Supporting InformationSeekell et al. 10.1073/pnas.1608438113SI Materials and MethodsMaterials. Poly(D,L-lactide-coglycolide) 50:50 (PLGA) (inherentviscosity, 0.66 dL/g) was purchased from Durect Corporation. Per-fluorooctylbromide (PFOB), dichloromethane (DCM), Plur-onic F-68, polyvinylpyrrolidone (PVP) (average Mr, 10,000),polyvinyl alcohol (PVA) (average Mr, 31,000−50,000), and so-dium bicarbonate were purchased from Sigma-Aldrich. Plasma-Lyte A was purchased from Baxter Corporation. Ultrapurewater was obtained by purifying deionized water with a Milli-QSystem (18 MΩ; Millipore).

Particle Characterization. The size distribution of PHMs was mea-sured by light obscuration (Accusizer 780A). Cross-sectional anal-ysis and surface morphology were assessed by scanning electronmicroscopy (SEM) (Supra55VP). Visualization of the core–shellstructure was accomplished with an ion beam polisher (JEOL).All samples were adhered to a holder with double-sided Cutape and sputter coated with a 5-nm-thick layer of Pt/Pd at aratio of 80:20 at 40 amp. All samples were imaged at a voltagerange 2.0–5.0 kV.

Quantification of Porosity and Shell Thickness.The porosity and shellthickness were determined by image analysis of SEM cross-sec-tional images using ImageJ.

Swelling Experiments. Swelling experiments were performed aspreviously described. Briefly, PHM powders (100 mg) wereplaced in vials and their total weight recorded (weight of PHM =weight of vial + PHM – weight of vial). Samples were hydratedwith Plasma-Lyte A (37 °C) and placed on a rocker in an in-cubator at 37 °C. At various time points, vials were removedfrom the incubator, the PHMs rapidly filtered, and theirweights recorded. Excess water was removed during the filtra-tion process. The water uptake was reported as the weight ofwater/weight of PHM.

Differential Scanning Calorimetry.The exothermic and endothermicheat flow of PLGA and hydrated PHMs were measured usingdifferential scanning calorimetry (DSC Model Q200; TA Instru-ments). Samples masses were determined using thermogravimetricanalysis. All samples were loaded into aluminum sample pans andhermetically sealed. Dry samples were cooled/heated from −10 °Cto 60 °C at 20 °C/min, and hydrated samples were cooled/heatedfrom 10 °C to 80 °C at 20 °C/min. Heat flow through the sampleswas measured as a function of temperature, relative to an emptyreference pan.

Instrumentation for in Vivo Animal Experiments. The left femoralartery and right femoral vein were cannulated (24- and 20-gaugeangiocatheters, respectively) and transduced for continuous centralarterial and venous blood pressure monitoring. A baseline arterialblood gas sample (ABG) was taken for analysis by cooximetry(ABL80 Flex CO-OX; Radiometer America). Plasma-Lyte A wascontinuously infused via both arterial (2mL/h) and venous (1mL/min)lines to maintain line patency and animal hydration. Full sternotomywas then performed to allow access for instrumentation. A pressurecatheter (Millar Mikro-Tip Pressure Catheter Transducer; modelSPR-671; 1.4F) was placed into the pulmonary artery via the rightventricular outflow tract to monitor pulmonary arterial pressure.A pressure–volume catheter (Millar Mikro-Tip Pressure–Vol-ume Catheter Transducer; 9-mm spacing; model SPR-847; 1.4F)was then placed transapically into the left ventricle to monitor

stroke volume (SV), left ventricular end diastolic volume, andleft ventricular end diastolic pressure (LVEDP). Finally, a pO2probe (E-Series Sensor; Oxford Optronix) was placed into theright ventricle for continuous measurement of the partial pres-sure of oxygen in the venous blood (PvO2). Continuous hemo-dynamic data were recorded using LabChart Pro-8 software(ADInstruments). Following instrumentation, FiO2 was decreasedfrom 1.0 to 0.3. An arterial blood gas was taken to determine thebaseline hemoglobin content of the blood. Controlled hemorrhagewas then performed via the arterial line to decrease the hemo-globin level of each animal to ≤9.6 g/dL. Sodium chloride (0.9%)was simultaneously infused via the femoral venous line to replaceintravascular volume and maintain adequate arterial blood pres-sure. A further 10-min stabilization period was then ensured beforeinitiation of the experiment protocol. Each animal received abolus injection (5 mL) of oxygenated Plasma-Lyte A via thevenous line over 1 min, followed by a bolus of Plasma-Lyte A(1 mL) to flush the injection line. PvO2 data were collected duringincrement and until return to baseline. This process was sequen-tially repeated for oxygen-loaded PHMs [1% and 2% (wt/wt)]resuspended in oxygen-saturated Plasma-Lyte A. Arterial bloodwas drawn between each injection and cooximetry performed todetermine baseline values for the subsequent injection. Ifnecessary, animals were again hemorrhaged as described tomaintain a hemoglobin level between 8.5 and 9.5 g/dL. Theobservation period for the final PHM infusion [i.e., 2% (wt/wt)]was extended to 60 min, and repeat ABG samples were drawnat 30 and 60 min.

Hemolysis Assay. All procedures were approved by the IRB atBoston Children’s Hospital. Venous blood was collected fromhealthy volunteers in tubes containing heparin (17 μL/mL). ThehRBCs were collected and washed four times by centrifugation at500 × g for 10 min and then resuspended with Plasma-Lyte A (fourparts hRBC to six parts Plasma-Lyte A). PHM dispersions wereprepared in Plasma-Lyte A with increasing particle concentrations(0.5–20 mg/mL). Aliquots of stock hRBCs (100 μL) was added tothe PHM dispersions (1 mL), and the solutions were incubated at37 °C with gently rocking. As a negative control, hRBCs (100 μL)were incubated with Plasma-Lyte A (100 μL; 0% hemolysis),whereas for a positive control, hRBCs were treated with purifiedwater (100 μL; 100% hemolysis). The solutions were incubated for5, 30, and 60 min, and the serum was collected by centrifugation at378 × g for 15 min. Each experiment was repeated in triplicate.Hemolysis was determined by measuring the release of lactatedehydrogenase (LDH) from the hRBCs using the LDH-basedTOX-7 kit (Sigma-Aldrich) according to the manufacturer’sinstructions.

SI CalculationsCalculation of Fractional Oxygen Released.

Fractional O2   released

=

�1.36 mL O2   gas

g  hgb × g HgbdL  blood×

ΔSO2100

�+ ð0.0031×PaO2Þ× dL  blood

total mL O2,

where Hgb is the hemoglobin concentration (grams per deciliter),ΔSO2 is the change in oxygen saturation from baseline (percent-age) at time (t), and PaO2 is the partial pressure of oxygen(millimeters of mercury).

Seekell et al. www.pnas.org/cgi/content/short/1608438113 1 of 4

Calculation of Oxygen Carrying Capacity of Blood from SO2.

Volume  of   oxygen  ðmLÞ=��

1.36 mL O2   gasg Hgb

×g Hgb

dL  blood×SO2

100

�

+ 0.0031×PaO2

�×dL  blood,

where Hgb is the hemoglobin concentration (grams per deciliter),ΔSO2 is the change in oxygen saturation from baseline (percentage)at time (t), and PaO2 is the partial pressure of oxygen (millimetersof mercury).

Calculation of the Actual Oxygen Carrying Capacity of Blood per Gramof hRBC.

Density  of   blood  cells  circulating= 1,125kgm3,

Concentration  of  RBC  in  blood  ≈ 5.0× 109RBCmL

,

Concentration  of  Hb  in  blood= 15g

100 mL  blood,

Oxygen  carrying  capacity  of  Hb= 1.34mL O2

g Hb.

Then we calculated the milliliters of O2 per RBC and the numberof RBCs per gram using the following equations:

mL O2

RBC=

15 g Hb100 mL  blood

×mL  blood

5× 109  RBC×1.34 mL O2

1 g Hb

= 4.02× 10−11  mL O2

RBC,

RBCg

=  5× 109RBCmL  blood

×mL  blood

1.125  g RBC= 4.3× 109

RBCg

.

The gas carrying capacity of RBCs per gram of RBC is given bythe following:

mL O2

g=4.02× 10−11mL O2

RBC×4.39× 109RBC

g= 0.17

mL O2

g.

Calculation of Cardiac Index and Pulmonary Vascular Resistance. Thecardiac index (CI) (milliliters per minute per kilogram) wascalculated using the following equation:

CI=COkg

=SV  ×  HR

kg,

where CO is the cardiac output (milliliters per minute), SV is thestroke volume (milliliters per beat), HR is the heart rate (beats perminute), and kg is the mass of the rat. The pulmonary vascular resis-tance (PVR) (millimeters of mercury·minute·kilograms per milliliter)was calculated using the following equation:

PVR=�MPAP−LVEDP

CI

�;          MPAP=  

PASP+ 2ðPADPÞ3

,

where MPAP is the mean pulmonary arterial pressure (millimetersof mercury), PASP is the pulmonary arterial systolic pressure (mil-limeters of mercury), PADP is the pulmonary arterial diastolic pres-sure (millimeters of mercury), LVEDP is the left ventricular enddiastolic pressure (millimeters of mercury), and CI is the cardiac in-dex (milliliters per minute per kilogram).

20 min6 min2 min0.5 min B CPost-freeze

dryingA

Fig. S1. (A) Phase separation of PLGA and PFOB in the absence of Pluronic F-68 results in nonporous shells. (B) Fluorescent microscopy confirms the solidnature of PLGA shell in the absence of F-68. (C) Freeze-drying results in persistence of core–shell particles with PFOB-filled cores as PFOB is not permeablethrough the PLGA shell.

Fig. S2. (A) Slow precipitation of PLGA (i.e., DF = 86) led to coalescence of nanoemulsions at the core and encapsulated the PFOB droplet. (B) Incorporation ofa water-soluble fluorescent dye during fabrication confirmed coalescence of the spontaneously formed nanoemulsions around the PFOB core.

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Fig. S3. Rehydration of PHMs results in gas-filled particles. (Scale bar, 10 μm.)

Fig. S4. Experimental time line and protocol for in vivo efficacy testing.

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1 100

2

4

6

Diameter (µm)

Num

ber %

DPF=0DPF=60

0.0

0.5

1.0

Gas

Car

ryin

g C

apac

ity

(mL

O2/

g PH

M)

DPF = 0 60

P > 0.05

Fig. S5. (A and B) Size distributions (A) and gas carrying capacity (B) for formulation 4 at 0 and 60 d postfabrication (dpf). Freshly made PHMs were eitheranalyzed immediately or placed in a vacuum desiccator at room temperature and analyzed at 60 dpf.

Table S1. List of manufacturing parameters and shell properties

FormulationHomogenization

speed, rpm* Dilution factor Mean diameter, μmMean shell

thickness, μmMean pore

diameter, μmMean pore

density, #/μm2

1 2,000 86 8.5 ± 0.24 2.3 ± 0.10 1.4 ± 0.2 0.3 ± 0.032 2,000 150 9.3 ± 0.29 1.1 ± 0.06 0.24 ± 0.03 6.5 ± 0.73 4,000 86 3.3 ± 0.84 0.2 ± 0.07 0.12 ± 0.07 9.0 ± 104 4,000 150 1.3 ± 0.99 0.31 ± 0.12 0.25 ± 0.05 6.1 ± 0.65

*Silverson Machines, Inc.; East Longmeadow, MA; model: L5M-A.

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