coalescence due to strong electrostatic repulsion between droplets. The TEM image (Figure 2a) show densely packed, spherical PFH-NEs with a multimodal size distribution consisting of mean sizes of 53 ± 8, 70 ± 14, and 101 ± 28 nm (Figure 2b, Table 2). The larger sizes are probably induced by surface interactions of these PFH-NEs. To test if the larger sizes are due to these surface interactions,

we

first

labelled

the

PFH

core

with

DiI-18C

(1,1’-Dioctadecyl-3,3,3’,3’-

Tetramethylindocarbocyanine, Scheme 2b) using a previously published method, in which the dye was first mixed with diethyl ether for solubilisation in PFH28. The dye-loaded PFH-NEs were immobilized on streptavidin-coated glass coverslips using a biotin-linker scheme (Scheme 2b & Supplementary Techniques, Scheme S1) and imaged using a custom built Total Internal Reflection Fluorescence (TIRF) microscope to demonstrate successful dye-encapsulation (Figure 2c) 29,30,31 . The hydrodynamic diameter (DH) of DiI-18C-PFH-NEs freely diffusing particles in phosphate buffered saline (PBS, 0.9% w/v salt) was estimated using fluorescence correlation spectroscopy (FCS) 32,33. The particle diameter estimated by fitting the FCS curve also includes a thin layer of water molecules, thus providing a more 5 ACS Paragon Plus Environment

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accurate measurement of the actual PFH-NE size in a physiological environment (Figure 2d). The hydrodynamic diameter was found to be DH = 54 ± 4 nm, very similar to DLS measurements, suggesting that the larger, broader distributed size peaks measured by TEM (Figure 2b, green and blue lines) are indeed most likely surface-perturbed artifacts. Also, the ability of DiI-18C to internalize in PFH droplets is encouraging for the ability of these nanoemulsions to encapsulate therapeutic drugs with similar hydrophobic and chemical characteristics. The insertion of the biotin-linker can potentially increase in-vivo signals from fluorescence and photoacoustic imaging of tissue by conjugating biotinylated nanoparticles with primary-antibodies which specifically attach to receptors on the surface of cells34. Functionalization of scAuNPs. The scAuNPs can also be functionalized to carry different therapeutic agents and fluorescent molecules using the hydroxyl group on its silica shell opening up the unique opportunity of multimodal imaging using fluorescence and photoacoustic imaging for theranostic applications (Scheme 3). The scAuNPs used in our experiments have a 5 nm diameter gold core and a 10 nm thick silica shell, allowing it to be functionalized with thiols and labelled with maleimide derivatives of fluorescent dyes (see Scheme 3 & Supplementary Methods). The absorption spectrum of these nanoparticles shows a maximum at 516 nm and the TEM data shows that their size ranges from 20 to 30 nm. The scAuNPs are negatively charged with a mean of 28 ± 3 mV and remain stable for several months (see Supplementary Methods, Figure S2). PFH-NEs with unfluorinated/fluorinated scAuNPs. PFH-NEs were mixed with unfluorinated and fluorinated silica coated gold nanoparticles to determine their spatial distribution and localization for efficient vaporization of the PFH-NEs. Unfluorinated scAuNPs were added after making nanoemulsions while fluorinated scAuNPs were added before sonication to potentially encapsulate the fluorinated nanoparticles in the PFH core of nanoemulsions. Nanoemulsions with unfluorinated scAuNPs had gold particle clusters (Figure 3a) ranging from a single-particle size (25 nm) to clusters as large as 200 nm in diameter (insert in Figure 3a). PFH nanoemulsions with fluorinated scAuNPs exhibited smaller gold particle clusters, which were localized at or near their surface. The darker regions seen in the TEM image for the fluorinated-scAuNP-PFH-NEs tandem might be caused by the highly electron dense PFH, given that fluorinated scAuNPs solubilize well in PFH 6 ACS Paragon Plus Environment

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(Figure 3c). The outer layer surrounding an individual PFH dense droplet containing fluorinated scAuNPs is more clearly seen in the zoomed-in image in Figure 3c. The formation of clusters was further probed by Fluorescence Cross-Correlation Spectroscopy (FCCS), which measured the co-diffusion of PFH-NEs and unfluorinated/fluorinated scAuNPs (Figure 3, b,d). The PFH nanoemulsions were labelled with BODIPY500/510 which has similar chemical properties as Zonyl-FSP (Scheme 2c) whereas scAuNPs were further functionalized with thiols and labelled with Texas Red (TR) maleimide (Scheme 3, & see Supplementary Methods, Figure S3). Fitting of the FCCS curves according to Supplementary equation S4 gave the fraction of co-diffusing nanoparticles (i.e., containing both fluorophores) of 74 ± 8 % and 44 ± 10 % for the unfluorinated and fluorinated samples, respectively (Figure 3b, d). Comparing these values to the negative control sample, we infer that the measured cross-correlation is indeed caused by specific interactions between the PFH nanoemulsions and scAuNPs (Supplementary Data, Figure S4). In terms of the size of the tandems, results from DLS suggest that when unfluorinated, larger clusters of PFH-NEs and scAuNPs are prevalent (DH = 160 ± 12 and 520 ± 40 nm), which is consistent with the 1component fit from FCCS (DH = 388 ± 36 nm). When fluorinated, smaller and larger PFH-NE and scAuNP clusters are formed shown by both DLS (DH = 111 ± 10 and 432 ± 39 nm), and a 2-component FCCS fitting (DH = 55 ± 18 and 230 ± 78 nm) (Table 3). The larger clusters are most likely formed due to strong electrostatic attractions between individual PFH-NEs and scAuNPs. Since the size of individual PFH-NEs and scAuNP clusters are on average less than 300 nm, these samples can be used to target a variety of cancer cells by extravasation through endothelial gaps, which are usually no smaller than 200-300 nm in size35. Since it has been shown that internalization of nanoparticles depends on size, it might be possible for scAuNPs and PFHNEs to be internalized in cancer cells through endocytosis, where PFH droplets can be vaporized and cause cell death36,37. The ability of these nanoparticles to be fluorescently labelled can provide the unique opportunity for spatial and temporal multimodal imaging of tumours using fluorescence, photoacoustics and ultrasound.


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PFH-NEs-scAuNPs for photoacoustic and ultrasound imaging. Since perfluorohexane has a high boiling point (56°C), it is difficult to directly vaporize PFH-NEs using laser irradiation. The use of silica-coated gold nanoparticles (scAuNPs) significantly reduces the vaporization threshold, transferring the required energy to nanoemulsions through non-radiative heat. Although bare gold nanoparticles can be used to vaporize PFHNEs, the silica coating has other advantages. It facilitates efficient and uniform transfer of heat to the nanoemulsions, and protects the gold core from melting under pulsed irradiation, improving quality and reproducibility of imaging38. Due to these two factors, the photoacoustic signal is greatly enhanced, enabling imaging of tissue at greater depths39. The ability of scAuNP-PFH-NEs to act as contrast agents in vivo was determined using a commercial photoacoustic and ultrasound imaging system (VEVO LAZR) with a block of gelatin-phantom as a mimic for biological tissue, as published previously40,41 (Figure 4a). The scAuNP-PFH-NEs were injected using a syringe through a microchannel ~1 mm in diameter (Figure 4b, d, white arrows). Imaging was performed under a laser fluence of ~20 mJ/cm2 and an ultrasound frequency of 21 MHz. Upon excitation with 680 nm light and a 21 MHz frequency, the scAuNP-PFH-NEs show good ultrasound and photoacoustic signal with averaged signal amplitudes of (3.07 ± 0.54) x104 (signal to noise ratio, SNR = 376 ± 59) and 9.50 ±1.14 (SNR = 29 ± 3), respectively (Figure 4b, d). For fluorinated scAuNP-PFH-NEs, the averaged signal amplitudes were (3.32 ± 0.14) x104 (SNR = 156 ± 22) and 4.31 ± 0.53 (SNR = 15 ± 2), for ultrasound and photoacoustic imaging respectively. The signals for both unfluorinated and fluorinated samples were well above blank microchannels filled with only Milli-Q water (see Supplementary Figure S5 for fluorinated samples and controls). The increases seen in the ultrasound signal (Figure 4d, e) arise due to scattering effects and acoustic impedance mismatches as a result of phase-converted PFH-NEs when compared to the coupling from the gelatin-phantom tissue42,43. The observed photoacoustic signal is due to the generation of pressure waves through the expansion of PFH-NEs as a result of heat transfer from excited scAuNPs and the signal produced from the direct excitation of the scAuNPs (Figure 4 b and c). These results suggest that when in tandem these PFH-NEs and scAuNPs can be used for enhancing contrast when monitoring tumor growth with time, and for in 8 ACS Paragon Plus Environment

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vivo tracking during drug delivery. Similar photoacoustic and ultrasound signals could also be achieved using excitation wavelengths up to 800 nm (data not shown) due to the plasmon resonance from scAuNP clusters surrounding PFH-NEs (Figure 3a, Figure S2b). Vaporization of PFH-NEs using unfluorinated/fluorinated scAuNPs. Vaporization of PFH-NE droplets was induced using a 532 nm laser source with a 330 picosecond (ps) pulse width and 4 kHz repetition rate (Figure 5, a, b). Focusing the laser beam through a 40x microscope objective resulted in a laser fluence of approximately 50 mJ/cm2 at the sample. The ability to vaporize PFH-NEs using a relatively low laser energy is caused by efficient heat transfer from proximal scAuNPs (Figure 5c). The laser fluence is comparable to those used for vaporizing larger, micron-sized PFH droplets44,45, at similar or lower particle concentrations, suggesting the benefit of these smaller NEs to both enhance bioavailability of drugs and contrast imaging at the target site (tumor) using biologically relevant laser fluencies. The surrounding environment in the optical images (Figure 5a-c) show the scAuNP-PFH-NE clusters, which are larger in the unfluorinated than in the fluorinated sample, possibly due to a higher free energy of formation due to ultrasonication during synthesis. When viewed under lower magnification, PFH microbubbles tend to cluster, expand and then collapse before abruptly bursting (Figure 5c, Supplementary Video 1). Such behaviour of PFH microbubbles during expansion could be due to the thermoelasticity of the zonyl-FSP shell of PFH-NEs41. The expansion of PFH microbubbles is most likely due to localized increases in pressure which in turn enhances the photoacoustic signal for imaging (as seen in Figure 4b,c) and also has the potential to generate large enough pressure waves to destroy cancer cells by perturbing their cell membranes46. The vaporization of nanoemulsions resulted in the formation of microbubbles with diameters ranging from 1 to 40 µm (Figure 5d). Using ideal gas law approximations and considering the density (1680 kg/m3) and molecular mass of PFH (0.338 kg/mol), a single 50 nm droplet should expand ~ 5 times its initial diameter after vaporization in vitro47. This suggests that the PFH microbubbles formed are a result of fusion of PFH nanobubbles. The size distribution tails towards larger values and it was fitted to a Generalized Extreme Value (GEV) model (see Supplementary equation S10). The fitting yielded a value of 8 ± 1 µm for the most probable diameter of the PFH microbubbles just before 9 ACS Paragon Plus Environment

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cavitation. Even though the majority of microbubbles are expected to be formed outside the vasculature in our application, the microbubble size is well below the mean size limit (45.4 ± 8.9 µm) required for safe intravenous microbubble administration stated by the Food and Drug Administration (FDA)48. To explore the potential of vaporisation and microbubble formation in the absence of a pulsed-laser radiation source, the PFH-NEs alone, and the two scAuNP-PFH-NE variants (i.e., unfluorinated/fluorinated) were each incubated separately with MCF-7 cells for 6 hours at 37 oC (5% CO2, in the dark) and imaged using a brightfield microscope (Figure 5e-g). Microbubbles were not observed in MCF-7 cells incubated with PFH-NEs alone (Figure 5, e). For the unfluorinated or fluorinated scAuNP-PFH-NE tandems however, vaporisation occurred (Figure 5, f, g) in an environment well below the vaporisation temperature threshold of perfluorohexane. In fact, DLS measurements of PFH-NEs alone in solution after 60 days of incubation at 37oC (5% CO2, in the dark) did not show the formation of microbubbles (DH = 99 ± 7 nm, Table 1). This suggests that heating of scAuNPs, followed by heat transfer to the immediate vicinity of PFH-NEs increases the local temperature to the boiling temperature of PFH-NEs without significantly affecting the bulk temperature. One potential source contributing to localized heating may be infrared light from the lamp of the microscope. Such phenomena has been previously observed with polymer-coated AuNPs, more formally known as surfacelocalized hyperthermia49. To further verify this, Milli-Q water was heated to 37 °C and PFH-NEs-scAuNPs were immediately added thereafter while being maintained in a dark environment. The formation of PFH microbubbles under these conditions were also observed when imaged under low-light illumination (Supplementary Data, Figure S6). Thus, it is clear that scAuNPs upon irradiation are major contributors for microbubble formation by increasing the efficiency of heat transfer from the surrounding environment to the PFH-NEs. Further studies will be performed in order to understand the underlying mechanism(s) of heat transfer in detail and will be studied quantitatively using a thermally-labile azo-linked fluorescence assay published previously49. The effect of nanodroplet-coalescence of PFH and scAuNP clusters on potentially reducing the theoretical vaporisation threshold of perfluorohexane droplets will also be further investigated using molecular dynamic simulations50. 10 ACS Paragon Plus Environment

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Cell viability studies (Supplementary Data, Figure S7) revealed that these PFH-NEs and scAuNPs were biocompatible with cells at very similar concentrations of perfluorohexane, Zonyl FSP and scAuNPs. MCF-7 cells with unfluorinated scAuNP-PFH-NEs were 96.4 ± 0.9 and 94.9 ± 1.8 % viable after 6 and 15 hours incubation, respectively. For the fluorinated scAuNP-PFH-NEs, the viability was 93.0 ± 1.5 and 90.1 ± 2.2 %, after the same respective time points above. PFH microbubbles can be used for enhancing contrast imaging of tumor growth by accumulating in the tumors site51 and can also adhere to the surface of cells or can be internalized, potentially leading to membrane disruption and cell death (Figure 5, f, g), as shown by the slightly lower cell viabilities than the control (Figure S7). This suggests that the scAuNP-PFH-NEs, once targeted to the tumor site, have the characteristics of a cancer therapeutic in the absence of small-molecule drugs, destroying cancer cells with time through pressure waves from microbubble cavitation. Localization of PFH-NEs and scAuNPs in breast cancer cells. Fluorescently-labelled PFH-NEs and scAuNPs (unfluorinated and fluorinated) were incubated with MCF-7 breast cancer cells to determine whether these nanoemulsions and nanoparticles could passively target tumors via superficial binding to tumour tissue. After 6 hours of incubation, both types of nanoemulsions displayed internalization and/or accumulation at the cell membranes (Figure 6). This could be due to the strong negative charge at the surfaces of PFH-NEs or to endocytosis into the cytoplasm of the cells. Therefore, adding therapeutic agents on the surface of PFH-NEs and/or scAuNPs could target important membrane receptors (i.e., endothelial growth factor receptors) leading to cancer cell death. TIRF images show that both PFH-NEs and scAuNPs tandem variants are co-localized as clusters either within or near the surface of cells after 2 hours of incubation (Figure 6, a-f). Also seen are nonfluorescent, scattering microbubbles52 (Figure 6, a,b, white arrows) after excitation with 532 nm radiation. The scattering was strongest under 473 nm laser excitation (Figure 6a). The ability of TIRF microscopy to detect microbubbles outside cells suggest their ability to strongly scatter light and serve as important ultrasound and photoacoustic contrast agents for imaging tumors. To assess the penetration depth of these PFH-NEs for suitability for cancer imaging and delivery of drugs, confocal imaging was used (Figure 6, g-n). After incubation for 6 hours, both the unfluorinated (Figure 6, g, h), 11 ACS Paragon Plus Environment

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and fluorinated (Figure 6, k, l) variants showed complete internalization into DiR-membrane-labelled-MCF-7 cells (Figure 6, i, m). The nanoemulsions were found distributed across the cytosol, cytoplasm, and within endosomes, as determined by the co-localization of each of dye-labelled species in the series of images (Figure 6, j, n). Using sequential laser excitation with appropriate fluorescence emission filters (see Supplementary Methods), a z-scan of several confocal sections within these MCF-7 cells were acquired. These images showed that PFH-NE-scAuNPs were uniformly localized throughout the volume of MCF-7 cells (Supplementary Video 2), considering blank MFC-7 cells displayed minimal autofluorescence (Figure S8).

MATERIALS & METHODS PFH-NEs and scAuNPs synthesis. PFH nanoemulsions with a PFH core and Zonyl FSP shell were created by vortexing PFHs (1100-3-70, Synquest Laboratories), Zonyl FSP (09988, Sigma Aldrich) and Milli-Q water to generate micron sized crude oil-in-water emulsions, followed by ultrasonication (see Supplementary Methods). Silica coated gold nanoparticles (scAuNPs) were either purchased from NanoHybrids (92318H250UL) or synthesized using the common reduction method53 (see Supplementary Methods). For characterizing scAuNPs in the ultraviolet-visible (UV-Vis) range, a Perkin Elmer Lambda 20 UV/Vis spectrometer was used. Detailed explanation and characterization of functionalized and fluorescently labelled scAuNPs and PFH nanoemulsions are described in the Supplementary Methods. Dynamic Light Scattering. The hydrodynamic size and stability of nanoparticles were determined using Dynamic Light Scattering (DLS) (Brookhaven 90Plus) with a 35 mW, 678 nm laser and a scattering angle of 90°. The intensity weighted size distributions were converted into number distributions using Mie theory and by inputting the complex refractive index of the nanoparticles. Each size distribution represents an average of independent measurements performed using a set mode of 8 runs (15 s each). The size distributions were then curve fitted using the multimodal Gaussian fitting algorithm in OriginLab. The average sizes reported are the mean ± half widths from the fitted Gaussian distributions. Zeta Potential. The surface charge of nanoparticles was determined by means of the zeta potential (Brookhaven ZetaPlus) using Henry’s equation for large double layer thickness (κ-1) and particle radius (a) (κa 12 ACS Paragon Plus Environment

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< 1) with the same operating conditions as DLS (See Supplementary Methods for further details). The zeta potentials reported are averages of multiple runs from several sample replicates. Measurements were carried out in triplicates, at room temperature and at low volume concentrations (< 1% v/v) to avoid multiple scattering effects. The Brookhaven instrument was calibrated using samples (Duke Scientific Corporation Nanosphere 500 nm and 90 nm size standards, Brookhaven BI-ZR3 zeta potential reference material) with known size/charge prior to measurements. Transmission Electron Microscopy. PFH-NEs and scAuNPs were imaged using a JEOL JEM-1200 electron microscope with a magnification range of 50-500,000x and maximal resolution of 0.35 nm. Images were recorded with an AMT charge coupled device (CCD) camera (Advanced Microscopy Techniques Corporation). Typically, 100 µL of unstained sample was placed on a carbon grid for imaging under 80 kV beam energy. The size distribution was obtained using the ‘imfindcircles’ function in MATLAB and the histogram was fit to the General Extreme Value (GEV) model (MATLAB), according to supplementary equation (S10). Photoacoustic and Ultrasound Imaging. For experiments, a gelatin phantom was made to mimic physical characteristics of biological tissue. To make the phantom, gelatin (Type A) (G2500, Sigma Aldrich) was mixed with water (10% w/v) at ~80 °C until all the gelatin was solubilized. Next 2% v/v of formaldehyde (252549, Sigma Aldrich) was added to solidify the matrix into gel before immediately pouring into a rectangular plastic mold. In order to allow the nanoparticles to be imaged, an inclusion 5 mm below the phantom surface was created using a metal rod placed through the side holes of the mold creating a microchannel for the PFH-NEs and scAuNPs to be injected. The phantom was left at room temperature to solidify and was stored in the refrigerator until further use. A commercial VEVO LAZR imaging system (FUJIFILM VisualSonics Inc.) with a tunable (680-970 nm) Nd:YAG laser was used to simultaneously get photoacoustic and ultrasound images of PFH-NEs with scAuNPs. The nanoparticles were imaged using 680 nm laser excitation and a maximum fluence of ~20 mJ/cm2 (30 mJ energy, 150 mm2 spot size), with a repetition rate of 20 Hz and pulse duration of 4-6 ns. For ultrasound, a transducer emitting 21 MHz frequency was used, with a focusing depth of 11 mm and a frame 13 ACS Paragon Plus Environment

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rate of 5 frames per second. The region of interest covered a depth of 15 mm and a lateral width of 14.08 mm. For photoacoustic and ultrasound imaging a gain of 70 dB and a 2D gain of 50 dB were used. All measurements were performed at physiological temperature of 37 °C, in a heated water bath. Data from ultrasound mode and photoacoustic mode were analyzed using MATLAB scripts provided by the company (FUJIFILM VisualSonics Inc.). Ultrasound and photoacoustic signal amplitudes were averaged over three independent trials after converting the radio-frequency (RF) signals using a Hilbert transform and taking maximum absolute signal values. Signal to noise (SNR) was calculated using the ratio of the averaged peak amplitudes of the sample and a region in the phantom without nanoparticles. The averaged signals reported are the mean ± standard deviation from more than 25 RF lines per replicate. Optical Light Microscopy. To get optical images of PFH microbubbles an IX81 Olympus inverted optical phase contrast microscope was used44. A 532 nm fiber optic laser (Microchip STG-03E, Teem Photonics) collimated through the side port was used to focus on the sample with a 330 ps pulse width, 4 kHz repetition rate, a 50 µm focusing spot size through a 40x objective and with 800 nJ per pulse (See Supplementary Methods). Fluorescence Microscopy. For multi-color localization studies of PFH-NEs and scAuNPs in MCF-7 breast cancer cells, a custom-built Total Internal Reflection Fluorescence (TIRF) and commercial confocal microscope (LSM700, Zeiss Microscopy) with on-stage incubation (37°C, 5% CO2) was used. For single-color localization studies of immobilized PFH-NEs onto a coverslip, a PEG-biotin-streptavidin linker scheme was used for imaging on the custom-built TIRF microscope54 (See Supplementary Methods and Techniques). Fluorescence Correlation Spectroscopy. For determining the colloidal hydrodynamic radius in solution of fluorescently labelled nanoparticles and the incorporation efficiency of scAuNPs onto PFH-NEs, fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS) was performed on a custom built confocal microscope (see Supplementary Methods). The raw multi-channel intensity-time traces are converted to (cross) correlation curves and fitted to (cross) correlation models to calculate the diffusion time (τD) and hydrodynamic radius from the Stokes-Einstein relation55,56 , 14 ACS Paragon Plus Environment

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=

∙

∙ ∙

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Equation (1)

where kB is the Boltzmann constant, T is the temperature, η is the viscosity of the medium, D is the diffusion coefficient and RH, is the hydrodynamic radius. In addition, the fraction of co-diffusing species can be estimated from the amplitudes of the auto- and cross-correlation curves for the Texas Red and BODIPY labelled nanoparticles. Differences in the co-diffusion fractions of Texas Red labelled scAuNPs and BODIPY-labelled PFH-NEs in different samples are related to the variability in the concentration of each nanoparticle species during synthesis (see Supplementary Methods).

CONCLUSIONS Using a combination of imaging and spectroscopy techniques, this study has shown that PFH nanoemulsions have the potential to be used as theranostic agents. These novel nanoemulsions have the capability to deliver hydrophobic drugs encapsulated in their PFH core, thus making them suitable for a wide range of therapies. The introduction of scAuNPs not only increases the potential of these nanodroplets to be used as contrast agents for ultrasound and photoacoustic imaging but also enables the addition of drugs onto the silica coated shell. We have shown that these PFH-NEs adhere to the cell surface and can be internalized actively or passively. Upon exposure to resonant radiation that heats up the scAuNPs, these nanoparticles can change from a liquid droplet to a gas microbubble and release their therapeutic payload. The rapid expansion and collapse of microbubbles not only increases ultrasound backscatter and contrast required for ultrasound and photoacoustic imaging but can also enable effective intracellular drug uptake57,58. A unique property of these PFH-NEs are their small (~100 nm) size and stability (~100 days) under physiological conditions. To date, these nanoemulsions are one of the smallest and most stable ultrasound and photoacoustic contrast agents. Due to their small size they could also serve potentially as a new class of nanoparticles for gene delivery in sub-cellular structures, evading some common extracellular and intracellular barriers59. For example, because these PFH-NEs are highly charged and avoid aggregation at physiological salt concentrations, they could be more efficient than some gene delivery vehicles, which have been shown to be 15 ACS Paragon Plus Environment

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easily cleared by phagocytic cells and the reticuloendothelial system60. Along with this comes the ability of PFH microbubbles to generate pressure waves which could damage cancer cells and further enhance drug tumor penetration, serving as a more potent treatment towards cancer. We have also demonstrated that our PFH-NEs have the potential to be highly effective drug carriers through encapsulation. The measured hydrodynamic radius of DiI-18C loaded PFH-NEs corresponds to the encapsulation of at least six drug molecules per PFH-NE with an effective concentration of ~125 µM which is representative for the lower limit of solubility in PFH (0.1 g/L), such as in the case of doxorubicin. For the higher limit of solubility in PFH (1,000 g/L) as in the drug paclitaxel, this number drastically increases to about 60 molecules corresponding to an effective concentration of 1000 µM (See Supplementary Calculations for both limits). Comparing these concentrations to literature IC50 values for the aforementioned drugs in MCF-7 cancer cells (which ranges from 1 to 10 nM)61, suggest that these PFH-NEs can be used as drug vehicles at very low particle concentrations, thus avoiding the adverse effects of high systemic toxicity. More in depth, in vitro and in vivo studies are presently being carried out to test the effectiveness of drug encapsulation as a function of solubility, as well as the therapeutic effect of a drug loaded PFH-NEs in cancer cells.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami. Detailed experimental procedures, characterization, supplementary videos, as well as the supporting data.

AUTHOR INFORMATION Corresponding Authors Email: [email protected] Email: [email protected] Notes The authors declare no competing financial interests. ACKNOWLEDGEMENTS 16 ACS Paragon Plus Environment

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This work was supported by grants from Canadian Institute of Health Research (CIHR) and CFI (Canadian Foundation for Innovation) to M.C.K and a National Sciences and Engineering Research Council (NSERC) grant to C.C.G. TEM and confocal fluorescence experiments were performed at the Advanced Bioimaging Centre and Imaging Facility, user facilities operated by SickKids and St. Michael’s hospital in Toronto. D.A.F and D.D.F were supported by a Ryerson Graduate Fellowship and an Ontario Graduate Scholarship, respectively.

Graphical Table of Contents. Mechanism for scAuNP-PFH-NEs drug-delivery via cellular internalization through the endothelial lining of carcinogenic tissue (endocytosis) and exposure to non-ionizing radiation.


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SCHEMES & SCHEME CAPTIONS Scheme 1. Synthesis of PFH nanoemulsions. Schematic of three step process used to synthesize PFH nanoemulsions with diameter below 100 nm.

Scheme 2. Schematic of PFH-NE constructs. Illustrations of synthesized (a) unlabelled PFH-NEs, (b) PFH-NEs with inserted biotin-linker and encapsulated DiI-18C, (c) BODIPY500/510 labelled PFH-NEs. Repeating units of Zonyl-FSP not shown for simplicity.

Scheme 3. Functionalization of silica-coated gold nanoparticles (scAuNPs). Schematic of the step by step functionalization of silica-coated AuNPs with hydroxyl termination (grey-colour coded coating) with (3-Mercaptopropyl)-trimethoxysilane (3-MPTMS), 1H,1H,2H,2H-perfluorodecyltriethoxysilane (PFDTES) and Texas Red (TR) maleimide.


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FIGURES & FIGURE LEGENDS

Figure 1. Size and stability from PFH nanoemulsion synthesis. (a) Averaged number-weighted DLS distributions of nanoemulsions (three replicates each) at day 1 made of 2% PFH/1% FSP (blue), 12% PFH/3%FSP (black), and 9% PFH/1%FSP (red, bimodal size distribution). (b) 12% PFH/3% FSP nanoemulsions in 0% (blue) and 0.9% w/v (150 mM) NaCl (green) both at day 30 (three replicates each) . (c) Zeta potential distribution at day zero and (d) over the course of 225 days for 12% PFH/3% FSP nanoemulsions. Yellow star over 180 days in (d) indicates the day an aliquot of PFH and Zonyl FSP were used to make nanoemulsions labelled with DiI-18C for TIRF imaging and Fluorescence Correlation Spectroscopy (FCS). Error bars represent standard deviation between three replicate measurements. Results are summarized in Table 1.


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Figure 2. PFH nanoemulsions as colloidally stable biological theranostic agents. (a) TEM image of nanoemulsions made from 12% v/v Perfluorohexane (PFH), 3% v/v Zonyl FSP fluorosurfactant (FSP) (0% NaCl, day 1) (scale bar: 200 nm). (b) Multicomponent Gaussian fitting of the size distribution of PFH nanoemulsions measured by TEM. (c) TIRF image (scale bar: 5 µm) of nanoemulsions with the PFH core containing the fluorescent dye DiI-18C. (d) FCS data of PFH nanoemulsions encapsulating DiI-18C (red circles) along with the hydrodynamic diameter (DH = 54 ± 4 nm) calculated from equation (1) by fitting the data to Supplementary equation (S2) (black line). Fitting parameters of TEM and FCS data are given in Table 2.


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Figure 3. Co-localization & co-diffusion of PFH-NEs and scAuNPs. TEM images of PFH (12% v/v) nanoemulsions with unfluorinated (a) and fluorinated (c) scAuNPs (scale bar: 200 nm). Dual-color FCCS data (symbols) and fitting according to Supplementary equation (S4) (black lines) for unfluorinated (b) and fluorinated (d) scAuNP-PFH-NE tandems. Inserts in (a) and (c) are zoomed-in regions of interest, with scale bars of 200 nm. Parameters of FCCS fitted curves are given in Table 3.


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Figure 4. Photoacoustic and ultrasound imaging of PFH-NEs and scAuNPs. (a) Schematic of VEVO LAZR system setup for photoacoustic and ultrasound imaging of scAuNPs and PFH-NEs. (b) Photoacoustic and (d) ultrasound images of scAuNP-PFH-NEs after excitation with 680 nm radiation and 21 MHz ultrasound frequency, respectively. Color-coded intensity bars in (b, d) are in units of decibels (dB). Signal amplitudes from simultaneous (c) photoacoustic and (e) ultrasound imaging as function of depth within the ~1mm microchannel inclusion loaded with scAuNP-PFH-NEs as indicated by the white arrows in (b, d). Signals for photoacoustic and ultrasound imaging in (c, e) are from a representative single RF line, whereas reported means are from at least 25 RF lines per replicate.


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Figure 5. Vaporization of the PFH-NE-scAuNP tandem. Representative time series images (optical phase contrast microscopy) of vaporisation of PFH nanoemulsions made from 12% Perfluorohexane (v/v) and 3% Zonyl FSP (v/v) with 5 nm (a) unfluorinated and (b) fluorinated scAuNPs, upon exposure to 532 nm radiation. Scale bar is 10 µm and elapsed time in (a) and (b) is ~30 s. (c) Representative image showing cluster formation and expansion of microbubbles after exposure to 532 nm radiation with 330 picosecond (ps) pulse width and 4 kHz repetition rate, scale bar 15 µm (See Supplementary Video 1). (d) Shows the distribution of droplet expansion into microbubbles before imploding, fit to the Generalized Extreme Value (GEV) model according to Supplementary equation (S10). (e) Optical images (brightfield) of MCF-7 cells with internalised PFH-NEs, (f) unfluorinated PFH-scAuNP-NEs, and (g) fluorinated PFH-scAuNP-NEs after 6 hours incubation at 37oC in the absence of pulsed-laser radiation. White arrows in (f, g) show PFH microbubbles.


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Figure 6. Cellular internalisation of PFH-NE-scAuNP tandem in MCF-7 cells. TIRF images of BODIPY labelled PFH-NEs (a, d), Texas-red labelled scAuNPs (b, e), and overlays from unfluorinated (c) and fluorinated (f) samples after 2 hours incubation. White arrows in (a, b) indicate the formation of PFHmicrobubbles. Fluorescence confocal images of BODIPY labelled PFH-NEs (g, k), Texas-red labelled scAuNPs (h, l), DiR-membrane-labelled MCF-7 cells (i, m) and corresponding overlays for unfluorinated (j) and fluorinated (n) samples after 6-hour incubation (See Supplementary Video 2). Scale bar: 10 µm.


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TABLES Table 1. Size and stability from nanoemulsion synthesis. Average hydrodynamic diameter a, DH of PFH/FSP nanoemulsions from number weighted size distributions from DLS at day 1, 30 and 60. PFH Zonyl FSP NaCl Temperature Day Concentration Concentration Concentration (oC) (n) (%v/v) (%v/v) (%w/v) 2 1 25 1 — 9 1 25 1 — 12 3 0.9 25 30 12 3 25 30 — 12 3 37 60 — a Error margins in peak sizes are reported as fitted Gaussian half-widths.

Peak 1 (nm)

Peak 2 (nm)

233 ± 2 129 ± 13 53 ± 5 64 ± 9 99 ± 7

— 520 ± 40 — — —

Table 2. PFH nanoemulsions as stable biological theranostic agents. Average diameter from TEM images and hydrodynamic diameter (nm), DH of fluorescently labelled 12% PFH/3% FSP-NEs. Sample

Diameter (nm) Species 1 Species 2 Species 3 50 ± 5 — — Non-labeled (DLS)a 53 ± 8 70 ± 14 101 ± 28 Non-labeled (TEM)a, b 54 ± 4 — — DiI-18C-labeled PFH core (FCS)c a Error margins in peak sizes are reported as fitted Gaussian half-widths. b The fractional contributions for species 1, 2, and 3 are 43, 42, and 15%, respectively. c Error margin represents standard error from FCS fitting algorithm.

Table 3. FCCS and DLS results of PFH nanoemulsions-scAuNPs tandems. Hydrodynamic diameters, DH, of PFH (12% v/v) nanoemulsions with 5 nm unfluorinated and fluorinated scAuNPs. FCCS a Diameter (nm) DLSb Diameter (nm) Sample Species 1 Species 2 Species 1 Species 2 388 ± 36 160 ± 12 520 ± 40 Unfluorinated — 55 ± 18 230 ± 78 111 ± 10 432 ± 39 Fluorinated a Error margin represents standard error from FCCS fitting algorithm. b Error margins in peak sizes are reported as fitted Gaussian half-widths.


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(61) Uhr, K.; Prager-van der Smissen, W. J.; Heine, A. A.; Ozturk, B.; Smid, M.; Göhlmann, H. W.; Jager, A.; Foekens, J. A.; Martens, J. W. Understanding Drugs in Breast Cancer Through Drug Sensitivity Screening. SpringerPlus 2015, 4, 1-11.


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SUPPLEMENTARY INFORMATION: SYNTHESIS OF STABLE MULTIFUNCTIONAL PERFLUOROCARBON NANOEMULSIONS FOR CANCER THERAPY AND IMAGING Authors: Donald A. Fernandes1, ‡, Dennis D. Fernandes2, 3, ‡, Yuchong Li2, 3, Yan Wang4, Zhenfu Zhang2, 3, Dérick Rousseau1, Claudiu C. Gradinaru2, 3,*, and Michael C. Kolios1,4,*

Author Affiliations: Department of Chemistry & Biology, Ryerson University, 2 Department of Physics, University of Toronto, 3 Department of Chemical and Physical Sciences, University of Toronto Mississauga, 4Department of Physics, Ryerson University. *Authors to whom correspondence may be addressed. ‡ These authors made equal contributions to this work. 1

CORRESPONDENCE: Michael C. Kolios Address: Department of Physics, Ryerson University, Toronto, 350 Victoria Street Toronto, Ontario M5B 2K3, Ontario, Canada E-mail: [email protected] • Phone: +1 (416) 979-5000 x3157 • Fax: +1 (416) 979-5343 Claudiu C. Gradinaru Address: Department of Chemical Physical Sciences, University of Toronto, Mississauga, 3359 Mississauga Road North, Mississauga, L5L 1C6, Ontario Canada. E-mail: [email protected]. • Phone: +1 (905) 828-3833 • Fax: (905) 828-5425.


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1. SUPPLEMENTARY METHODS Synthesis of PFH-NEs and scAuNPs. PFH nanoemulsions with a PFH core and Zonyl FSP shell were created by vortexing the appropriate amount of PFH (1100-3-70, Synquest Laboratories), Zonyl FSP (09988, Sigma Aldrich) and Milli-Q water for 1 minute to create micron sized crude oil-in-water emulsions. Nanoemulsions were then prepared by ultrasonication using a conical tip sonicator instrument (Digital Model 250, Branson). Sonication cycles (alternations between 10 seconds with sonication and 20 seconds without) were performed for 2 minutes at 10 W power (20% maximum acoustic power) and at 4°C in an ice water bath. Silica coated gold nanoparticles were initially purchased from NanoHybrids Inc., Austin, Texas (92318H250UL) and later synthesized through the use of the reduction method for further experiments. Silica coated gold nanospheres from NanoHybrids were synthesized using a modified Stöber method involving tetraethyl orthosilicate (TEOS)1. Optical density of the silica-coated nanospheres was determined by spectrophotometry measurements and particle concentrations were determined and confirmed by inductively-coupled plasma mass spectrometry (ICP-MS) by NanoHybrid Inc. Synthesis of gold nanoparticles (AuNPs) using the common reduction method2 involved using sodium citrate and chloroauric acid, heating the mixture to boiling and then slowly cooling to 90°C. To create a silica coating on gold nanoparticles, 3-aminopropyl-trimethoxysilane (APS) and sodium silicate were added under vigorous magnetic stirring3. The polymerization reaction was carried out by changing pH from 11 to 8.5 by adding hydrogen chloride with the mixture allowed to stand for 24 hours. Silica coated gold nanoparticles were fluorinated by the addition of 1H,1H,2H,2H-perfluorodecyltriethoxysilane (658758-25G, Sigma Aldrich), ammonium hydroxide (320145-500ML, Sigma Aldrich) and methanol ((34860-1L-R, Sigma Aldrich) with the solution allowed to mix for 24 hours4,5,6. For characterizing scAuNPs in the ultraviolet-visible (UV-Vis) range, a Perkin Elmer Lambda 20 UV/Vis spectrometer was used. Size and stability of PFH nanoemulsions. To test for stability at physiological saline, PFH-NEs with 12 % (v/v) PFH and 3 % (v/v) zonyl FSP were prepared using ultrasonication with 10 W of power, with a sonication time of 2 minutes (pulsing of 10 sec on and 20 sec off). Once made, the droplet solution was diluted 31 ACS Paragon Plus Environment

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2x with 1.8% w/v NaCl to get the final concentration of 0.9% w/v NaCl. The PFH-NEs were then stored at room temperature until use for TEM measurements. For TEM, a few drops (~ 100 uL) of the concentrated solution was placed on a copper grid for imaging (Figure S1). Characterization of 5 nm silica coated gold nanoparticles. To characterize 5 nm scAuNPs, an initially concentrated solution (initial density of 6.4 x 1015 nanoparticles/mL) was diluted down (~ 200 x) to get sufficiently spaced out particles for imaging under TEM (Figure S2a). The nanoparticle density and absorption properties for vaporization of PFH-NEs (Figure S2b) was determined using UV-Vis spectroscopy and knowing the absorption extinction coefficient for gold from literature. For absorption measurements, a Perkin Elmer Lambda 20 UV/Vis spectrometer was used with a resolution of 1 nm, scan speed of 240 nm/min and slit width of 2 nm. For calculating zeta potential (Figure S2c), a Brookhaven 90 Plus instrument was used, connected to ZetaPlus software. The sample was diluted down through serial dilution to a concentration (~ 1.1 x 1013 nanoparticles/mL) giving both stable and reproducible readings. Zeta Potential measurements. Particles depending on their charge either move to the positive end or negative end of the electrode upon application of an electric field. The magnitude of charge also determines the velocity, which is then used to compute mobility and zeta potential. The mobility depends on the velocity of the particles and applied electric field, whereas the zeta potential depends on mobility and viscosity of liquid. The intensity profiles for the zeta potentials was measured from the Doppler shift of particles with respect to a reference beam (i.e., light scattered from the particles moving in the direction of the electric field). The sign (positive or negative) of the difference between the frequencies of scattered light and the reference beam is related to the sign of charge of the particle (i.e., the zeta potential), which is none other than the mean (peak) zeta potential intensity. The Debye-Hückel approximation was used for calculating zeta potential at the slipping plane relative to a point in the bulk fluid away from the interfacial double layer. Since the ratio of the particle radius to double layer thickness (κa) is less than 1 (considering the NPs are in ultrapure water and a radius of 12.5 nm), Henry’s equation was used knowing the electrophoretic mobility of the particles, whose magnitude and sign depends on the magnitude of the electric field and frequency shift of scattered light. 32 ACS Paragon Plus Environment

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Co-localization of PFH nanoemulsions-scAuNPs tandem. For co-localization studies of fluorescently unlabelled PFH-NE-fluorinated scAuNP samples using TEM (inserts in Figure 3), a 2 mL solution of unfluorinated scAuNPs (3.2 x 1013 nanoparticles/mL, 0.5 OD) were centrifuged (16,000xg for 90 minutes) to get a concentrated solution. The scAuNPs were redispersed by vigorous stirring in 250 µL methanol with 80 µL of 1H,1H,2H,2H-perfluorodecyltriethoxysilane and 70 µL of ammonium hydroxide added 5 minutes later. The final solution was then left for stirring for 1 hour and left sitting for 24 hours. The methanol was then evaporated using nitrogen gas and solubilized in 300 µL PFH. This solution was spun down 2 times at 16,000xg for 30 minutes to remove excess reactants. The fluorinated solution was then stored at room temperature until further use in 300 µL PFH. Half of the fluorinated scAuNPs sample (150 µL) was first diluted down to 600 µL using PFH with Zonyl FSP (150 µL) and Milli-Q water (4250 µL) added to make crude emulsions using a vortex. These PFH microemulsions were then sonicated for 2 minutes (pulsing of 10 sec on and 20 sec off) and 20% acoustic power amplitude to create nanodroplets. To make the PFH-NE-unfluorinated scAuNP sample, 1.4 mL of unfluorinated scAuNPs (3.2 x 1013 nanoparticles/mL, 0.5 OD) were centrifuged (16,000xg for 90 minutes) and then redispersed in 0.7 mL Milli-Q water to get a final nanoparticle concentration of 6.4 x 1013 nanoparticles/mL. A fraction of this solution (0.5 mL) was added to 0.5 mL of already prepared 12%PFH/3%FSP NEs to make the final solution for TEM. TEM measurements involving PFH-NEs were made without any further dilution to avoid vaporization of PFH-NEs. For optical imaging of vaporization of NEs, the remaining amount of unfluorinated/fluorinated scAuNPs were diluted down to final scAuNP concentrations used for fluorescence microscopy (7.5 x 1012 nanoparticles/mL and 1.5 x 1012 nanoparticles/mL, respectively) before sonication to create PFH-NE-scAuNP samples. The final unfluorinated/fluorinated samples were diluted 2x prior to imaging using optical light microscopy. For co-localization studies using fluorescence microscopy, the scAuNPs were surface functionalized with thiol groups (-SH) prior to covalent labelling with Texas Red maleimide (T-6008, Life Technologies). To functionalize scAuNPs, 2 mL of nanoparticle solution (3.2 x 1013 nanoparticles/mL, 0.5 OD) was centrifuged (16,000xg for 60 minutes) and redispersed under vigorous stirring for 30 minutes in 800 µL ethanol. Then 200 33 ACS Paragon Plus Environment

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µL Milli-Q water and 25 µL (3-Mercaptopropyl)-trimethoxysilane (3-MPTMS, 175617-25G, Sigma Aldrich) were added for thiol functionalization and the final solution stirred further for 30 minutes. The solution was then centrifuged (10,000xg, 99 minutes) and then redispersed in 1 mL ethanol for 5 minutes. This washing and centrifugation step was repeated two more times. The functionalized scAuNPs were then stored at 4°C in 1 mL Milli-Q water until further use. A portion of this solution (250 µL) was then used for fluorescent labelling with Texas Red (T-6008, Life Technologies) by adding 30 µL (8 µM) of Texas Red maleimide and 790 µL of MilliQ water. The fluorescent dye was added dropwise while stirring the scAuNPs in Milli-Q water. This reaction was allowed to react for 2 hours at room temperature before continuous washing and centrifugation (at least three times) to remove unbound dye. Concentration of Texas Red was determined using a UV-Vis spectrometer from the Lambert-Beer law. For fluorination of Texas Red labelled scAuNPs, 250 µL of labelled solution was centrifuged (16,000xg, 60 minutes) and redispersed in 1 mL of methanol. Then 20 µL of 1H,1H,2H,2Hperfluorodecyltriethoxysilane and 20 µL of ammonium hydroxide were added 5 minutes later and left to stir for 30 minutes. The methanol solvent was then evaporated using nitrogen gas and the fluorinated nanoparticles solubilized in 600 µL PFH. The amount of 1H, 1H, 2H, 2H-perfluorodecyltriethoxysilane and Texas Red maleimide to add for functionalization and fluorescent labelling were determined by calculating the amount of silica present on the surface of nanoparticles from the surface area of scAuNPs and silica. Functionalization and fluorescent labelling of scAuNPs was verified using absorption and fluorescence spectroscopy with shifts in the maximum absorption/fluorescence wavelengths (Figure S3) seen due to changes in the refractive index at the surface of nanoparticles and changes in structure of the fluorescent molecule7. To label the shell of PFH-NEs, 1 mg of BODIPY500/510 (D-3793, Life Technologies) was first dissolved in 1 mL of ethanol to give a final dye concentration of 2.5mM. To make fluorescently labelled PFH-NEfluorinated scAuNP samples BODIPY (20 µL) was first vigorously mixed for ~5 minutes with Zonyl FSP (150 µL) to get BODIPY labelled vesicles. These vesicles were then mixed with 600 µL of Texas Red labelled, fluorinated scAuNPs (1.5 x 1012 nanoparticles/mL) and 4230 µL of Milli-Q water using a vortex prior to 34 ACS Paragon Plus Environment

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sonication (2 minutes, 10 sec on/ 20 sec off). This sample was diluted 1,000x for fluorescence measurements. For making fluorescently labelled PFH-NE-unfluorinated scAuNP samples, the previously made Texas Red labelled, unfluorinated scAuNPs (7.5 x 1012 nanoparticles/mL) were diluted 1,000x (500 µL) and mixed with 1,000x diluted BODIPY labelled PFH-NEs (500 µL).

PFH-NEs and scAuNPs in cancer cells. Passive targeting and internalization of PFH-NEs-scAuNPs tandems and microbubbles in the MCF-7 (breast adenocarcinoma) cell line from ATCC was determined using Total Internal Reflection Fluorescence Microscopy (TIRFM), confocal microscopy and optical microscopy. The cells were grown and maintained in a humidified cell incubator at 37 ͦC and 5 % CO2 with Dulbecco’s Modified Eagle Media (DMEA) comprising 4500 mg glucose/L, L-glutamine, NaHCO3, and sodium pyruvate with 10 % fetal bovine serum. MCF-7 cells were first incubated for 32 hours at an initial concentration of 100,000 cells/mL in poly-d-lysine glass bottom dishes (MatTek, P35GC-1.5-14-C) prior to fluorescence imaging. To label the membranes of cells, 1 µL of the 2 mM cell-labelling solution (DiD, & DiR, i.e, 1,1’-Dioctadecyl3,3,3’,3’-Tetramethylindodicarbocyanine Perchlorate and Iodide, respectively) was added per mL of cell media and left to incubate for 20 minutes. The cell media was then washed three times to remove free dye from solution prior to adding 1-2 mL of fresh DMEA. To determine localization of nanoparticles and microbubbles in cells, unfluorinated and fluorinated samples were added to the cell sample at similar concentrations used for Fluorescence Cross-Correlation Spectroscopy (FCCS). The cells were then incubated for 2 or 6 hours before being imaged using fluorescence or optical microscopy.

Optical light microscope. A 40x PlanFLN phase contrast objective (Ph2, Olympus) with 0.6 numerical aperture (~500 nm theoretical lateral resolution) was used to control the power density for excitation. A dichroic mirror (Chroma) was used to reflect 500-650 nm wavelength of light towards the sample, transmitting all other wavelengths of light for optical viewing. Images were captured from a CCD camera (Lumenera) at a frame rate of 4 frames per second. All measurements were made under physiological conditions (i.e., pH 7.4, at 36°C).


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Images of MCF-7 cells incubated with unfluorinated and fluorinated PFH-NEs and scAuNPs were taken using a ZOE Fluorescent Imager, with gain setting of 8, exposure time of 460 ms and LED intensity of 30.

TIRF microscopy. Fluorescent images of cellular internalisation of unfluorinated and fluorinated scAuNPNEs were acquired using a custom-built total internal reflection fluorescence (TIRF) microscope used previously in literature8. Fluorescent images were obtained using sequential alternating excitation with a 633 nm laser for DiD/DiR fluorescence, a 532 nm laser (B&W Tek, USA) for Texas-Red fluorescence and a 473 nm laser (Laser Glow, Canada) for BODIPY500/510 fluorescence with excitation intensity controlled by an acousto-optic tunable filter (TF625-350-2-11-BR1A, Gooch & Housego), reflected by a dichroic mirror (FF495-Di02, Semrock) and focused through an oil-immersion objective (1.45NA/60X Plan-Apochromat, Olympus, USA) . To filter out the laser scattering, a long-pass (LP-488-RS, Semrock) and a band-pass filter (HQ512/25, Chroma) were used. An electron-multiplied charge-coupled device (EMCCD, DU-897BV, Andor Technology) was used to image samples with an illumination area of ~2500 µm2 and an exposure time of 30 ms per frame for a total of 500 frames. The same optical filter set for fluorescence emission was used as in FCCS for BODIPY and Texas-Red fluorescence. For detecting DiD/DiR fluorescence, a long pass 647 (LP-647-RS, Semrock) and a 708/80 nm (HQ-708/80, Chroma) bandpass filter were used. Images found in Figure 6 were processed, normalised, summed, and colour-coded using ‘Image J’. Fluorescence confocal microscope. Fluorescently labelled PFH nanoemulsions and scAuNPs were measured on a custom-built confocal microscope with plasma cleaned glass coverslip (VMR, cat. No. CA48366-249-1) and diluted down to sufficient amounts (~10 nM) to improve the signal-to-noise and avoid detector saturation effects. Samples were excited using solid state 473 nm laser (Laser Glow, Canada) and a solid state 532 nm laser (B&W Tek, USA). Along the excitation path, scattered light was reflected using a dichroic mirror (FF585-Di01, Semrock) and focused onto the sample using oil-immersion objective (1.4NA/100X UplanSApo, Olympus). Additional scattered light along the emission path was filtered out with a


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long-pass (LP-488-RS, Semrock) and fluorescence from BODIPY500/510 and Texas Red was split into two channels using an emission dichroic mirror (FF585-Di02, Semrock, USA). To improve signal to noise, a 512/25 nm (FF01-512/25, Semrock) and a 610/25 nm (FF01-620/25, Semrock) band pass filter were placed along the emission paths of BODIPY500/510 and Texas Red, respectively. Fluorescence emission was collected using set avalanche photodiodes (PerkinElmer, Canada & COUNT, Laser Components GmbH, Germany), and raw photonic data was processed using a custom-made LabVIEW program and converted into FCS and FCCS curves using a commercial correlator (Mad City Labs). To minimize vaporisation of PFH-NE-scAuNPs over the course of a measurement, laser intensities used at the sample never exceeded 1 kW/cm2, and gave relatively consistent intensities for FCS. For experiments on internalization of PFH-NEs and scAuNPs in MCF-7 breast cancer cells, a Zeiss LSM 700 confocal microscope was used. Samples were sequentially excited using the multitrack setup using solid state 488 (10 mW), 555 (10 mW) and 639 nm (5 mW) lasers and focused using a 63X (1.4 N.A.) oil immersion objective (i.e., starting with 639 nm and ending with 488 nm). Emission from BODIPY500/510 and DiR were separated using a 554 nm dichroic beam splitter with 640 nm short and long pass filters. For Texas Red and DiR, a 630 nm dichroic beam splitter was used with 630 nm short and long pass filters. To avoid excessive vaporization, only a maximum of only 4% of laser power was used for experiments. Confocal Z stack scans were taken using the Z-stack option at 5 frames per second, 4 averages per frame and pixel dwell time of 1.58 µs.

2. SUPPLEMENTARY DATA Negative and positive controls for FCCS experiments. To show that the interaction between the two types of nanoparticles (PFH NEs and scAuNPs) were not influenced by the fluorescent molecules (BODIPY and Texas Red) percent co-diffusion was calculated from dual color FCS by mixing the two molecules in equal molar amounts (Figure S4a). To correct for spectral-crosstalk and mis-overlap of lasers from the FCS confocal setup, a correction factor from dually labelled DNA with Texas Red and Fluorescein was determined (Figure 37 ACS Paragon Plus Environment

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S4b). Since in these constructs, the dye pair are tethered to the same molecule, theoretically, all three correlation curves from the FCCS plot should overlap, which was not observed. This observation however, may be attributed to slight deviations in overlap of the respective detection volumes, and can be corrected for by determining individual correction factors for each autocorrelation curve (red and green) such that 100% codiffusion in the crosscorelation curve (blue) is attained. This correction factor was then multiplied by codiffusion values determined from each multi-exponential fitting. Cell viability of MCF-7 cells with PFH-NEs and scAuNPs. Cell viability was carried out using a haemocytometer with 4x4 square grids (Figure S7). A 25 µL aliquot of a 0.4% Trypan Blue solution (Gibco) was added to a 250 µL cell suspension at a density of ~ 1.9 x 106 cells/mL. Cells were then diluted to get proper spacing on the haemocytometer grid for cell counting. Cells were imaged immediately on an Olympus CKX41 microscope under 4x objective. Cells that were blue were counted as dead under brightfield light. Over 10,000 cells were averaged for each of the unfluorinated and fluorinated samples to get final cell viability values. Images were captured using the software, Q Capture.

3. SUPPLEMENTARY EQUATIONS Model fitting of FCS curves. The diffusion of the nanoparticles in a continuous medium is based on Fick’s law where the mean-square displacement of the diffusing particle in three dimensions can be expressed as, 〈 〉 = 6

Equation (S1)

where is the particles diffusion coefficient, which depends on its hydrodynamic radius and solvent viscosity through the Stokes-Einstein equation (equation (1), from main text). However in some cases, molecular crowding cannot be avoided and an anomalous diffusion model for the autocorrelation function must be fitted to the experimental data. Autocorrelation functions using both the normal (α =1) and anomalous diffusion models (0 < α < 1) were fitted. It was found that the anomalous diffusion model gave the better fit for the FCS data based on the residuals and χ2 values.


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Our nanoemulsions most likely diffuse anomalously due to electrostatic interactions (i.e., van der Waals, see Supplementary Video 1) resulting in the diffusion of larger PFH-NE and scAuNP clusters during measurements. Because the mean-square displacement follows a power law, 〈 〉 ~ the expression of the autocorrelation function from single channel FCS can be written as follows,

"/

= 〈〉 1 + ! !

"/

1 + ! !

"/

1 + %! ! ! $

∙ &1 +

"

'

"

( ()

*

Equation (S2)

where + is the aspect ratio (height to width) of the ellipsoidal detection volume, , is the average number of fluorophores and - their diffusion time. The diffusion coefficient and hydrodynamic radius of the nanoparticles can be determined knowing the half-width (./ ) of the detection volume, where is the triplet state relaxation time, and 0 is the average fraction of fluorophores found in the triplet state.

- =

12 %

Equation (S3)

3-

Similarly, the anomalous diffusion model can be fit to the crosscorelation curve (blue) using the following mathematical expression, where 〈,4 〉 and 〈,5 〉 are the number of diffusing fluorescent species from the green and red channels, 〈,6 〉 the number of co-diffusing species and -,6 is the diffusion time of co-diffusing nanoparticles. 〈8 〉 &1 9 〉〈: 〉

6 = 〈

"

+ & * * ,8

"/

&1 + $% ! & * * ,8

∙ &1 + " '

"

()

*

Equation (S4)

Calibration of detection volume for FCS. The dimensions (lateral and axial) of the detection volume, approximated by a three dimensional Gaussian function, can be determined by fitting the correlated data from a fluorescent sample with known diffusion coefficients. The blue and green detection volumes were determined using Rhodamine 101 and Rhodamine 6G, respectively. The diffusion coefficients from these fluorescent molecules were determined from literature and the FCS data was fitted to the single channel, autocorrelation model to determine the width of the detection volumes. The effective volume, ;

Synthesis of Stable Multifunctional Perfluorocarbon Nanoemulsions

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