Highly Uniform Perfluoropropane-Loaded Cerasomal Microbubbles

Subscriber access provided by University of Otago Library

Article

Highly uniform perfluoropropane loaded cerasomal microbubbles as a novel ultrasound contrast agent Chunyang Zhang, Zhu Wang, Chunan Wang, Xiongjun Li, Jie Liu, Ming Xu, Shuyu Xu, Xiaoyan Xie, Qing Jiang, Wei Wang, and Zhong Cao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b03668 • Publication Date (Web): 26 Jun 2015 Downloaded from http://pubs.acs.org on July 2, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Highly uniform perfluoropropane-loaded cerasomal microbubbles as a novel ultrasound contrast agent Chunyang Zhanga, Zhu Wangb, Chunan Wanga, Xiongjun Lia, Jie Liua, Ming Xub, Shuyu Xua, Xiaoyan Xieb, Qing Jianga, Wei Wangb*，Zhong Caoa*

a

Department of Biomedical Engineering, School of Engineering, Sun Yat-sen University, Guangzhou, Guangdong 510006, China

b

Department of Medical Ultrasonics, Institute of Diagnostic and Interventional Ultrasound, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510080, China

*E-mail: [email protected] *E-mail: [email protected]

Abstract: Microbubbles are widely used as ultrasound contrast agents owing to their excellent echoing characteristics under ultrasound radiation. However, their short sonographic duration and wide size distribution still hinder their application. Herein, we present a hard-template approach to produce perfluoropropane-loaded cerasomal microbubbles (PLCMs) with uniform size and long sonographic duration. The preparation of PLCMs includes deposition of Si-lipids onto functionalized CaCO3 microspheres, removal of their CaCO3 cores and mild infusion of perfluoropropane (PFP). In vitro and in vivo experiments showed that PLCMs had excellent echoing characteristics under different ultrasound conditions. More importantly, PLCMs could be imaged for much longer than SonoVue (commercially used microbubbles) under the same ultrasound parameters and concentrations. Our results demonstrated that PLCMs have great potential for use as a novel contrast agent in ultrasound imaging.

Keywords: microbubbles, Si-lipid, stability, ultrasound contrast agent, uniform size distribution, hard-template 1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1. Introduction Ultrasound imaging has attracted tremendous interest in recent years due to its favorable characteristics, including its real-time data acquisition, non-invasiveness, cost-effectiveness, portability and wide availability1, 2. Moreover, to enhance backscattering signals between different tissues for better visualization of a specific tissue, microbubbles have been utilized because the compressibility of their gas cores is greater than that of normal tissues3, 4. In addition to their gas cores, the sizes and shell materials of microbubbles have significant effects on the sensitivity and duration of ultrasound imaging5-8. The size of a microbubble affects its ability to travel through vasculature and its resonant frequency in an applied ultrasound field9, 10. On the one hand, the diameter of a microbubble must be smaller than 7 µm for it to cross the pulmonary capillary bed. However, it should not be too small, as smaller microbubbles also have lower reflectivity11, 12. The optimal microbubble size is between 2 and 5 µm. On the other hand, the resonant frequency of a microbubble is directly related to its size and size distribution13, 14. Since current ultrasound imaging systems have limited frequency bandwidth, a clinical imaging system is only optimal for a small percentage of traditional contrast agents with polydisperse populations, while monodisperse microbubbles can enhance the sensitivity of ultrasound imaging because of their consistent echogenic response14. Unfortunately, conventional techniques for fabricating microbubbles that involve sonication or mechanical agitation generally result in broad size distribution and batch-to-batch variation15, 16. Therefore, improved techniques for fabricating monodisperse microbubbles are extremely desirable in developing this highly efficient ultrasound contrast agent. Various microbubble shell materials have been studied, such as lipids and silica. Lipid microbubbles have low interfacial tension and good biocompatibility, which facilitates their commercialization and has led to the extensive use of lipid-coated ultrasound contrast agents, such as SonoVue17,

18

. However, lipids alone do not

provide a strong enough barrier to slow bubble dissolution, which is the underlying cause behind the inability of lipid microbubbles to retain their enhanced ultrasound 2


Page 2 of 23

Page 3 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


imaging capabilities for a sufficient length of time under high-power irradiation19, 20. Therefore, lipid microbubbles need to be repeatedly injected into patients (at least twice) when they are being subjected to high-power irradiation for the detection of superficial organs. Recently, a great deal of attention has been paid to research focused on increasing the stability of microbubble contrast agents because microbubbles with sufficient stability in the bloodstream can have improved blood circulation times and strong scattered signals in a region of interest21-23. An inorganic silica material has been introduced to produce novel hollow microspheres as ultrasound contrast agents, which have the advantages of shell rigidity and highly controllable size distribution24, 25

. Recently, Hall and Yang

26, 27

reported that hollow silica microspheres produce a

strong ultrasound signal in aqueous solution. However, Hall's hollow silica microspheres required a minimal imaging mechanical index (MI) of approximately 0.5, which is much higher than the clinically used level; a high mechanical index causes a high rate of lung hemorrhages and broken capillaries. In comparison, the multifunctional hollow silica microspheres that were fabricated by Yang could enhance ultrasound imaging under low mechanical index, but their ultrasound signal in vitro was not strong relative to that of SonoVue. Additionally, the preparation of hollow silica microspheres in both reports required extreme conditions to remove their core templates, such as the use of tetrahydrofuran (THF) 26, 28. Consequently, it is highly beneficial and desirable to develop a simple strategy to create uniform microbubbles, which can provide enhanced contrast of backscattering signal and high structural stability in circulation. Herein, we developed a novel microbubble system using Si-lipid as a shell material, which combined the advantages of both lipid and silica microbubbles. Hard template was used to generate gas perfluoropropane (PFP)-loaded cerasomal microbubbles as ultrasound imaging contrast agents (Scheme 1). It was expected that PFP-loaded cerasomal microbubbles (PLCMs) would show good stability, controllable size and uniform size distribution, which together would provide enhanced contrast for backscattering signal in ultrasound imaging. The PLCMs were 3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 23

characterized and investigated in vivo to reveal their potential as an efficient ultrasound contrast agent.

Scheme 1. Illustration of the preparation of perfluoropropane-loaded cerasomal microbubbles (PLCMs).

2. Materials and methods 2.1 Materials N-[N-(3-Triethoxysilyl) propylsuccinamoyl] dihexadecylamine (Si-lipid) was synthesized

according

to

a

procedure

that

was

reported

previously29.

1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-DOPE) was obtained from Avanti Polar Lipids (USA). Poly(allylamine hydrochloride) (PAH; ~15,000 MW) and poly(sodium 4-styrenesulfonate) (PSS; ~70,000 MW) were purchased from Sigma-Aldrich (USA). Calcium nitrate tetrahydrate (Ca(NO3)2

·

4H2O), sodium carbonate (Na2CO3) and disodium

ethylenediaminetetraacetate dihydrate (EDTA) were purchased from Guangzhou Chemical Reagent Factory (China). Perfluoropropane(C3F8) was obtained from Dalian Special Gases (China). Organic solvents, such as ethyl alcohol and dichloromethane, were of analytical grade. The water used throughout the experiment was purified using a Milli-Q synergy purification system. Unless otherwise stated, all reagents and chemicals were used as received without further purification.

4


Page 5 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2.2 Preparation of PAH-coated CaCO3 microspheres PSS-doped CaCO3 microspheres with uniform diameters were synthesized via colloidal aggregation of Na2CO3 and Ca(NO3)2 as previously reported30, 31. Briefly, 100 mL of a Ca(NO3)2 (0.025 M) aqueous solution containing either 0.4% (w/v) or 0.8% (w/v) PSS was created, into which 100 mL of an Na2CO3 (0.025 M) solution was rapidly added under magnetic agitation (600 rpm) for 15 seconds at room temperature. After allowing the solution to stand for 15 min, the CaCO3 microspheres were collected and washed by centrifugation. The adsorption of PAH onto the CaCO3 microspheres was conducted in a 0.1 M NaCl solution for 20 min followed by three washes with deionized water. 2.3 Preparation of PFP-loaded cerasomal microbubbles (PLCMs) PLCMs were prepared using an ethanol injection method. Briefly, 10 mg Si-lipid dissolved in ethanol (pH=3) was injected slowly and continually into 5 mL CaCO3-PAH (10 mg/mL) microsphere aqueous solution under water-bath sonication for 5 min. Then, the suspensions were incubated for 12 h at room temperature to allow siloxane networks to develop on the surfaces of the microspheres. Excess Si-lipid was removed by three repeated centrifugation/water-wash cycles. To obtain hollow cerasomal microcapsules (CMs), the core-shell microspheres were exposed to an EDTA solution (0.2 M) for 20 min. This process was repeated 3 times to ensure the core was completely removed. Following this, the obtained CMs were washed an additional 3 times with deionized water, and the samples were lyophilized in ampoule bottle with mannitol (10% by wt.), which was included as a freeze-drying protective additive.Finally, the bottle was filled with perfluoropropane to obtain the PFP-loaded cerasomal microbubbles, which can be then used directly by injecting normal sodium solution through the septum. 2.4 The stability of CMs and PLCMs The morphological structural stability of CMs and PLCMs is critical for their application as ultrasound contrast agents in vivo. A lyophilization process was performed as previously described: the obtained CMs were resuspended in 2 mL of an aqueous solution containing 10% mannitol and were lyophilized for 48 h (mannitol was used to prevent CMs aggregation during lyophilization). Following this, the morphological structure of the CMs was investigated before and after lyophilization 5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

using an optical microscope (Olympus LX71, Olympus Co., Japan). In addition, PLCMs with two size distributions (2 µm and 4 µm) were dispersed in PBS solution containing 10% FBS and were incubated at 37 ℃. After 24 hours, the PLCMs were characterized by optical microscopy. Furthermore, ultrasound contrast enhancement change before and after incubation of PBS containing 10% FBS was investigated. Moreover, the morphological structures of the PLCMs (250 µg mL-1) and the commercial ultrasound contrast agent SonoVue (250 µg mL -1) were investigated after ultrasonic irradiation (Aplio500 system, Toshiba Medical Systems; Tokyo, Japan) under the same ultrasound conditions (MI=0.2, frequency=7.0 MHz, irradiation time=5 min). 2.5 Hemolysis assay Fresh whole human blood stabilized with heparin was kindly provided by Sun Yat-Sen University Cancer Center. Human red blood cells (HRBCs) were obtained by removing the serum for a hemolysis assay according to the literature32. Briefly, HRBCs were separated from whole blood by centrifugation at 1500 rpm for 10 min and were purified via five successive washes with sterile isotonic PBS. Following this, the packed red blood cells were suspended in 4 mL PBS. Then, 300 µL of the diluted HRBC suspension was added to 1.5 mL water (positive control), sterile isotonic PBS (negative control), or PBS buffer containing PLCMs-2µm with a concentration ranging from 50 to 800 µg mL-1. After gentle shaking, the samples were left to stand for 3 h at 37 ℃ . Finally, the supernatants were measured at 541 nm using a UV-DU730 absorption spectrophotometer to analyze the release of hemoglobin. The percent hemolysis of each sample was calculated by the following formula33, 34:

The percent

hemolysis =

ASample − ANegative APositive − ANegative

× 100 %

ASample : Absorption value of sample’s supernatant at 541 nm. Apositive : Absorption value of positive group’s (deionized water) supernatant at 541nm. Anegative: Absorption value of negative group’s (PBS buffer) supernatant at 541 nm. 2.6 Cytotoxicity of PLCMs Cell toxicity caused by PLCMs was evaluated against different cell lines, including human cervical carcinoma cells (HeLa cells) and human liver L02 cells, 6


Page 6 of 23

Page 7 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


obtained from the Shanghai Institute of Material Media. The cells were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS) at 37 °C in 5% CO2. The cells were seeded in a 96-well plate at a density of 3000 cells per well and were cultured in 5% CO2 at 37 °C for 12 h. Following this, different concentrations of PLCMs diluted in RPMI 1640 media were used to replace the previous media. After incubation for a predetermined time (12 h or 24 h), the media was removed, and the cells were rinsed with PBS. Thereafter, 100 µL fresh media and 20 µL MTT (5.0 mg mL-1) were added into each well, and the cells were incubated for an additional 4 h. After removing the media, the resultant formazan salt crystals were dissolved in 150 µL DMSO and analyzed on a microplate reader (BioTek, Synergy 4, USA) at a 570 nm wavelength. The percent cell viability is determined using MTT assay. 2.7 In vitro ultrasound imaging In vitro ultrasound imaging was performed on PLCMs in a plate of reconstructive Wilkins-Chalgren agar (with an inner diameter of 10 mm) using an Aplio500 (Toshiba Medical Systems, Tokyo, Japan) with a 375BT convex transducer (frequency range, 1.9-6.0 MHz) and a PLT-805AT linear probe (frequency range, 6.2-12 MHz). Typically, 3 mL plastic pasteur pipettes included normal saline containing PLCMs-2µm in a series of concentrations (50-500 µg mL-1), and then the tube was immersed in a de-aerated water tank. Contrast-enhanced ultrasound imaging was obtained with different frequencies and mechanical indexes. The de-aerated normal saline was used as a blank control for all of the vitro ultrasound imaging experiments. In addition, the ultrasound contrast signal duration of PLCM was investigated under 3.5 MHz and 7.0 MHz (MI=0.2) in normal saline solution and whole blood. The commercially available ultrasound contrast agent SonoVue (Bracco, Milan, Italy) was used as a control. 2.8 In vivo ultrasound imaging To assess the acoustic behavior of PLCMs in vivo, ultrasound imaging was performed on the kidney of a New Zealand white rabbit using a contrast-enhanced ultrasound imaging mode at frequencies of 3.5 MHz (MI of 0.2) and 7.0 MHz (MI of 0.2). The animal experiments were approved by a regional animal care committee. The New Zealand white rabbit (2.5 kg of body weight) was anesthetized using pentobarbital sodium (4.0 mL, 2% w/v, 0.9% saline), which was administered though the auricular vein. Meanwhile, a warm blanket was used to maintain the rabbit’s body 7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 23

temperature within normal levels during the experiment. After activation of the contrast imaging function, a bolus injection of 2 mL PLCMs-2 µm in normal saline solution at a concentration of 800 mg mL-1 was administered via the auricular vein. Digital contrast-enhanced ultrasound cine clips or images of the kidney were stored on digital disks. The scanning parameters were optimized based on the in vitro experiments conducted at 3.5 MHz (MI=0.2) and 7.0 MHz (MI=0.2). 2.9 Characterization of PLCMs The zeta potential of PLCMs was estimated by measuring dynamic light scattering (DLS) in aqueous media using a Malvern Zetasizer (Nano ZS90, Malvern instruments Ltd., UK) at room temperature. PLCM morphology was observed by optical microscopy (CKX41, Olympus Co., Japan) and field emission scanning electron microscopy (FE-SEM, Zeiss-Ultra 55, Zeiss Co., Germany). The SEM specimens were prepared by casting an aliquot of PLCM suspension onto copper foil. The specimens were sputtered with gold for 1 min and then inspected using a FE-SEM. The hybrid Si-lipid shell was investigated by confocal laser scanning microscopy (CLSM, Olympus -IX71, Olympus Co., Japan) with 1% NBD-DOPE mixed into the Si-lipid as a lipid-fluorescent probe. A drop of microbubbles was deposited on a thin glass slide for CLSM testing. The polymerization degrees of the surfaces of the PLCMs were detected using matrix-assisted

laser

spectrometry. Spectra

desorption/ionization were

collected

time-of-flight using

(MALDI-TOF) mass

a Bruker UltrafleXtreme mass

spectrometer equipped with a 337 nm N2 laser in the reflector mode and 25 kV acceleration voltage. Dithranol (Aldrich, 97%) was used as a matrix. 3. Results and Discussion 3.1 Fabrication and characterization of PLCMs The synthetic process for making PLCMs is shown in Scheme 1. Monodisperse CaCO3 microspheres were coated with Si-lipid using a combined sol-gel and self-assembly process in aqueous solution. The diameters of the resulting capsules were determined by the sizes of the CaCO3 templates that were used to form the cores. As the size of a microbubble affects its ability to cross into pulmonary microcirculation and its reflectivity on ultrasound, the ideal diameter of a microbubble is between 2 and 5 µm. The diameters of the two CaCO3 templates that were used in 8


Page 9 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


this study were 2 µm and 4 µm. Homogeneous CaCO3 microspheres (2 µm) have a zeta potential of -11.8 mV in water, and the templates were coated with PAH polyelectrolytes to modify their surfaces for Si-lipid deposition. After the above steps, the zeta potential of the CaCO3 template shifted from -11.8 mV to +9.3 mV. Following Si-lipid shell formation, the surface potential changed to -15.2 mV from +9.3 mV. The changes in surface zeta potential indicated successful coating with the Si-lipid. The complete removal of the template core was confirmed by thermo-gravimetric (TG) analysis. As shown in Figure S1, calcium carbonate, cerasome and cerasomal microcapsule weight loss were estimated to be 6.0%, 68.1% and 65.9% respectively within a temperature range of 150-600°C. Almost all calcium carbonate core have been eleminated (less than 5% residual). SEM images showed a smooth sphere surface, implying homogeneous deposition of Si-lipid on the template’s surface (Figure S2a and S2c). After the CaCO3 template cores were removed, two types of microcapsules were prepared in the present study, which were approximately 2 µm (Figure 1a and 1b) and 4 µm in diameter (Figure 1c and 1d): slightly smaller than that of the template spheres. Furthermore, the uniform diameter of PLCMs (Figure 1e and 1f) was determined from the images by ImageJ software (at least 100 microcapsules per sample). Meanwhile, the perforated particles accounted for about 20% (PLCMs-4µm) and 6% (PLCMs-2µm) of the total. We believed that these perforated particles were formed due to the vacuum drying in SEM measurement, which seems to be more reasonable when considering the gas-cored structure. CLSM was used to observe the incorporation of NBD-DOPE into the microbubble membranes. As shown in Figure 2c, homogeneous fluorescence cycles were observed, which indicated the presence of homogeneously distributed Si-lipids in the membranes of the PLCMs (4 µm). The membranes of the PLCMs (2 µm) appeared as fluorescent solid dots rather than as cycles, most likely due to the insufficient resolution of the CLSM imaging technique (Figure 2a).

9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. SEM images of (a, b) PFP-loaded cerasomal microbubbles (PLCMs) of 2.0 µm and (c, d) 4.0 µm in diameter. Histograms with size distribution of (e) 2.0 µm and (f) 4.0 µm PLCMs.

Figure 2. CLSM images of (a, b) 2.0 µm and (c, d) 4.0 µm PLCMs.

10


Page 10 of 23

Page 11 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3.2 Stability of CMs and PLCMs It is well known that stability is critical for the application of microbubbles as an ultrasound contrast agent. Ultrasound microbubbles must remain intact in the bloodstream until they reach a region of interest and must avoid aggregation when passing through the pulmonary capillary bed. However, conventional lipid microbubbles do not have sufficiently strong shells to withstand bubble dissolution due to the high fluidity of bilayer membranes, which accounts for the poor colloidal stability of lipid microbubbles in storage. Thus, it is important to prepare novel microbubbles with increased stability. In the present work, the stability of CMs was investigated in PBS containing 10% FBS and were incubated at 37°C for 24 hours. It was found that there is no significant difference in mean particle size or size distribution between the samples before and after freeze-drying (Figure S3). In addition, it was found that there was nearly no CMs collapse even under a high-pressure vacuum during the lyophilization process (Figure S3b and S3d), possibly due to the Si-O-Si framework formed on the surfaces of the microbubbles, which retains their spherical, hollow shape. Remarkably, the lyophilized CMs revealed almost no changes in microcapsule size or morphological structure over a prolonged storage time of up to 14 days at 25°C. The good stability of CMs during lyophilization ensures their application in future experiments. Furthermore, the stability of PLCMs was then investigated in PBS buffer containing 10% fetal bovine serum (FBS). No obvious changes were observed over time (Figure 3b and 3d), which suggested that the existence of FBS (10%) could not induce aggregation of PLCMs, probably due to their negative surface potential. In addition, the ultrasound contrast enhancement did not change significantly during the incubation time (30 min), which indicated that the PLCMs could keep its imaging effectiveness in PBS containing 10% FBS(Figure S4).

11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3. Optical microscope images of 2 µm and 4 µm PLCMs before (a, c) and (b, d) after being incubated in PBS media with 10% FBS. It is well known that good stability can prolong the lifetime of ultrasound contrast agents under acoustic irradiation. To improve the stability of microbubbles, researchers have made tremendous efforts35, 36. Here, the morphological structures of the PLCMs and a commercial ultrasound contrast agent were investigated before and after ultrasound irradiation for 5 min. As shown in Figure S5a, the SonoVue microbubble had a broad size distribution, ranging from 2 to 10 µm before irradiation, and most of the SonoVue microbubbles disappeared after ultrasound irradiation (FigureS5b). Meanwhile, the SonoVue microbubble solution changed from opaque to transparent. However, the PLCM solution maintained its previous appearance, and the microbubbles still retained their initial spherical structures with ultrasound irradiation, as shown in the optical images of Figure S5c-5f. These results prove once again that the stability of PLCMs is much higher than that of conventional liposomal microbubbles. The high morphological stability of PLCMs has been attributed to their surface siloxane framework, which is absent in the lipid bilayers of conventional microbubbles. To verify this hypothesis, MALDI-TOF-MS was performed to confirm the existence of an inorganic siloxane framework on the PLCMs. As shown in Figure S6, dimers, trimers and other higher molecular weight oligomers, such as hexamers, were detected in the sample. These results indicated that the surfaces of the PLCMs were comprised of a siloxane framework, which essentially contributed to their high 12


Page 12 of 23

Page 13 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


stability. Apparently, PLCMs can serve as a promising novel ultrasound contrast agent while avoiding the colloidal instability problem of lipid microbubbles. 3.3 In vitro cytotoxicity and hemolysis assay of PLCMs The cytotoxicity of an ultrasound contrast agent is vital to its biomedical application. In vitro cytotoxicity was tested on L02 and HeLa cell lines. An MTT assay was used to estimate the cytotoxicity of PLCMs-2µm. As shown in Figure 4, HeLa cells retained 95% cell viability after incubation with PLCMs for 12 h at a high concentration of 800 µg mL-1. When the incubation time was prolonged to 24 h, the cell viability was still maintained above 90%, suggesting that PLCMs exhibit little cytotoxicity in either of the cell lines, and the differences in cell viability after 12 and 24 h of incubation were negligible. The prominent biocompatibility of PLCMs is favorable for their in vivo application as ultrasound contrast agents (UCAs).

Figure 4. In vitro cell viability of (a) L02 cells and (b) HeLa cells incubated with PLCMs at different concentrations for 12 h and 24 h at 37 °C (n=3). In their use as an ultrasound contrast agent, the blood biocompatibility of PLCMs must also be considered. Herein, a hemolysis experiment was conducted to evaluate blood compatibility. Once hemolysis occurs, the hemoglobin that is present in red blood cells will be released into solution, turning it red. The density of this red color correlates with hemolytic activity, which can be estimated by measuring the UV absorbance of a supernatant at 541 nm. As presented in Figure 5, PLCMs did not have obvious hemolytic effects under the experimental concentrations that were tested (50-800 µg mL-1). Even at the highest experimental concentration of 800 µg mL-1, the PLCMs only experienced approximately 1.3% hemolysis at a prolonged exposure time of 3 h. Consequently, it can be concluded that PLCMs have negligible hemolytic 13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

activity.

Figure 5. Hemolysis assay (+) using deionized water as a positive control and (-) PBS as a negative control. Hemolysis caused by PLCMs at different concentrations (n=3). 3.4 In vitro ultrasound imaging In vitro US images of PLCMs in normal saline with different parameters are shown in Figure 6. First, the effects of ultrasound frequency on acoustic agents were investigated. Figure 6a revealed that clear signals could be obtained at frequency values from 3.0 to 7.0 MHz, brightening gradually. The gray-scale intensities on the images were increased analyzed by ImageJ software (Figure 6c). These results indicated that PLCMs serve as a novel ultrasound contrast agent that can be used both in low (3.0 and 3.5 MHz) and in high (6.5 and 7.0 MHz) frequency fields. Furthermore, MI has great influence on ultrasound image quality. As shown in Figure 6b, backscatter signals increased when the MI was raised from 0.04 to 0.4, suggesting that PLCMs can retain an integrated morphological structure within a wide amplitude of insonation. However, no obvious increase of gray value was observed when the MI increased to 0.4, as confirmed in Figure 6d. Although Food and Drug Administration (FDA) ultrasound regulations stipulate that the maximum MI is 1.9 in clinical examination, a lower MI is safer and more favorable for clinical use. Thus, an MI in the range of 0.08 to 0.2 is applicable for ultrasound imaging of PLCMs. Meanwhile, contrast enhancement was found to be concentration dependent (Figure 6e-6h). A remarkably brighter US imaging can be obtained with increasing concentration. Signal enhancement could clearly be observed even at a very low concentration of 14


Page 14 of 23

Page 15 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


31.25 µg mL-1, which is not attainable with hollow silica microspheres under similar experimental conditions 24, 26.

Figure 6. In vitro ultrasound imaging of PLCMs at a concentration of 250 µg mL-1 at different frequencies (a and c; MI = 0.2) and MI values (b and d; frequency of 7.0 MHz). In vitro ultrasound images of different concentrations of PLCMs in normal saline solution under (e) 7.0 MHz (MI=0.08) and (f) 7.0 MHz (MI=0.2). (g) The average gray values obtained from Figure 6e. (h) The average gray values obtained from Figure 6f. Furthermore, the in vitro ultrasound images of PLCMs in normal saline solution and whole blood were compared to those of SonoVue microbubbles to investigate imaging lifetime (Figure 7 and 8). The lifetime of a SonoVue microbubble is only 4 min under ultrasound irradiation in both normal saline solution and whole blood (Figure 7d and 7e); the results were confirmed by gray-scale value analysis in Figure 15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

7f. However, the imaging lifetime of PLCMs can last as long as over 20 min under the same conditions. As shown in Figure 7c, there is no remarkable decrease in the gray-scale intensity in either normal saline solution or whole blood. The intensity decreased by only 12% after 16 minutes. In clinical settting, ultrasound irradiation at various frequencies are commonly used for detecting tissue at different depths within the body. Therefore we compared the performance of PLCMs with SonoVue at higher frequencies (7.0 MHz). As shown in Figure 8, PLCMs exhibited the same ultrasound efficiency as SonoVue initially. But SonoVue could only last for 4-5 min. However, the PLCMs were very stable under the same ultrasound settings (7.0 MHz, MI=0.2) and could be imaged for 10 min. The signal intensity of PLCMs stayed around 60% even after 10 min, which was confirmed by gray-scale value analysis in Figure 8c. As a control study, the gray-scale value of SonoVue dramatically decrased to 25% of its initial intensity (Figure 8f). The imaging lifetime of a contrast agent is crucial to its clinical application. The favorable imaging lifetime of the PLCMs was probably due to the existence of silica networks on the surfaces of their gas cores even under ultrasound irradiation. In contrast, a lipid bilayer alone cannot provide a barrier strong enough to resist bubble dissolution. Our results confirmed that PLCMs possess significantly high stability for long-term ultrasound imaging.

Figure 7. In vitro ultrasound imaging of 250 µg mL-1 PLCMs in (a) normal saline 16


Page 16 of 23

Page 17 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


solution and (b) whole blood recorded at different times. (c) The average gray values obtained from (a) and (b). In vitro ultrasound imaging of 250 µg mL-1 SonoVue in (d) normal saline solution and (e) whole blood recorded at different times. (f) The average gray values obtained from (d) and (e) (frequency of 3.5 MHz, and MI of 0.2).

Figure 8. In vitro ultrasound imaging of 250 µg mL-1 PLCMs in (a) normal saline solution and (b) whole blood recorded at different imaging times. (c) The average gray values obtained from (a) and (b). In vitro ultrasound imaging of 250 µg mL-1 SonoVue in (d) normal saline solution and (e) whole blood recorded at different imaging times. (f) The average gray values obtained from (d) and (e) (frequency of 7.0 MHz, and MI of 0.2). 3.5 In vivo ultrasound imaging In vivo imaging experiments were performed using a New Zealand white rabbit. As shown in Figure 9a and 9c, the image of its kidney was completely black when only normal saline was injected into the rabbit. However, as shown in Figure 9b and 9d, significantly positive contrast enhancement of the rabbit’s kidney was observed a few seconds after the intravenous injection of PLCMs, indicating that PLCMs have the ability to cross pulmonary capillaries to achieve systemic circulation. As revealed in 17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 9d, the local intensity of the acoustic signal is obviously increased after PLCM injection. The above results proved the outstanding acoustic effect of PLCMs under both low and high ultrasound power, which is important for ultrasonic imaging. In addition, the high resolution of ultrasound imaging under high frequency and MI value is important for ultrasonography of superficial organs. Notably, no arrhythmia or other side effects were observed during the experimental procedure, which suggested that the PLCMs had no acute toxicity. These results indicate that PLCMs have great potential as an ultrasound contrast agent.

Figure 9. In vivo ultrasound images of male rabbit kidney before (a) and after (b) intravenous injection of PLCMs in a normal saline suspension (frequency of 3.5 MHz, and MI of 0.2). Images taken before (c) and after (d) intravenous injection of PLCMs in a normal saline suspension (frequency of 7.0 MHz, and MI of 0.2). 4. Conclusion Size polydispersity and colloidal instability in aqueous media represent two major obstacles for commercial ultrasound contrast agents. We have successfully developed novel PFP-loaded cerasomal microbubbles (PLCMs), which combine high colloidal stability and size homogeneity. The PLCMs exhibited good biocompatibility and could encapsulate perfluoropropane. They also showed excellent echoing characteristics under different ultrasound conditions. More importantly, PLCMs could be imaged for much longer periods of time than SonoVue under the same ultrasound parameters and concentrations, owing to a silica framework being present on the surfaces of the PLCMs. These results reveal the potential of PLCMs as a novel ultrasound contrast agent. Furthermore, PLCMs offer a biocompatible platform for the synthesis of multifunctional ultrasound contrast agents via their conjugation with organic dyes, drugs and photothermal materials. Further experiments to explore these 18


Page 18 of 23

Page 19 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


potentials are currently underway. 5. Supporting Information Thermo gravimetric analysis of CMs, SEM images of CaCO3-PAH microspheres coated with Si-lipids; optical images of CMs before and after lyophilization, in vitro ultrasound imaging and the average gray values of PLCMs incubation of PBS containing 10% FBS recorded at various incubation time, optical images of the SonoVue and PLCMs microbubbles before and after ultrasound irradiation, and the MALDI-TOF mass spectra of PLCMs and species of lipid oligomers from the PLCMs. The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org. 6. Acknowledgments This work was supported by the National Natural Science Foundation of China (81101142, 81301238, U1401242), Natural Science Foundation of Guangdong Province (2014A030312018) and Guangdong Innovative Research Team Program (No. 2009010057).

References 1.

Liang, H. D.; Blomley, M. J. K. The Role of Ultrasound in Molecular Imaging. Br. J. Radiol.

2003, 76, 140-150. 2.

Schutt, E. G.; Klein, D. H.; Mattrey, R. M.; Riess, J. G. Injectable Microbubbles as Contrast

Agents for Diagnostic Ultrasound Imaging: The Key Role of Perfluorochemicals. Angew. Chem. Int. Ed. 2003, 42, 3218-3235. 3.

Diaz-Lopez, R.; Tsapis, N.; Santin, M.; Bridal, S. L.; Nicolas, V.; Jaillard, D.; Libong, D.;

Chaminade, P.; Marsaud, V.; Vauthier, C.; Fattal, E. The Performance of PEGylated Nanocapsules of Perfluorooctyl Bromide as an Ultrasound Contrast Agent. Biomaterials 2010, 31, 1723-1731. 4.

Diaz-Lopez, R. R.; Tsapis, N.; Libong, D.; Chaminade, P.; Connan, C.; Chehimi, M. M.;

Berti, R.; Taulier, N.; Urbach, W.; Nicolas, V.; Fattal, E. Phospholipid Decoration of Microcapsules Containing Perfluorooctyl Bromide Used as Ultrasound Contrast Agents. Biomaterials 2009, 30, 1462-1472. 5.

Chlon, C.; Guedon, C.; Verhaagen, B.; Shi, W. T.; Hall, C. S.; Lub, J.; Bohmer, M. R. Effect

19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

of Molecular Weight, Crystallinity, and Hydrophobicity on the Acoustic Activation of Polymer-Shelled Ultrasound Contrast Agents. Biomacromolecules 2009, 10, 1025-1031. 6.

Gorce, J.-M.; Arditi, M.; Schneider, M. Influence of Bubble Size Distribution on the

Echogenicity of Ultrasound Contrast Agents: A Study of SonoVue. Invest. Radiol. 2000, 35, 661-671. 7.

Martinez, H. P.; Kono, Y.; Blair, S. L.; Sandoval, S.; Wang-Rodriguez, J.; Mattrey, R. F.;

Kummel, A. C.; Trogler, W. C. Hard Shell Gas-filled Contrast Enhancement Particles for Colour Doppler Ultrasound Imaging of Tumors. MedChemComm. 2010, 1, 266-270. 8.

Poehlmann, M.; Grishenkov, D.; Kothapalli, S. V. V. N.; Harmark, J.; Hebert, H.; Philipp, A.;

Hoeller, R.; Seuss, M.; Kuttner, C.; Margheritelli, S.; Paradossi, G.; Fery, A. On the Interplay of Shell Structure with Low and High-frequency Mechanics of Multifunctional Magnetic Microbubbles. Soft Matter 2014, 10, 214-226. 9.

Hettiarachchi, K.; Talu, E.; Longo, M. L.; Dayton, P. A.; Lee, A. P. On-chip Generation of

Microbubbles as a Practical Technology for Manufacturing Contrast Agents for Ultrasonic Imaging. Lab. Chip. 2007, 7, 463-468. 10. Dalla-palma, L.; Bertolotto, M.; Quaia, E.; Locatelli, M. Detection of Liver metastases with Pulse Inversion Harmonic Imaging: Preliminary Results. Eur Radiol. 1999, 9, 382-387. 11. Segers, T.; Versluis, M. Acoustic Bubble Sorting for Ultrasound Contrast Agent Enrichment. Lab. Chip. 2014, 14, 1705-1714. 12. Sirsi, S.; Feshitan, J.; Kwan, J.; Homma, S.; Borden, M. Effect of Microbubble Size on Fundamental Mode High Frequency Ultrasound Imaging in Mice. Ultrasound. Med. Biol. 2010, 36, 935-948. 13. Hettiarachchi, K.; Zhang, S.; Feingold, S.; Lee, A. P.; Dayton, P. A. Controllable Microfluidic Synthesis of Multiphase Drug-carrying Lipospheres for Site-targeted Therapy. Biotechnol. Progr. 2009, 25, 938-945. 14. Talu, E.; Hettiarachchi, K.; Zhao, S.; Powell, R. L.; Lee, A. P.; Longo, M. L.; Dayton, P. A. Tailoring the Size Distribution of Ultrasound Contrast Agents: Possible Method for Improving Sensitivity in Molecular Imaging. Mol. Imaging. 2007, 6, 384-392. 15. Cohen, J. L.; Cheirif, J.; Segar, D. S.; Gillam, L. D.; Gottdiener, J. S.; Hausnerova, E.; Bruns, D. E. Improved Left Ventricular Endocardial Border Delineation and Opacification with 20


Page 20 of 23

Page 21 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


OPTISON (FS069), a new echocardiographic contrast agent: Results of a phase III multicenter trial. J. Am. Coll. Cardiol. 1998, 32, 746-752. 16. Stride, E.; Edirisinghe, M. Novel Microbubble Preparation Technologies. Soft Matter 2008, 4, 2350-2359. 17. Cox, D. J.; Thomas, J. L. Ultrasound-Induced Dissolution of Lipid-Coated and Uncoated Gas Bubbles. Langmuir 2010, 26, 14774-14781. 18. Sijl, J.; Dollet, B.; Overvelde, M.; Garbin, V.; Rozendal, T.; de Jong, N.; Lohse, D.; Versluis, M. Subharmonic Behavior of Phospholipid-coated Ultrasound Contrast Agent Microbubbles. J. Acoust. Soc. Am. 2010, 128, 3239-3252. 19. Kwan, J. J.; Borden, M. A. Lipid Monolayer Collapse and Microbubble Stability. Adv. Colloid Interface Sci. 2012, 15, 82-99. 20. Szijjarto, C.; Rossi, S.; Waton, G.; Krafft, M. P. Effects of Perfluorocarbon Gases on the Size and Stability Characteristics of Phospholipid-coated Microbubbles: Osmotic Effect versus Interfacial Film Stabilization. Langmuir 2012, 28, 1182-1189. 21. Pisani, E.; Tsapis, N.; Galaz, B.; Santin, M.; Berti, R.; Taulier, N.; Kurtisovski, E.; Lucidarme, O.; Ourevitch, M.; Doan, B. T.; Beloeil, J. C.; Gillet, B.; Urbach, W.; Bridal, S. L.; Fattal, E. Perfluorooctyl Bromide Polymeric Capsules as Dual Contrast Agents for Ultrasonography and Magnetic Resonance Imaging. Adv. Funct. Mater. 2008, 18, 2963-2971. 22. Zhang, K.; Chen, H.; Guo, X.; Zhang, D.; Zheng, Y.; Zheng, H.; Shi, J. Double-scattering/reflection in a Single Nanoparticle for Intensified Ultrasound Imaging. Sci. Rep. 2015, 5, 1-11. 23. Zhao, X.; Zhang, X.; Xue, L.; Wang, J.; Shen, B.; Luo, C.; Wang, Q. Preparation and In Vivo Evaluation of Ligand-conjugated Polymeric Microbubbles as Targeted Ultrasound Contrast Agents. Colloids Surf. A. 2014, 452, 59-64. 24. Hu, H.; Zhou, H.; Liang, J.; An, L.; Dai, A.; Li, X.; Yang, H.; Yang, S.; Wu, H. Facile Synthesis of Amino-functionalized Hollow silica Microspheres and Their Potential Application for Ultrasound Imaging. J. Colloid. Interf. Sci. 2011, 358, 392-398. 25. Wang, X.; Chen, H.; Chen, Y.; Ma, M.; Zhang, K.; Li, F.; Zheng, Y.; Zeng, D.; Wang, Q.; Shi, j. Perfluorohexane-Encapsulated Mesoporous Silica Nanocapsules as Enhancement Agents for Highly Efficient High Intensity Focused Ultrasound (HIFU). Adv. Mater. 2012, 24, 785-791. 21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

26. Hu, H.; Zhou, H.; Du, J.; Wang, Z.; An, L.; Yang, H.; Li, F.; Wu, H.; Yang, S. Biocompatiable Hollow Silica Microspheres as Novel Ultrasound Contrast Agents for In Vivo Imaging. J. Mater. Chem. 2011, 21, 6576-6583. 27. Lin, P.-L.; Eckersley, R. J.; Hall, E. A. H. Ultrabubble: a Laminated Ultrasound Contrast Agent with Narrow Size Range. Adv. Mater. 2009, 21, 3949–3952. 28. Yang, P.; Ding, J.; Guo, J.; Shi, W.; Hu, J. J.; Wang, C. A Strategy for Fabrication of Uniform Double-shell Hollow Microspheres as Effective Acoustic Echo Imaging Contrast Agents Through a New Polymer-backbone-transition Method. J. Mater. Chem. B. 2013, 1, 544-551. 29. Katagiri, K.; Hashizume, M.; Ariga, K.; Terashima, T.; Kikuchi, J.-i. Preparation and Characterization of a Novel Organic–Inorganic Nanohybrid “Cerasome” Formed with a Liposomal Membrane and Silicate Surface. Chem-Eur. J. 2007, 13, 5272-5281. 30. Tong, W.; Dong, W.; Gao, C.; Mohwald, H. Charge-Controlled Permeability of Polyelectrolyte Microcapsules. J. Phys. Chem. B. 2005, 109, 13159-13165. 31. Zhang, C.; Cao, Z.; Zhu, W..; Liu, J.; Jiang, Q.; Shuai, X. Highly Uniform and Stable Cerasomal Microcapsule with Good Biocompatibility for Drug Delivery. Colloids Surf., B. 2014, 116, 327-333. 32. Schellinger, J. G.; Pahang, J. A.; Johnson, R. N.; Chu, D. S. H.; Sellers, D. L.; Maris, D. O.; Convertine, A. J.; Stayton, P. S.; Horner, P. J.; Pun, S. H. Melittin-grafted HPMA-oligolysine Based Copolymers for Gene Delivery. Biomaterials 2013, 34, 2318-2326. 33. Mingwu Shen; Hongdong Cai; Xifu Wang; Xueyan Cao; Kangan Li; Su He Wang; Rui Guo; Linfeng ; Guixiang Zhang; Shi, X. Facile One-pot Preparation, Surface Functionalization, and Toxicity Assay of APTS-coated Iron Oxide Nanoparticles. Nanotechnology 2012, 23, 1-10. 34. Saxena, V.; Diaz, A.; Clearfield, A.; Batteas, J. D.; Hussain, M. D. Zirconium Phosphate Nanoplatelets: A Biocompatible Nanomaterial for Drug Delivery to Cancer. Nanoscale 2013, 5, 2328-2336. 35. Boase, N. R. B.; Blakey, I.; Thurecht, K. J., Molecular Imaging with Polymers. Polym. Chem-UK. 2012, 3, 1384-1389. 36. Liu, B.; Zhou, X.; Yang, F.; Shen, H.; Wang, S.; Zhang, B.; Zhi, G.; Wu, D. Fabrication of Uniform Sized Polylactone Microcapsules by Premix Membrane Emulsification for Ultrasound Imaging. Polym. Chem-UK. 2014, 5, 1693-1701. 22


Page 22 of 23

Page 23 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table of Contents:

23


Highly Uniform Perfluoropropane-Loaded Cerasomal Microbubbles

Recommend Documents