Supplemental material for

Title: Establishment of a total liquid ventilation system using a saline-based oxygen micro/nano bubble dispersion in rats

Authors: Kenta Kakiuchi1, Kenichi Matsuda2*, Norikazu Harii2, Keitaro Sou3, 4, Junko Aoki2, Shinji Takeoka1*

Affiliations:

1. Department of Life Science and Medical Bioscience, Graduate School of Advanced Science and Engineering, Waseda University (TWIns), Shinjuku-ku, Tokyo 162-8480 Japan.

2. Department of Emergency and Critical Care Medicine, University of Yamanashi School of Medicine, 1110 Shimokato Chuo, Yamanashi 409-3898, Japan.

3. Waseda Bioscience Research Institute in Singapore (WABIOS),11 Biopolis Way, #05-02, Helios, Singapore, 138667 Republic of Singapore.

4. Organization for University Research Initiatives, Waseda University, 513 Wasedatsurumaki-cho, Shinjuku-ku, Tokyo, 162-0041 Japan.

Corresponding author:

Shinji Takeoka, Department of Life Science and Medical Bioscience, Graduate School of Advanced Science and Engineering, Waseda University (TWIns), Shinjuku-ku, Tokyo 162-8480 Japan. Phone: +81-3-5369-7324. Fax: +81-3-5369-7324. E-mail:

Kenichi Matsuda, Department of Emergency and Critical Care Medicine, University of Yamanashi School of Medicine, 1110 Shimokato Chuo, Yamanashi 409-3898, Japan.

Japan. Phone: +81-5-5273-9812. Fax: +81-5-5273-6716. E-mail:

Keywords: micro/nano bubbles, oxygen, liquid ventilation, lung

Preparation of Hb solution for measurement of oxygen content

The 40 mL fresh bovine blood samples (Shibaura Organ Co., Ltd., Tokyo, Japan) were separated into plasma and cellular components by centrifugation (2000 ´ g, 10 min, 4°C). After centrifugation, the layers of plasma containing platelets and white blood cells were carefully removed. An equal volume of Dulbecco's phosphate-buffered saline (DPBS) was added to the red blood cell (RBC) layer to resuspend and then centrifuged again at the same conditions (repeated twice). An equal volume of ultrapure water was added to the washed RBC layer and then put on ice for 30 min. After hemolysis, the membrane stroma materials of RBC were separated by ultracentrifugation (10000 ´ g, 10 min, 4°C). The Hb dispersion collected from the supernatant was diluted 100 fold with DPBS and filtered (DISMIC-25CS; Toyo Roshi Co. Ltd., Tokyo, Japan).

Fig. S1 Oxygen binding curve of an Hb solution at 23.2°C and [Hb] 0.0028mM.

Fig. S2 (a) Size distribution of bubbles generated by generator A. (b) Size distribution of bubbles generated with generator A and PVA. (c) Size distribution of bubbles generated with generator B. (d) Average size distribution of the bubbles generated with generator C. All dispersions were generated in TLV system and picked from the same point as experiment 1. Insets showed the picture of the generated bubbles by each generator (red: average result of first measurement (n=3). blue: average result of second measurement (n=3)). When the micro bubbles are generated in liquid, the liquid turns white generally and its color changes completely in a few second. We also confirmed such color change in (b), (c) and (d)

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Fig. S3 The absorption spectra of an Hb solution of samples in different oxygen contents. The spectrum of Ai shows a peak at 430 nm as a soret band of deoxyHb. The spectrum of Af has a peak at 415 nm as a soret band of oxyHb. The spectrum of As showed a broad peak from 415 nm to 430 nm because the part of deoxyHb was converted to oxyHb by the addition of the sample liquid.

a

b

Fig. S4 (a) Oxygen content in micro/nano bubble dispersions generated with different generators. (**p<0.01) (b) Oxygen content in macro bubble dispersions generated with generator A at different PVA concentration (0 - 6.4 ´10-3 wt%). The oxygen content was increased to 4.8 ´ 10-3 wt% from 1.6 ´ 10-3 wt% PVA concentration. It is suggested that this effect is due to the increase of micro bubble density by PVA addition.

There was no change when PVA was added to the sample solution prepared by using generators B and C (data not shown). This difference would probably be caused by a different bubble generation mechanism. Generator A adopts a gas-liquid mixing system to generate micro bubbles. Therefore, we assume PVA participates in the outbreak phase of micro bubbles. On the other hand, generators B and C adopt a pressurized dissolved system for bubble generation. This system makes oxygen gas on over-dissolved state under high pressure, where bubbles are generated by quickly reducing the pressure. Even if the added PVA participates in the stabilization of those bubbles, the oxygen content does not increase.

Fig. S5 Relationship between the oxygen content measured by two different methods over a time period (n = 3). (Opened circle: oxygen dissolved meter, closed circle: our new method) The result from using our method showed higher oxygen content than the result measured with a dissolved oxygen meter within a min. However, there was no significant difference between the results after a min. It means that large micro bubbles worked as oxygen carried for a min. After a min, smaller bubbles remained in a liquid and kept the liquid super-saturation.

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