Readily accessible multifunctional fluorous emulsions

Mixtures of perfluorocarbon and water containing functionalized polymer surfactants and fluorous-tagged small molecules yield multifunctional emulsions with defined functionality on the inside and outside of the droplets.


Supporting Figures
. Stability of emulsions prepared from hydrophilic polymers. Emulsions were synthesized by dissolving the indicated polymer in PBS (2.8 wt%, except for 21 where solubility limited the polymer to 1.6 wt%) and sonicating in the presence of 7:3 PFD/PFTPA (20 wt%). Error bars represent the polydispersity as measured by DLS. The surface charge of the resulting emulsions was measured. Error bars represent the average of five zeta potential measurements. (C) The emulsions were dried to a polymer residue and analyzed by infrared spectroscopy. The spectra were normalized to the dominant carbonyl stretch of 9. Figure S3. Pluronic-F68 control for the modification of perfluorocarbon emulsions using the adamantane-β-cyclodextrin association. (A) Schematic for the control experiment. (B) Pluronic-F68 nanoemulsions were prepared and varying amounts of 25 or 26 in acetonitrile were added. The acetonitrile was removed by evaporation and the surface charge of the treated nanoemulsions was measured. Error bars represent the average of five zeta potential measurements. Figure S4. Different size nanoemulsions can be prepared by varying the amount of surfactant. (A) Schematic for Pluronic-F68 nanoemulsion formation. Varying amounts of Pluornic-F68 were dissolved in phosphate buffered saline (PBS) and sonicated in the presence of 7:3 perfluorodecalin (PFD)/perfluorotripropylamine (PFTPA) (20 wt%). (B) Dynamic light scattering (DLS) data for emulsions prepared as described in A. (C) The stability of nanoemulsions prepared in A over two months. The error bars represent the polydispersity of the sample as measured by DLS.

Emulsion Preparation and Characterization
Reagents Perfluorodecalin (PFD) and perfluorotripropylamine (PFTPA) were purchased from Synquest and used without further purification. Pluronic-F68 was purchased from Sigma-Aldrich. Phosphate buffered saline (PBS) was purchased from Mediatech Inc.

Sonication
Bath sonication was performed in a Branson 3510 sonicator. Probe sonication was performed with a Microson Ultrasonic Cell Disruptor.

Dynamic light scattering and zeta potential
Dynamic light scattering (DLS) data was obtained on a NanoBrook Omni (Brookhaven) instrument. Samples were diluted 1:100 in 0.1x PBS and equilibrated (5 min) to 25 o C before measurement collection. Light scattering measurements were performed at 90 o and size distribution was determined by the NNLS algorithm. Error bars for DLS data represent one polydispersity unit (+/-0.5 polydispersity) as determined from the average of five DLS experiments (100 s collection each). Zeta potential measurements were obtained on a NanoBrook Omni (Brookhaven) instrument or a Zetasizer Nano ZS (Malvern). Samples were diluted 1:100 in 0.01x PBS and equilibrated (5 min) to 25 o C before measurement collection. Five replicate measurements (20 scans each) were collected and averaged for all zeta potential data. The Smoluchowski model was employed. Error bars represent two standard deviation units (+/-1 standard deviation).

Photophysical data
Absorbance spectra were obtained on an Cary 4000 UV/Vis spectrophotometer (Agilent Technologies) with a scan rate of 2000 nm/min. The instrument was blanked on the solvent prior to obtaining a spectrum. Photoluminescence spectra were obtained on a Jobin Yvon/Horiba Instruments spectrophotometer. Absorbance and photoluminescence data were collected in quartz cuvettes.

Confocal Microscopy
Confocal microscopy was performed on a Leica TCS SP2 Confocal Laser Scanning Microscope. All images were acquired with a 63X oil objective. Microscopy samples were prepared on glass slides that were cleaned with acetone (1 h bath sonication), isopropanol (1 h bath sonication) and dried with nitrogen. A "window" of scotch tape was then applied. The sample was dropcast inside the window, a coverslip was placed ontop and secured with clear nail polish.

Infrared spectroscopy
Infrared (IR) spectroscopy was performed on a Thermo Scientific Nicolet 6700 Fourier transform infrared spectrometer using the attenuated total reflectance (ATR) mode on a germanium crystal.

Figure 2
Stock solutions in acetone (10 mM) containing aniline 4, triazine 5, trityl 6, or benzimidazole 7 were prepared. 100, 10, 1, or 0 µL of these stock solutions were added to eppendorf tubes and the acetone was allowed to evaporate overnight. The following morning, PFD (18 µL) and PFTPA (8 µL) were added to each tube and sonicated (bath) until dissolved, at which point 28 mg/mL Pluronic-F68 in PBS (250 µL) was added. Each mixture was sonicated (probe, 0.02 watts) for 15 minutes at 0 o C. Dynamic light scattering, zeta potential, and absorbance measurements were obtained as described above.

Figure 4B
A stock solution of poly(methyl vinyl ether-alt-maleic anhydride) in acetone (40 mg/mL) was prepared and 100 µL was placed in six eppendorf tubes. The acetone was evaporated overnight. The following morning, PBS (200 µL) was added and these mixtures were sonicated (bath) until the solutions were transparent (~4.5 h), S9 at which point PFD (18 µL) and PFTPA (8 µL) were added. These four samples were then differentiated by the addition of glycine (22, 25 µL of 150 mg/mL solution in water plus 25 µL DMSO), methyl glycine (23, 50 µL of 126 mg/mL solution in 1:1 DMSO/PBS ), methyl arginine (24, 50 µL of 261 mg/mL methyl arginine in 1:1 DMSO/PBS ), or nothing (50 µL of 1:1 DMSO/PBS). The mixtures were then sonicated (probe, 0.02 watts) for 15 minutes at 0 o C. The zeta potential of each emulsion was measured as described above. The average of five zeta potential measurements is plotted in Figure 4B.

Figure 4C
The emulsions prepared and analyzed in Figure 4B were dried in a vacuum oven at 40 o C overnight. The resulting residue was analyzed by ATR-FTIR. Plotted in Figure  4C are the IR spectra from 1800 to 1200 cm -1 normalized to the dominant carbonyl stretch in the polymer (1712 cm -1 ).

Figure 5B
A solution of 28 mg/mL poly(β-cyclodextrin) in PBS (500 µL) was combined with PFD (36 µL) and PFTPA (16 µL) and sonicated (probe, 0.02 watts) for 15 minutes at 0 o C. The emulsion solution was aliquoted into five portions (50 µL each). A solution of 1-adamantaneamine (26, 5 mg/mL) in CH3CN was prepared. A differing amount of 1-adamantaneamine solution was added to each aliquot (0, 1, 2.5, 5, 10 µL) followed by CH3CN so that each sample contained 10 µL total CH3CN. The solutions were mixed, incubated at room temperature for 10 minutes, and the CH3CN was removed via gentle blowing with nitrogen. The zeta potential of the treated emulsions were measured as described above. The above procedure was repeated with 1-adamantyl carboxylic acid (25, 5 mg/mL solution).
Using the above solutions 6 samples were prepared in triplicate as follows: ( The solutions were incubated at room temperature for 15 minutes in the dark. The samples were trice washed by centrifugation (3 min at 5000 rpm) and resuspension in PBS (250 µL). Following the third wash, the emulsions were resuspended in PBS (75 µL). The photoluminescence of the resulting perfluorocarbon emulsion solution was measured. Excitation was at 415 nm, emission was collected from 450-650 nm with an integration time of 0.25 s and slit width of 7 nm. Plotted in Figure 5C is the photoluminescence at 520 nm (λmax) for samples A-F. Samples G and H were diluted 1:10 in PBS and 5 µL was dropcast onto a clean microscope slide. A cover slip was affixed and these samples were analyzed by confocal microscopy. Laser power = 25%, excitation = 458 nm, emission collection 480-600 nm, gain = 721, offset = -24.8, scale bar = 1 micron. No settings were changed in between imaging sample G and H.
Figure S1, Figure 3C-H Emulsions were prepared as described in Figure 3. Dynamic light scattering of each sample was acquired at various timepoints over two months as described above.

Figure S2C
The emulsions prepared and analyzed in Figure S2B were dried in a vacuum oven at 40 o C overnight. The resulting residue was analyzed by ATR-FTIR. Plotted in Figure  S2C are the IR spectra from 1800 to 1200 cm -1 normalized to the dominant carbonyl stretch in the polymer (1712 cm -1 ).

Figure S3
A solution of 28 mg/mL Pluronic-F68 in PBS (500 µL) was combined with PFD (36 µL) and PFTPA (16 µL) and sonicated (probe, 0.02 watts) for 15 minutes at 0 o C. The nanoemulsion solution was aliquoted into five portions (50 µL each). A solution of 1-adamantaneamine (26, 5 mg/mL) in CH3CN was prepared. A differing amount of 1-adamantaneamine solution was added to each aliquot (0, 1, 2.5, 5, 10 µL) followed by CH3CN so that each sample contained 10 µL total CH3CN. The solutions were mixed, incubated at room temperature for 10 minutes, and the CH3CN was removed via gentle blowing with nitrogen. The zeta potential of the treated nanoemulsions were measured as described above. The above procedure was repeated with 1-adamantyl carboxylic acid (25, 5 mg/mL solution).

General Procedure
Solvent was removed by reduced pressure with an IKA RV-10 Rotovapor equipped with a Welch self-cleaning dry vacuum or house vacuum. Products were further dried by reduced pressure with a Maxima D2A high vacuum. Thin layer chromatography was performed with Baker-flex Silica Gel 1B-F plates (JT Baker). Flash chromatography was performed using technical grade silica gel with 60Å pores and 230-400 mesh particle size (Sigma-Aldrich, 717185). All 1 H and 13 C spectra are reported in ppm and referenced to solvent peaks. NMR spectra were obtained on Bruker Avance 400 or 600 instruments. High resolution electrospray ionization (ESI) mass spectra were obtained from the MIT Department of Chemistry Instrument Facility.