TY - JOUR
AU1 - Kodama,, Tetsuya
AU2 - Tomita,, Noriko
AU3 - Horie,, Sachiko
AU4 - Sax,, Nicolas
AU5 - Iwasaki,, Hiroko
AU6 - Suzuki,, Ryo
AU7 - Maruyama,, Kazuo
AU8 - Mori,, Shiro
AU9 - Manabu,, Fukumoto
AB - Abstract Sonoporation is achieved by ultrasound-mediated destruction of ultrasound contrast agents (UCA) microbubbles. For this, UCAs must be tissue specific and have good echogenicity and also function as drug carriers. Previous studies have developed acoustic liposomes (ALs), liposomes that encapsulate phosphate buffer solution and perfluoropropane (C3F8) gas and function as both UCAs and drug carriers. Few studies have examined the co-existence of gas and liquid in ALs. This study aims to elucidate AL structure using TEM. The size, zeta potential and structure of ALs were compared with those of two other UCAs, human albumin shell bubbles (ABs; Optison) and lipid bubbles (LBs). ABs and LBs encapsulate the C3F8 gas. Particle size was measured by dynamic light scattering. The zeta potential was determined by the Smoluchowski equation. UCA structure was investigated by TEM. ALs were ~200 nm in size, smaller than LBs and ABs. ALs and LBs had almost neutral zeta potentials whereas AB values were strongly negative. The negative or double staining TEM images revealed that ~20% of ALs contained both liquid and gas, while ~80% contained liquid alone (i.e. nonacoustic). Negative staining AB images indicated electron beam scattering near the shell surface, and albumin was detected in filament form. These findings suggest that AL is capable of carrying drugs and high-molecular-weight, low-solubility gases. nanobubbles, drug delivery system, sonoporation, ultrasound contrast agent, cavitation Introduction Ultrasound contrast agents (UCAs) are nano/ microbubbles that contain air or a high-molecular-weight, low water-solubility gas (e.g. C3F8, C4F10 and SF6) encapsulated in a lipid or albumin shell [1–6]. The diameters of UCAs vary from 100 nm to 10 μm and their behavior primarily depends on the ultrasound characteristics. The behavior of UCAs is described by an equation of motion that consists of external force, viscosity and stiffness terms [7]. When the external force is small, i.e. ultrasound pressure is small, UCAs undergo small volumetric oscillation (linear). With increasing ultrasound pressures, the amplitude of volumetric oscillation increases and oscillation becomes aperiodic (nonlinear), resulting in destruction of UCAs [1,8]. Drug delivery via sonoporation using ultrasound and UCAs is a technique used for diagnosis and treatment [1,8] and is based on bubble destruction modes. During sonoporation, primary UCAs and subsequent bubble cavitation generated by the collapse of the UCAs induce mechanical forces such as liquid jets and shock waves [8]. These forces interact with the surrounding cells, resulting in the permeation of exogenous molecules into cells [1,8]. Sonoporation is a noninvasive, nonimmunogenic and tissue-specific procedure that has been used to treat cancer and many other diseases [1,3,9]. However, the efficiency of molecular delivery is relatively low; therefore, it has not been recognized as a clinically valuable approach. One strategy towards improving the efficiency of molecular delivery is to develop UCAs that are tissue-specific and that can function as drug carriers. Suzuki et al. [9] developed a novel form of liposome containing the C3F8 gas and phosphate buffer solution and demonstrated that it functions as an acoustic liposome (AL) applicable to a nonvirus molecular delivery system [5,6]. The liposome surface was covered with polyethyleneglycol (PEG); therefore, it was assumed that this molecule would not be incorporated by the reticuloendothelial system, thereby allowing a longer retention in the blood [10]. In addition, the tumor-targeting potential and drug-carrying capability are significantly improved by conjugating PEG with ligands specific for the target tissue and by producing bubbles with diameters of <100 nm, which allows for enhanced permeability and retention (EPR) effects [11,12]. These studies concluded that the liposome would be acoustic due to the differences between ultrasound backscatter intensities in the presence/absence of ultrasound. However, the coexistence of gas and liquid in the liposome has not been examined, neither have its size and structure been discussed. The present study investigated the size, zeta potential and structure of ALs and compared these values with those of two other types of UCAs: a single human albumin shell (ABs; Optison) and lipid bubbles (LBs). Both UCAs encapsulated the C3F8 gas, which was identical to the liposome gas. Transmission electron microscopy (TEM) was used to assess the structure of UCAs, and the TEM images were obtained using either negative or double staining. The TEM findings will be used as parameters to evaluate biodistribution, safety and efficacy of micro/nanoparticulate systems. Methods Nano/microbubble preparation Three types of UCA—ABs (5.0–8.0 × 108 bubbles mL−1; Optison™, Amersham Health Plc, Oslo, Norway), LBs and ALs—were used. LBs were created in an aqueous dispersion of 2 mg mL−1 1,2-distearoyl-sn-glycero-phosphatidylcholine (DSPC) (Avanti Polar Lipids, Alabaster, AL, USA) and 1 mg mL−1 polyethylene glycol (PEG) distearate (Sigma- Aldrich) using a 20 kHz stick sonicator (130 W, Vibra Cell, Sonics & Materials Inc., Danbury, CT, USA) at 50% amplifying strength for 1 min, in the presence of C3F8 gas in a sterilized 7 mL Bijou vial [8,13,14]. The vial cap has two openings that serve as a gas inlet and outlet. During sonication, the C3F8 gas was kept under the condition of inflow and outflow through the openings. The LB concentration was 3.4 × 108 bubbles mL−1 [13]. ALs were prepared by modifying the protocol of Suzuki et al. [9]. First, DSPC (NOF Co., Tokyo, Japan) and 1,2-distearoyl- sn-glycero-3-phosphatidyl-ethanolamine-methoxy-polyethyleneglycol (DSPE-PEG2000-OMe) (NOF Co.) (94:6 [mol/mol]) were dissolved in 10 mL of 9:1 (v/v) chloroform/methanol. Next, 5 mL of phosphate-buffered saline (PBS) without Mg2+ and Ca2+ (pH 7.2 at room temperature, Sigma) was added to the solution. The solution was then sonicated using a 20 kHz stick sonicator (Sonics & Materials). Liposomes were obtained by reverse phase evaporation at 65°C. The organic solvent was completely removed, and the size of the liposomes was adjusted to <100 nm using extruding equipment (Northern Lipids Inc., Vancouver, BC, Canada) with three sizing filters (pore sizes: 100, 200 and 600 nm) (Nuclepore Track-Etch Membrane, Whatman plc, UK). The resulting liposomes were passed through a 0.45 μm pore size filter (MILLEX HV filter unit, Durapore polyvinylidine-difluoride (PVDF) membrane, Millipore Corporation, MA, USA) for sterilization. Lipid concentration was measured using the Phospholipid C-test Wako (Wako Pure Chemical Industries, Ltd, Osaka, Japan). To produce AL, a liposome suspension of 1 mL (lipid concentration: 1 mg mL−1) was sonicated using a bath sonicator (42 kHz, 100 W; Bransonic 2510J-DTH, Branson Ultrasounics Co., Danbury, CT, USA) and a 20 kHz stick sonicator (130 W, Sonics & Materials, Inc.) at 50% amplifying strength for 1 min, in the presence of C3F8 in a sterilized 7 mL Bijou vial, as described above. Dark field microscopy Immediately after sonication, 20 μL drops of either AL or LB were put on a glass cover and were observed under an inverted microscope (IX81, Olympus, Tokyo, Japan) equipped with an illuminator (Darlkite Illuminator, Micro Video Instruments, Avon, MA, USA). Echogenicity measurement The air inside the 5 mL vials containing 1 mL of liposome suspension (lipid concentration: 1 mg mL−1) sealed with a rubber cap together with an aluminium jacket was replaced with 12 mL of air or C3F8 gas and supercharged with another 12 mL of each gas. The suspension in the vial was sonicated in a bath sonicator (Branson Ultrasounics) for 2 min. The suspension was transferred to a 7 mL Bijou vial and further sonicated by a 20 kHz sonicator (Sonics & Materials) at 50% amplifying strength for 1 min while 5 mL of each gas was injected at a rate of 300 mL h−1 using a syringe pump (model KDS 100, KD Scientific, Holliston, MA, USA). Four milliliters of a 40-fold dilution with PBS were added to a well of a 6-well plate and the B-mode image was acquired with a high-frequency ultrasound imaging system with a center frequency of 55 MHz (VEVO 770, Visualsonics Inc., Toronto, Canada). The grayscale histogram of a selected ROI was measured using the implemented software of the US imaging system. The ROI circle was set to 1.00 mm2, 1 mm above the bottom of the well. Size and zeta potential The size and zeta potentials of the bubbles were measured using a zeta potential & particle size analyzer (zeta potential range: −200 to +200 mV, particle size/distribution range: 0.6 nm to 7 μm, laser source: laser diode (660 nm), ELSZ-2, Otsuka Electronics, Osaka, Japan). The size was measured using the dynamic light scattering. The zeta potential was automatically calculated on the basis of the electrophoretic mobility using the Smoluchowski equation: ζ = 4πηu/ε, where ζ is the zeta potential, u is the electrophoretic mobility and η and ε are the viscosity and dielectric constant of the solvent, respectively. The Smoluchowski equation is applicable to a solid surface on which a surface-charge layer exits and electrolyte ions do not penetrate through the surface, i.e. hard particles [15]. In the present study, the three types of bubbles were assumed to be hard particles. The bubble solutions were diluted in PBS to ~107 bubbles mL−1 at room temperature (21–23°C). The average values of the sizes and zeta potentials were calculated using four to nine independent measurements on each sample. TEM Either negative or double staining was used for AL. Negative staining was used for LB and AB. The stained samples were examined with a JEM-2000EX operated at 100 kV (JEOL Datum, Tokyo, Japan) at the Hanaichi UltraStructure Research Institute, Aichi, Japan; or with a H-7600 operated at 80 kV (Hitachi Tokyo, Japan) at Tohoku University, Sendai, Japan. For the negative staining, a 400-mesh grid (EM fine-grid F-400, Nisshin EM Co., Tokyo, Japan) with a carbon support film (10–20 nm in thickness) was used, and was given a hydrophilic treatment. Samples were stained at either room temperature or at 80°C. For the former case, a drop of sample solution, distilled water and phosphotungstic acid (Merck, Tokyo, Japan) were put on a parafilm (Pechiney Plastic Packaging Co., Menasha, WI, USA). The grid was put into the sample drop (30 s), then in a distilled water drop for washing (10 s) and finally in a phosphotungstic acid drop for staining (10 s). Any excess solution was removed with filter paper. For the latter case, a parafilm was floated on water heated at 80 °C, and the procedure outlined above was then followed. For the double staining, an AL solution generated in the presence of C3F8 in a sterilized 7 mL vial was immediately added to 1 mL of 2% agarose (Cambrex Bio Science Rockland, Inc., Rockland, USA) to obtain a stable solution that did not release gas. Then, the AL solution was mixed with the same amount of 2% osmium tetroxide solution, and was fixed at 4°C for 6 h. Dehydration in an ethanol series (50–100%) at room temperature followed, and the solution was embedded in an EPON812 resin mixture at 60°C for 48 h. Thin sections were obtained using an ultramicrotome (Power Tome XL, RMC, Boekeler Instruments, Tucson, AZ, USA). They were stained with 2% uranyl acetate (Merck) for 15 min, washed with rinse solution and were finally stained with a lead stain solution (Sigma, Tokyo, Japan) for 5 min. Histogram of the absolute frequency distribution was obtained from 10 TEM images. The diameter of each AL was measured with rulers. Brightness analysis Two sets of TEM images (ALs, and non-gas-containing liposomes [LSs]) were analyzed to assess the average brightness value of the inside of each kind of liposome. The inner area of each liposome on the images was individually selected and its mean brightness value obtained by the ImageJ software (Rasband, W. S., Image J, U. S. NIH, Bethesda, MD, USA, http://rsb.info.nih.gov/ij/, 1997–2009.). For each image, a brightness value of the background was measured and used for normalization. Overstained areas were left out for both types of measurements. Relative brightness values (measured mean brightness/background brightness) were obtained for 106 LSs and 83 ALs. Statistical analysis All measurements were represented as either mean ± SD (standard deviation) or SEM (standard error of the mean). Statistical analysis was performed by using Student's t-test. Difference with P < 0.05 was considered significant. The statistical analysis was performed using Excel 2000 (Microsoft, USA) with the add-in software Statcel 2 [16]. Results First, the data obtained for ALs and LBs using dark light microscopy were examined, given that both have similar membrane components (Fig. 1a and b). Figure 1a shows that each AL was captured clearly. ALs with a diameter of up to 30 μm existed. Figure 1c shows the percentage of scattering intensity distribution and cumulative absolute frequency of ALs. Two peaks were observed indicating diameters of ~200 nm and 15 700 nm. Figure 1d shows the number distribution (%) and cumulative absolute frequency (%), which have been converted from Fig. 1c. Results show that most ALs have diameters of ~200 nm, while ALs with diameters exceeding a few micrometers accounted for <0.01% (Fig. 1c and d). Figure 1b shows the overall LB view. Although large bubbles were visible, the tiny bubbles that were observed in Fig. 1a were not detected in Fig. 1b. The mean diameters for the ALs, LBs and ABs are summarized in Table 1, with AL diameter being one digit smaller than that of the LBs and ABs. Fig. 1. Open in new tabDownload slide Dark field images of ALs and LBs and size distribution of ALs. (a) AL dark field image. (b) LB dark field image. (c) Scattering intensity distribution (%) and cumulative absolute frequency (%) of ALs, measured using dynamic light scattering. There are two peaks indicating diameters of ~200 nm and 15 700 nm ⁠. (d) Number distribution (%) and cumulative absolute frequency (%) of AL, measured by dynamic light scattering. Approximately 100% of ALs were ~200 nm in diameter ⁠. ALs with diameters exceeding a few micrometers accounted for <0.01%. The arrows (↓) in (c) and (d) indicate the line of the cumulative absolute frequency (%). Fig. 1. Open in new tabDownload slide Dark field images of ALs and LBs and size distribution of ALs. (a) AL dark field image. (b) LB dark field image. (c) Scattering intensity distribution (%) and cumulative absolute frequency (%) of ALs, measured using dynamic light scattering. There are two peaks indicating diameters of ~200 nm and 15 700 nm ⁠. (d) Number distribution (%) and cumulative absolute frequency (%) of AL, measured by dynamic light scattering. Approximately 100% of ALs were ~200 nm in diameter ⁠. ALs with diameters exceeding a few micrometers accounted for <0.01%. The arrows (↓) in (c) and (d) indicate the line of the cumulative absolute frequency (%). Table 1. Bubble characteristics Nano/microbubble . Shell . Gas . aSize (nm) . bZeta potential (mV) . AL DSPC/DSPE-PEG2000 Perfluoropropane 199 ± 84.4 (n = 8) −2.1 ± 0.9 (n = 4) LB DSPC/PEG Perfluoropropane 1222 ± 442.7 (n = 9) −4.2 ± 1.3 (n = 5) AB (Optison) Albumin Perfluoropropane 1689 ± 299.8 (n = 4) −40 ± 6.9 (n = 4) Nano/microbubble . Shell . Gas . aSize (nm) . bZeta potential (mV) . AL DSPC/DSPE-PEG2000 Perfluoropropane 199 ± 84.4 (n = 8) −2.1 ± 0.9 (n = 4) LB DSPC/PEG Perfluoropropane 1222 ± 442.7 (n = 9) −4.2 ± 1.3 (n = 5) AB (Optison) Albumin Perfluoropropane 1689 ± 299.8 (n = 4) −40 ± 6.9 (n = 4) aSize was measured using dynamic light scattering. Approximately 100% of ALs were ~200 nm in diameter. ALs with diameters larger than a few micrometers accounted for <0.01% (see Fig. 1). Further, 90% of the LBs were ~1200 nm in diameter (data not shown). bThe zeta potential was calculated using the Smoluchowski equation. Values are represented as mean ± SD. Open in new tab Table 1. Bubble characteristics Nano/microbubble . Shell . Gas . aSize (nm) . bZeta potential (mV) . AL DSPC/DSPE-PEG2000 Perfluoropropane 199 ± 84.4 (n = 8) −2.1 ± 0.9 (n = 4) LB DSPC/PEG Perfluoropropane 1222 ± 442.7 (n = 9) −4.2 ± 1.3 (n = 5) AB (Optison) Albumin Perfluoropropane 1689 ± 299.8 (n = 4) −40 ± 6.9 (n = 4) Nano/microbubble . Shell . Gas . aSize (nm) . bZeta potential (mV) . AL DSPC/DSPE-PEG2000 Perfluoropropane 199 ± 84.4 (n = 8) −2.1 ± 0.9 (n = 4) LB DSPC/PEG Perfluoropropane 1222 ± 442.7 (n = 9) −4.2 ± 1.3 (n = 5) AB (Optison) Albumin Perfluoropropane 1689 ± 299.8 (n = 4) −40 ± 6.9 (n = 4) aSize was measured using dynamic light scattering. Approximately 100% of ALs were ~200 nm in diameter. ALs with diameters larger than a few micrometers accounted for <0.01% (see Fig. 1). Further, 90% of the LBs were ~1200 nm in diameter (data not shown). bThe zeta potential was calculated using the Smoluchowski equation. Values are represented as mean ± SD. Open in new tab To confirm that the C3F8 gas was actually encapsulated by the AL shell, we measured the echogenicity of liposomes sonicated in the presence of either atmospheric air or C3F8 gas. Figure 2a shows characteristics of liposomes under either atmospheric air or C3F8 gas. Photos show that liposome suspension sonicated in the presence of C3F8 is cloudier than that of air and original liposome suspension (NONE). Next we measured echogenicity of each bubble by the method indicated in Fig. 2c. The US B-mode images show that liposome sonicated in the presence of the C3F8 gas have a high echogenicity. This tendency is confirmed by the brightness histogram of the liposome sonicated in the presence of the C3F8 gas that displays a shift to the right of the brightness levels compared to that with air. Figure 2b indicates the difference of brightness value between liposome sonicated in the presence of either atmospheric air or C3F8 gas. The values were normalized by that of NONE. There is a highly significant difference between them (P < 0.01). Fig. 2. Open in new tabDownload slide Confirmation of gas entrapment into liposome. Photos, US B-mode images and brightness histograms of original liposome suspension (none), liposome sonicated under either atmospheric air (air) or C3F8 gas (C3F8) indicate encapsulation of gas under the presence of the C3F8 gas but not in the presence of air (a). The US B-mode images were captured as shown in the scheme for ultrasound imaging (c). There was a highly significant difference in brightness value between liposome sonicated under atmospheric air and C3F8 gas. The values were normalized with that of liposome without gas. n = 4, mean ± S.E. **P < 0.01. Fig. 2. Open in new tabDownload slide Confirmation of gas entrapment into liposome. Photos, US B-mode images and brightness histograms of original liposome suspension (none), liposome sonicated under either atmospheric air (air) or C3F8 gas (C3F8) indicate encapsulation of gas under the presence of the C3F8 gas but not in the presence of air (a). The US B-mode images were captured as shown in the scheme for ultrasound imaging (c). There was a highly significant difference in brightness value between liposome sonicated under atmospheric air and C3F8 gas. The values were normalized with that of liposome without gas. n = 4, mean ± S.E. **P < 0.01. The zeta potential is one of the primary parameters indicative of drug delivery efficiency, since it informs about dispersivity, aggregability and mutual interaction inside the colloidal suspension. Zeta potential values are summarized in Table 1. ALs and LBs possessed neutral values since neutral lipid phosphatidylcholine was the primary component of their shells and the PEG distributed on their shell surfaces is a nonelectrolyte, water-soluble polymer. ABs had a strong negative charge, indicating that the AB colloid is the most stable of the three bubble types. Next, ALs were stained using negative staining, and their structures were examined by TEM (Fig. 3). In general, when a lipid bilayer is negatively stained, the stain solution penetrates the lipid bilayer. Existence of gas within certain areas of ALs will prevent that area from being stained effectively, resulting in a reduction in net electron density in that area. The black arrows in Fig. 3a and b indicate the presence of gas within the ALs. Decreased electron density in the central area was apparent in 69 out of 345 ALs, i.e. 20%. The shape of LBs (Fig. 3c and d) was not always spherical as compared to the shapes of ALs. A significant decrease in electron density was not observed in the interior making it difficult to determine whether gas existed in the LB. Figure 3d shows that some LBs had a bag configuration suggesting that an LB may potentially contain both gas and liquid. The AB shell structure caused strong electron beam scattering around the shell (Fig. 3e). As shown in the magnified figure (Fig. 3f), albumin was observed in filament form (indicated by the black arrow), with the layer being several hundred nanometers thick. The interior gas was assumed to be packed in a stable manner and covered with the thick albumin shell. The internal electron density was relatively low, indicating the existence of gas. Figure 4 shows the histogram of the absolute frequency distribution obtained from 10 TEM micrographs. The maximum value was obtained within the class interval of 91–120 nm. This value was about half that measured with dynamic light scattering (see Table 1). Figure 5 shows the distribution of relative brightness values in original liposomes (LSs) and ALs. The statistical distribution of ALs is slightly shifted to relative brightness values closer to 1 compared to the distribution of LSs, indicating that C3F8 gas bubbles are actually present inside some of the ALs. Fig. 3. Open in new tabDownload slide Three types of ultrasound contrast agent from negative staining. AL: (a) ×50 000, (b) ×100 000. LB: (c) ×15 000, (d) ×10 000. AB: (e) ×3500, (f) ×20 000. The black arrows in (a) and (b) show where electron density was relatively low, indicating the presence of gas. The black arrow in (f) indicates albumin in filament form. (a)–(f) were stained at room temperature. (a), (b) JEOL JEM2000EX operated at 100 kV. (c)–(f) H-7600 operated at 80 kV. Fig. 3. Open in new tabDownload slide Three types of ultrasound contrast agent from negative staining. AL: (a) ×50 000, (b) ×100 000. LB: (c) ×15 000, (d) ×10 000. AB: (e) ×3500, (f) ×20 000. The black arrows in (a) and (b) show where electron density was relatively low, indicating the presence of gas. The black arrow in (f) indicates albumin in filament form. (a)–(f) were stained at room temperature. (a), (b) JEOL JEM2000EX operated at 100 kV. (c)–(f) H-7600 operated at 80 kV. Fig. 4. Open in new tabDownload slide Histogram of the absolute frequency distribution. The data were obtained from 10 TEM images. The maximum value was obtained within the class interval of 91–120 nm. Fig. 4. Open in new tabDownload slide Histogram of the absolute frequency distribution. The data were obtained from 10 TEM images. The maximum value was obtained within the class interval of 91–120 nm. Fig. 5. Open in new tabDownload slide Relative brightness range. AL and LS TEM micrographs were analyzed to assess the average brightness value of the inside of each kind of liposome. The inner area of each liposome image was digitally selected to measure its mean brightness value. Relative brightness values (measured mean brightness/background brightness) were obtained for 106 LSs and 83 ALs. The statistical distribution of ALs is slightly shifted to relative brightness values closer to 1 compared to the distribution of LSs, indicating that gas bubbles are actually present inside some of the ALs. Fig. 5. Open in new tabDownload slide Relative brightness range. AL and LS TEM micrographs were analyzed to assess the average brightness value of the inside of each kind of liposome. The inner area of each liposome image was digitally selected to measure its mean brightness value. Relative brightness values (measured mean brightness/background brightness) were obtained for 106 LSs and 83 ALs. The statistical distribution of ALs is slightly shifted to relative brightness values closer to 1 compared to the distribution of LSs, indicating that gas bubbles are actually present inside some of the ALs. Figure 6 shows a magnified image of the AL, stained at 80°C with the negative staining. The fluidity of lipid layers increases due to heat, and results in the enhanced penetration of the staining solution. The shell thickness was 5.6 nm, which accords with a biomembrane with a thickness of 7–10 nm. Thus, the AL shell is assumed to be a single lipid bilayer. Fig. 6. Open in new tabDownload slide Shell structure of AL. TEM micrograph of AL, negatively stained at 80°C. The distance between two lines in the magnified figure was 5.6 nm, indicating a single lipid bilayer. Original magnification, ×50 000. JEOL JEM2000EX operated at 100 kV. Fig. 6. Open in new tabDownload slide Shell structure of AL. TEM micrograph of AL, negatively stained at 80°C. The distance between two lines in the magnified figure was 5.6 nm, indicating a single lipid bilayer. Original magnification, ×50 000. JEOL JEM2000EX operated at 100 kV. In order to investigate the AL structure in detail, we observed its cross-section, obtained from the double staining (Fig. 7). The black arrows in Fig. 7a indicate the presence of gas, while the white arrow indicates the presence of liquid. The percentage of AL was 24% (17 out of 70 liposomes). This value was similar to the 20% obtained and illustrated in Fig. 3a and b. Figure 7b shows that some ALs have an equal volume occupied by liquid and gas. The white arrows indicate the outside boundary, while the black arrows indicate the inside boundary. G shows the presence of gas, and L the presence of liquid. It is hard to judge whether the interface between the gas and the liquid within the AL is a gas/liquid interface or a lipid interface. Figure 7c shows an AL primarily occupied by gas. The proportion of gas relative to liquid is likely to vary depending on how the cross-section is cut. Figure 7d shows a liposome which was not sonicated, with a liquid-filled inside. Fig. 7. Open in new tabDownload slide Structure of AL from double staining. (a) The black arrows indicate the presence of gas in AL, while the white arrow indicates liquid. Original magnification: ×20 000. (b) AL occupied by ~50% (v/v) gas (G) and 50% (v/v) liquid (L). The white arrows indicate the outside boundary, while the black arrows indicate the inside boundary. Original magnification: ×30 000. (c) AL occupied mainly by gas (G). The liquid (L) portion was small. The white arrow indicates the outside boundary, while the black arrow indicates the inside boundary. Original magnification: ×20 000. (d) Liposome, which was not sonicated. The inside was filled with liquid (L). The white arrows indicate the outside boundary. Original magnification: ×50 000. (a)–(d) were obtained with JEOL JEM2000EX operated at 100 kV. Fig. 7. Open in new tabDownload slide Structure of AL from double staining. (a) The black arrows indicate the presence of gas in AL, while the white arrow indicates liquid. Original magnification: ×20 000. (b) AL occupied by ~50% (v/v) gas (G) and 50% (v/v) liquid (L). The white arrows indicate the outside boundary, while the black arrows indicate the inside boundary. Original magnification: ×30 000. (c) AL occupied mainly by gas (G). The liquid (L) portion was small. The white arrow indicates the outside boundary, while the black arrow indicates the inside boundary. Original magnification: ×20 000. (d) Liposome, which was not sonicated. The inside was filled with liquid (L). The white arrows indicate the outside boundary. Original magnification: ×50 000. (a)–(d) were obtained with JEOL JEM2000EX operated at 100 kV. Discussion The structure of an AL was investigated using TEM, and was compared with that for LB and AB. First we measured the diameter of AL by dynamic light scattering. The diameter of AL was ~200 nm (Table 1), which was about double the diameter calculated from the analysis of 10 TEM micrographs (Fig. 4). With dynamic light scattering, the size was measured immediately after AL production. TEM measurement indicated that the size of AL may have been influenced by the staining process and repeated electron beam exposure. These external factors might shift the frequency distribution to the lower value. The zeta potential was derived from the hypothesis that ALs, LBs and ABs are hard particles [15]. ALs and LBs were found to be almost neutral, whereas AB had strong negative values (Table 1). As can be seen in TEM images (Fig. 3e and f), the electron beams were strongly scattered around the shell surface of the ABs. The key component of AB, albumin, was detected in its filament form. Ohshima [15] reported that the Smoluchowski equation cannot be applied to soft particles such as red blood cells, i.e. particles with an electric surface charge boundary in which a slip line exists. ABs are most likely to be a type of soft particle, for which this equation cannot be applied. Equations taking into account the properties of this kind of particles should be investigated. From negative staining observations, it was assumed that AL have a single lipid bilayer as a shell structure (Fig. 6). The percentage of AL in which the presence of gas was detected was ~20%, and the proportion of volume occupied by gas and liquid varied depending on how the cross-sections were cut. Although it was hard to quantify the percentage of gas occupying the interior of AL due to the limited number of TEM images, it was clear from echogenicity that the C3F8 gas was actually encapsulated in ALs (Figs. 2 and 5). Several acoustic liposome structures have been suggested [17,18]. Huang et al. [17] proposed that the internal volume was occupied by air and liquid compartments, and that the interface between the air and liquid compartments was a lipid monolayer. Suzuki et al. [18] suggested that both liquid and unilamellar lipids containing air were encapsulated by a single lipid bilayer. In the present study, we observed that gas and liquid seemed to be encapsulated together by a single lipid bilayer. However, we could not judge whether the interface between the gas and the liquid was the gas/liquid interface or the lipid interface. The co-existence of gas and liquid in ALs provides evidence of its echogenicity and drug-carrying capabilities. Further, the tissue specificity of ALs can be improved by conjugating ligands against the target tissue with PEG on the AL surface. Recently, a high-frequency ultrasound system with ALs has been developed and applied so far to the imaging of anterior segment of the eye [19], skin [20] and tumor vasculature [21]. Studies have shown that the permeability of the tumor vasculature is enhanced, and the phenomenon is recognized as the EPR effect [11]. Most anticancer drugs have diameters of 10– 120 nm: Genexol-PM (20–50 nm in diameter), Doxil (80–90 nm in diameter), Abraxane (120 nm in diameter) [12]. Sonoporation delivery efficiency, in vivo behavior and tissue-specificity of ALs would possibly be enhanced if the diameter was controlled within the range of 10–120 nm, the surface was positively or negatively charged, and ligands against the tumor were conjugated to PEG on the surface [22–24]. Concluding remarks In summary, the findings of the present study indicate that AL have a shell consisting of a single lipid bilayer and can encapsulate both drugs and gas. The PEG distributed over the surface can be conjugated with tissue-specific ligands. Developing functional AL will assure the effectiveness of sonoporation. Funding This work was supported by a Grant-in-Aid for Scientific Research (B) [20300173 to T.K., 19390507 to S.M.]; a Grant-in-Aid for Scientific Research on Priority Area, MEXT [20015005 to T.K.]; a Grant for Research on Advanced Medical Technology, the Ministry of Health, Labor and Welfare of Japan [H19-nano-010 to T.K.]; a Grant for Research on Development of Systems and Technology for Advanced Measurement and Analysis, JST [T.K.]; and a Grant-in-Aid for JSPS Fellows [21-7271 to S.H.]. The authors would like to thank Yukiko Watanabe, Rui Chen and Li Li, for their technical assistance. References 1 Kodama T , Tomita Y , Koshiyama K , Blomley M J . Transfection effect of microbubbles on cells in superposed ultrasound waves and behavior of cavitation bubble , Ultrasound Med. Biol. , 2006 , vol. 32 (pg. 905 - 914 ) Google Scholar Crossref Search ADS PubMed WorldCat 2 Lindner J R . Microbubbles in medical imaging: current applications and future directions , Nat. Rev. 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TI - Morphological study of acoustic liposomes using transmission electron microscopy
JF - Journal of Electron Microscopy
DO - 10.1093/jmicro/dfp056
DA - 2010-06-01
UR - https://www.deepdyve.com/lp/oxford-university-press/morphological-study-of-acoustic-liposomes-using-transmission-electron-agvdkVnlqI
SP - 187
EP - 196
VL - 59
IS - 3
DP - DeepDyve
ER -