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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers
Size-controlled Preparation of Micro-sized Perfluorocarbon Emulsions as Oxygen Carriers via SPG Membrane Emulsification Technique Xiaoting Fu, Seiichi Ohta, Masamichi Kamihira, Yasuyuki Sakai, and Taichi Ito Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.9b00194 • Publication Date (Web): 21 Feb 2019 Downloaded from http://pubs.acs.org on March 4, 2019
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Size-controlled Preparation of Micro-sized Perfluorocarbon Emulsions as Oxygen Carriers via SPG Membrane Emulsification Technique Xiaoting Fu†, Seiichi Ohta‡, Masamichi Kamihira§, Yasuyuki Sakai†, Taichi Ito*†‡
†Department of Bioengineering, The University of Tokyo, 7-3-1 Hongo, Bunkyoku, Tokyo 113-8656, Japan ‡Center for Disease Biology and Integrative Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan §Department of Chemical Engineering, Kyushu University, 744 Motooka, Nishiku, Fukuoka 819-0395, Japan
ABSTRACT: We have developed micro-sized perfluorocarbon (PFC) emulsions with different sizes as artificial oxygen carriers (OCs) via Shirasu porous glass (SPG) membrane emulsification. Monodispersed PFC emulsions with narrow size distribution were obtained. By changing the membrane pore size, we were able to precisely control the size of emulsions, and fabricate emulsions similar in size to human red blood cells (hRBCs). Behaviors of Pluronics with different physiochemical properties (F127, F68,
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P85, and P103) as surfactants we are also investigated, which evidenced that the type and concentration of Pluronics have a major impact on the size of emulsions and the response to different thermal conditions. The F127-stabilized micro-sized PFC emulsions were stable even during autoclave sterilization. The emulsions were loaded with Ru(ddp)—an oxygen-sensitive probe—on their surfaces to indicate oxygen concentration. Finally, incubations with HeLa cells that show fluorescence in response to hypoxia cultured in 2D and 3D suggested the promising potential of our emulsions for OCs.
INTRODUCTION Perfluorocarbon (PFC) emulsions have long been investigated for use in biomedical applications including contrast agent,1,2 oxygen carriers (OCs),3-5 and cancer therapy.6-8 Among them, the development of OCs has been aiming to provide adequate distribution of oxygen to treat ischemic diseases and facilitate tissue growth in 3D cell culture system to overcome the worldwide shortage of donor tissues. To date, a variety of PFC-based OCs have been developed with sizes ranging from nanometers to micrometers.4,9-11 Owing to the low polarizability, PFC can dissolve significant volume of gases (e.g. O2, CO2, and N2) by physically entrapping the molecules. The amount of dissolved gases can be described by Henry’ Law, in which the solubility is linearly dependent on the partial pressure. In addition to their oxygen carrying capacity,
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PFC is of chemically inert with low side effects. The use of nano-sized PFC emulsions as OCs was proposed as early as 1966, and they have been developed commercially for more than 20 years.12,13 Other than nano-sized emulsions, micro-sized PFC-based OCs have also attracted attention.10,14-16 One of the key problems in previously reported PFC-based OCs is size control,17-20 because it relates to pivotal issues such as biological interactions and durability in practical applications. Although precise size control has been proposed as a requirement for OCs development, it is yet difficult to be achieved by traditional methods, which result in a wide size distribution.4,21-23 Shirasu porous glass (SPG) membrane emulsification technique, as illustrated in Figure 1, is an effective approach enabling to produce monodispersed emulsions with low energy comsuption.24-28 Uniform-sized oil-in-water (O/W) or water-in-oil (W/O) emulsions can be produced by ejecting the disperse liquid through pores of membrane into continuous liquid via pressure-forcing. Moreover, the size of the emulsions can be controlled in numerous ways (e.g., by altering the type or concentration of surfactant, or the membrane pore size). Although the fabrication of PFC emulsions using microsieves was previously reported, 29 to the best of our knowledge, none of the current papers has described the preparation of PFC emulsions by SPG membrane emulsification. Herein, we have, for the first time, successfully prepared PFC emulsions as OCs using
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SPG membrane emulsification. The size of the emulsions was controlled by various means: altering the pore sizes of the membranes; varying the surfactant concentration; and using different types of Pluronic copolymers (F127, F68, P85, and P103) as surfactants. The size distribution and stability of the emulsions at different temperatures were evaluated. The formed PFC emulsions were further functionalized with an oxygensensitive probe to indicate oxygen concentration. Furthermore, improved oxygen supply using the PFC emulsions was demonstrated by culturing them with HeLa cells that had been genetically engineered to show hypoxia-responsive EGFP expression.
Figure 1. Schematic illustration for preparation of PFC emulsions via SPG membrane emulsification EXPERIMENTAL SECTION Materials. Pluronics P85 and P103 were kind gifts from Adeka Co. (Tokyo, Japan).
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Pluronics F127 and F68 were purchased from Sigma-Aldrich (St. Louis, U.S.A.). Tris (4,7-diphenyl-1,10-phenanthroline) ruthenium(II) dichloride complex—abbreviated as Ru(ddp)—was purchased from Santa Cruz Biotech. (Texas, U.S.A.). Perfluorodecalin (FDC), sodium chloride, high glucose Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin-amphotericin B suspension (PSA), and phosphate buffer saline (PBS) were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Human red blood cells (hRBCs) from donated blood were provided by Japanese Red Cross Society, and were approved by Ethics Committee of the Faculty of Medicine, The University of Tokyo and the Ministry of Health, Labour and Welfare, Japan. Emulsion fabrication. Emulsions of FDC, which is a member of PFCs, were prepared via SPG membrane emulsification technique using an internal pressure-type module (SPG Technology, Miyazaki, Japan). Before membrane emulsification, hydrophilic SPG membrane (SPG Technology, Miyazaki, Japan) with pore sizes of 2, 3, 4 or 5 μm were immersed in continuous phase that comprised 0.9% NaCl aqueous solution with the desired amount of Pluronics copolymers as surfactants (0.2, 0.5, 1, 2, 3 wt%), then sonicated at room temperature for 1h. To obtain the FDC emulsions, 2 mL of FDC (the disperse phase) was pressurized into 20 mL of the continuous phase through SPG
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membrane while stirring at 150 rpm. Interfacial tension measurement. Interfacial tension was measured by the pendant drop method using a contact angle meter (PGx 13220-001, Buchel BV, Veenendaal, Netherlands). A droplet of FDC was formed within saline containing Pluronics (F127, F68, P85, P103) with various concentrations, and then the interfacial tension was measured. We conducted ten independent experiments for each condition. Data were expressed as average ± standard deviation. Optical observation. The FDC emulsions was observed using an inverted phase contrast microscope (IX73, Olympus, Tokyo, Japan). Size distribution. The size distribution of emulsions was measured by a laser diffraction particle size distribution analyzer (LA-950V2, Horiba, Kyoto, Japan). Thermal and long-term stability tests. For evaluating the stability of FDC emulsions under various thermal conditions, the size distributions of emulsions were measured before and after keeping the emulsions at 4 °C for 1 day and at 37 °C for 1 day and after sterilizing 6 mL of each sample at 120 °C and 100 kPa for 20 min followed by preservation at room temperature for 1 day. We conducted three independent experiments for each condition. Data were expressed as average ± standard deviation. For long-term stability, the samples were preserved at both 4 °C and room temperature.
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Size distributions of emulsions were measured at certain period. Loading oxygen sensitive probe to emulsions. In order to enable the emulsions to indicate oxygen concentration, 1 mL 0.1 mg/mL Ru(ddp)-in-ethanol solution was well mixed with 20 mL of 10 vol% FDC emulsions stabilized with 2% of F127 after emulsification followed by washing twice with pure water. The samples were divided into two groups: one was further deoxygenated by bubbling nitrogen gas for 20 min (the hypoxia group), whereas the other received no extra treatment (the normoxia group). Confocal microscopic images of Ru(ddp)-loaded FDC emulsions were taken by a confocal laser scanning microscope using a Kr laser (568 nm) for excitation light (LSM510 META NLO; Carl Zeiss AG, Jena, Germany). Oxygen loading and release. The oxygen loading and release of emulsions were evaluated by measuring the time change of the dissolved oxygen (DO) concentration. For oxygen loading, 20 mL of the 10 vol% Ru(ddp)-loaded FDC emulsions was first deoxygenated by nitrogen and then oxygenated by air (50 cm3/min). For oxygen release, same amount of the sample was first oxygenated by air and then deoxygenated by nitrogen (50 cm3/min). During the oxygen loading and release, 0.2 mL of the sample was taken out at various time points, and then fluorescence from loaded Ru(ddp) was measured using spectrofluorometer (FP-8200, JASCO, Tokyo, Japan). The obtained
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fluorescent intensities were converted to DO concentrations using a calibration curve prepared via DO meter (Visiferm DO ARC 120, Hamilton, Reno, USA). DO concentration during the oxygen loading and release was also monitored by inserting DO meter in the emulsion samples. We conducted three independent experiments for each condition. Data were expressed as average ± standard deviation. Oxygen supply. HeLa cells that had been genetically engineered to express EGFP in response to hypoxia were used to examine oxygen supply performance.30 For the 2D cell culture, the cells were seeded on a 24-well glass bottom plate (Iwaki, Asahi Glass Co., Tokyo, Japan) at a density of 2 ×104 cells per well with 750 μL of culture medium (DMEM, 10 % FBS, 1 % PSA) and kept overnight at 37 °C in 20 % O2 and 5 % CO2. For 3D cell culture, cells were seeded on the 24-well glass bottom plate at a density of 2 ×105 cells per well and kept at 37 °C in 20 % O2 and 5 % CO2 for 48 h. Then, 500 μL of 10 vol % FDC emulsions was added into the cells, which were kept at 37 °C in 2 % O2 and 5 % CO2 for 48 h. Fluorescent pictures were taken immediately after taking out the cells from the incubator, using fluorescent microscope (U-HGLGPS, Olympus, Tokyo, Japan) for 2D culture and confocal laser scanning microscope (TCS-SP2, Leica, Wetzlar, Germany) for 3D culture with a green emission filter at an excitation wavelength of 488 nm. The thickness of the formed multilayer was measured by randomly counting the number of
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cells at 144 points in the cross sections of the confocal microscopic images. Statistical analysis. Statistical test was performed using analysis of variation (ANOVA). Significant differences were considered as P
