Supplementary Materials and Methods
Nanoparticle Fabrication: These methods were adapted from the water-in-oil-in-water emulsion
solvent evaporation technique.15 Prior to NP fabrication, a 2% solution of polyvinyl alcohol
(PVA) (Acros Organics, Morris Plains, NJ) was made by dissolving PVA (20 g) in H2O (1,000
mL) with low heat. On the day of NP fabrication, methylene chloride [(MC), 50 µL, CH2Cl2,
Fisher Scientific)] was added to the PVA solution (2%, 24 mL). The PVA-MC solution was
centrifuged at 1,000 rpm for 10 min and filtered with a 0.22µm sterile filter to remove any
residual undissolved PVA. A polymer solution was made by dissolving high-molecular weight
85:15 uncapped poly(lactic-co-glycolic acid) (PLGA) (180 mg, Lakeshore Biomaterials,
Birmingham, AL) in MC in a glass scintillation vial. An aqueous solution of BSA (15 mg,
Jackson Laboratories in PBS (1.5mL)) or BSA and 5-fluorouracil [(5FU), MB Biomedical,
15mg] was added and vortexed. The PLGA-MC-BSA±(5FU) solution was placed in an ice bath
for 5 min and then sonicated at 45W for 150 s using a high-power sonicator (Misonix S-3000
Sonicator, Farmingdale, NY). The PLGA-MC-BSA ± (5FU) solution was added in two portions
with intermittent vortexing to the PVA solution (24 mL). The PVA-PLGA-MC-BSA ± (5FU)
solution was placed on an ice bath for 5 min and then sonicated again with 60W of energy for
150 s. This solution was stirred overnight. The suspension was then transferred into a thickwalled polycarbonate centrifuge tube and centrifuged at 20,000 rpm for 60 min at 4 ºC. After
discarding the supernatant, the pellet was resuspended in distilled water, repetitively vortexed,
and sonicated for 10 s. The resulting solution was centrifuged again at 20,000 rpm for 60 min at
4 ºC, pouring off the supernatant after centrifugation. The NP pellet was resuspended again in
distilled water (8 mL) and sonicated for 10 s. To remove large aggregates, the NP solution was
centrifuged at 1,000 rpm for 10 min at 4 ºC. A sample of the NP suspension was analyzed by a
submicron-particle sizer (Multisizer IIe, Beckman Coulter, Inc., Palo Alto, CA), Zetasizer 3000
(Malvern Instruments, Southborough, MA) and by scanning electron microscopy (SEM). The
supernatant was collected, frozen at -80 ºC for 45 min, and lyophilized for 2 days. The dry NP
powder was stored in a desiccator at -80 ºC until needed.
Conjugation of VivoTMTag 680 to Nanoparticles: BSA-PLGA NPs were conjugated to
fluorochrome–hydroxysuccinimide (VivoTag™680) using the protocol adapted from VisEn
Chemistry Notes.
Briefly, a NP solution was prepared at 5.0 mg/mL in 100mM sodium
phosphate buffer, pH 7.5. Next, the NP solution was added to VivoTag™680 such that the
concentration of the fluorochrome was approximately 2 mg/mL. The solution was mixed gently,
covered from light and allowed to react for 2 hours. Fluorochrome-labeled NPs (VT680-NPs)
were purified by centrifugation at 20,000 rpm for 60 min at 4 ºC, pouring off the supernatant
afterwards. VT680-NPs (0.1mg) were dissolved in MC (100uL), added to DPBS (1mL), and
stirred under a vacuum hood to allow the solvent to evaporate. Two hours later, the sample was
resuspended in DPBS (1mL) and the concentration of fluorochrome per NP was determined
spectrophotometrically. The combination of the number of NPs (determined by the mass,
density, and mean diameter) and the total absorbance enabled the determination of the number of
fluorochromes per NP.
This absorbance value was then subtracted from the absorbance of
“blank” BSA-PLGA NPS to obtain the number of fluorochromes per VT680-NPs.
Microbubble-Nanoparticle Composite Agent Fabrication: To fabricate MB-NP composite
agents, VT680-NPs or 5FU-NPs were combined with 1-Ethyl-3-(3 dimethylaminopropyl)carbodiimide (EDC) (1mg) and N-hydroxy-sulfosuccinimide (Sulfo-NHS) (2.2 mg) in 0.1 M
MES buffer, pH 6. The solution was allowed to react for 1 hr at room temperature. Next,
activated NPs were purified from excess EDC by centrifuging at 20,000 rpm for 60min at 4 ºC,
pouring off the supernatant after centrifugation. While particles were spinning, albumin MBs
were washed three times by differential centrifugation in degassed PBS to remove excess BSA.
Purified VT680-NPs or 5FU-NPs were incubated with washed MBs for 2 hr at room
temperature.
Subsequent to incubation, MBs conjugated to VT680-NPs or 5FU-NPs were
purified from unbound NPs by washing the suspension three times with degassed PBS. UV
spectroscopy measurements were performed on the MNCA supernatant to confirm that virtually
all NPs were bound to MBs. Subsequent to washing, the concentration and mean diameter of
MNCAs was determined using electrozone sensing (Multisizer-III, Beckman Coulter, Fullerton,
Calif) and the number of bound VT680-NPs per MB was determined spectrophotometrically.
Nanoparticle binding to MBs was assumed to be equivalent for 5FU-NPs and VT680-NPs.
Cytometry Imaging: For cytometry imaging, MBs were incubated with or conjugated to VT680NPs (MNCA), or imaged alone. Imaging was performed on an ImageStream (Amnis
Corporation, Seattle, WA) utilizing 658 laser (emission filter, 660-735nm). Data were collected
using INSPIRE 3.0, and analyzed in IDEAS 3.0 software (Amnis Corporation). Focused single
MBs were gated and the colocalization of VT680-NP fluorescence with MBs was assessed.
In Vitro 5-Fluorouracil Loading and Release: The encapsulation efficiency is defined as the
ratio of amount of drug encapsulated to that of the drug used in nanoparticle preparation. To
determine the drug content in the nanoparticles, lyophilized microspheres were dissolved in MC
and the MC-PLGA-5FU solution was diluted with simulated body fluid buffer saline (SBF, pH
7.4). The mixture was then vortexed vigorously for 5 min and MC was evaporated under a fume
hood while stirring for 4hrs. The final solution was taken for high-performance liquid
chromatography (HPLC) analysis. For in vitro release studies, 7 mg of lyophilized PLGA NPs
were suspended in 5 mL PBS at 37°C for two weeks. 0.5 mL of the suspension media was
removed with replacement at each time point and immediately frozen. After all samples had been
collected, the samples were rapidly thawed and centrifuged for 20 minutes at 20,000 rpm and 4°
C. Supernatants were carefully drawn off and allowed to reach room temperature before being
analyzed. 5-FU drug concentration was determined using a UV spectrophotometer (NanoDrop
2000C, Thermo Scientific, Wilmington DE). The absorbance spectrum of a stock solution of 0.1
mg/mL of 5-FU in room temperature PBS was measured and a peak was discovered at 266 nm.
Drug concentration in the samples was measured at 266 nm and calculated using a standard
curve.
Tissue Processing: Four hours after treatment with VT680-NPs and/or MBs or VT680-MNCAs,
animals were euthanized and the left ventricle was cannulated. Blood was exsanguinated with
10mL of 2% Heparinized Tris Buffered Saline (TBS) with CaCl2 (0.68mM), followed by 10mL
of TBS with CaCl2 (0.68mM), each for 10 minutes at 100 mm Hg. Following blood removal,
tissues were perfusion-fixed with 4% paraformaldehyde in PBS for 30 min. Specimens were
excised, embedded in O.C.T. medium, flash frozen, and cut into 5-micron thick sections.
Capillaries were labeled with Bandeiraea simplicifolia lectin (BSI-lectin) conjugated to Alexa
488 (1:200; Sigma Biosciences). Digitized images of cross-sectioned specimens were acquired
using a Nikon Eclipse TE2000-E microscope equipped with confocal accessories (Nikon DEclipse C1) using a ×60 Nikon oil immersion objective.