(M1230-08-52) Potential of Ultra Fine Bubbles to Enhance Insulin Membrane Permeability

Purpose: More than 100 years have passed since the discovery of insulin, yet it still cannot be administered orally. A major reason why insulin has not been developed as an oral or intranasal formulation is its poor membrane permeability due to aggregation. We are attempting to address this issue using ultra fine bubble (UFB). UFB is defined as bubble smaller than 1 μm. As shown in Fig. 1, bubble is classified according to its size. Millibubble and microbubble, which is larger than UFB, rises to the surface and collapse, whereas UFB remains stable for a long period because of its extremely small size, low buoyancy, and dominant Brownian motion. We have successfully observed, via electron microscopy, that insulin molecules adsorb onto the surface of UFBs. The report was presented at the Annual Meeting of the Society of Separation Process Engineers, Japan (Shimane, December 2024). We have also demonstrated the potential of UFB to suppress insulin aggregation. The report was presented at the 145th Annual Meeting of the Pharmaceutical Society of Japan (Fukuoka, March 2025). Furthermore, our experimental results suggest that insulin may permeate artificial membranes in the presence of UFB.The report was presented at the 68th Annual Meeting of the Japan Diabetes Society (Okayama, May 2025).  In this study, we further investigate whether insulin, whose aggregation is suppressed by UFBs, can permeate artificial membranes.

Methods: Conventional UFB generators typically produce UFBs at concentrations of several billion per milliliter. In this study, high concentration UFB water was obtained by evaporative concentration of UFB water prepared from ultrapure water. Solutions were prepared using ultrapure water, PBS, and saline as base solvents, each with and without UFBs. UFB containing PBS and saline were prepared by diluting each with high concentration UFB water. In the case of UFB containing solvents, additional preparations were made with different concentrations of UFB. Human insulin was mixed with these solvents, and membrane permeability tests were conducted using PAMPA (Parallel Artificial Membrane Permeation Assay) and μFLUX systems to determine the permeability coefficients.

Results: We successfully observed the adsorption of insulin molecules around UFBs in UFB-containing water using liquid-phase transmission electron microscopy (liquid-phase TEM) and scanning electron microscopy (SEM). This phenomenon is shown in Fig. 2: (a) represents images from liquid-phase TEM, and (b) represents SEM images of freeze-dried samples. Panels (a-1) and (b-1) show samples without UFBs, while (a-2) and (b-2) show samples containing UFBs. In the presence of UFBs, insulin molecules were observed to adsorb along circular or arc-like structures, as indicated by the red circles in (a-2) and (b-2). Such adsorption behavior was not observed in the absence of UFBs. The membrane permeability coefficient, P_e (cm·s⁻¹), is defined by Equation (1): P_e = J / (60C_donor) (1) where C_donor (µg·mL⁻¹) is the concentration of the donor solution, and J (µg·cm⁻²·min⁻¹) is the flux.　The flux J is calculated using Equation (2): J = V / A × dc/dt   (2) where V (mL) is the acceptor volume, A (cm²) is the membrane surface area, dc (µg·mL⁻¹) is the change in concentration in the acceptor, and dt (min) is the change in time. Based on these equations, the permeability coefficient was determined. Fig. 3 shows the relative membrane permeability coefficient, with the value in saline without UFBs set to 1. In all solvents tested—ultrapure water, PBS, and saline—a significant increase in membrane permeability was observed in the presence of high concentrations of UFB.

Conclusion: The membrane permeability of insulin was compared in the presence and absence of UFB using aqueous solution, PBS, and saline as solvents. The results suggest that high concentrations of UFB promote insulin membrane permeability.

Acknowledgements: This study was partially supported by collaborative research funding from Pharstoma Inc. We gratefully acknowledge PHYSIO MCKINA Co., Ltd. for providing equipment and valuable technical advice.

Classification and properties of bubble: Milli Bubble, more than 100 μm ; Micro Bubble, 1 - 100 μm ; Ultra Fine Bubble, less than 1 μm.

Electron microscopy of insulin in ultrapure water with and without UFB: (a-1) TEM without UFB ; (a-2) TEM with UFB ; (b-1) SEM without UFB ; (b-2) SEM with UFB.

Relative membrane permeability Pe in saline with and without UFB.