Polymer Hybrid Microbubble with MRI

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Self-Assembled Fe3O4/Polymer Hybrid Microbubble with MRI/ Ultrasound Dual-Imaging Enhancement Sheng Song,† Heze Guo,† Zequan Jiang,† Yuqing Jin,‡ Zhaofeng Zhang,*,‡ Kang Sun,*,† and Hongjing Dou*,† †

The State Key Laboratory of Metal Matrix Composites, School of Materials Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240, PR China ‡ Department of Plastic and Reconstructive Surgery, Shanghai First People’s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200080, PR China S Supporting Information *

ABSTRACT: An Fe3O4 nanoparticle/polymer hybrid microbubble was developed using a facile self-assembly approach. This approach involves two steps, including the initial fabrication of the iron oxide nanoparticle (IONP)/polymer hybrid microcapsules via self-assembly and a subsequent gas-filling process to yield the final microbubbles. Both in vitro and in vivo experiments demonstrated that the composite gas-filled microbubbles exhibit excellent T2-weighted magnetic resonance imaging (MRI) enhancement as well as ultrasound (US) imaging enhancement capabilities. Besides, this flexible approach allows the facile control of the microbubbles’ size and thus the imaging capabilities of the microbubbles through the tuning of the molar ratio between the precursors.

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microbubbles. Therefore, the need for a facile and flexible approach for the fabrication of MRI/US dual-imaging microbubbles remains. As a route through which NPs or other discrete components spontaneously organize via noncovalent interactions, polymermediated nanoparticle self-assembly provides a flexible approach for fabricating a diverse range of multifunctional composite materials.12,13 In particular, polymer-mediated Fe3O4 nanoparticles hold great promise for the development of multifunctional composite materials or devices.14 Among various polymer-mediated nanoparticle self-assembly approaches, the polyamine−salt aggregate (PSA) assembly strategy developed by Wong et al. has provided a flexible approach to fabricating microcapsules that incorporate SiO2 or Au nanoparticles.15−20 In this approach, the PSAs formed through ionic interactions between a cationic polyamine and multivalent anionic salts serve as templates that subsequently

INTRODUCTION In biomedical diagnostic applications, ultrasound (US) imaging and magnetic resonance imaging (MRI) are considered to be complementary techniques due to the sensitivity provided by US and the high spatial resolution of MRI.1 To improve the signal-to-noise ratio (SNR) of an image, contrast agents are usually necessary for both MRI and US techniques.2−4 For example, superparamagnetic iron oxide nanoparticles (IONPs) can shorten the T2 and T2* values of a tissue and thus provide a negatively enhanced contrast signal for MRI techniques.5 Meanwhile, US imaging contrast agents are usually based on gas-filled microbubbles with diameters ranging between 100 nm and 10 μm that allow them to provide stronger backscattered signals from blood.2,6 However, these two classes of contrast agents were usually developed in two different ways, and there have been only a few reports on the development of the MRI/ US dual-imaging contrast agents.7−11 In addition, the reported microbubble contrast agents with an IONP/polymer shell were all fabricated by an emulsification method, although the emulsification method was usually time-consuming and could lead to the possibility of residual emulsifiers remaining in the © 2014 American Chemical Society

Received: May 30, 2014 Revised: August 13, 2014 Published: August 19, 2014 10557

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hybrid aggregates (IPHAs) in which the IONPs are held together by the oppositely charged poly(allylamine) chains. In the third substep, negatively charged poly(acrylic acid) (PAA) chains are combined with the IONP-deposited aggregates to absorb the interior cationic poly(allylamine) chains along with their associated IONPs on the periphery of the aggregates, leading to the formation of microcapsules. On the basis of the fabrication of the hybrid microcapsules through step A, the inner cavity of the microcapsule was filled with a gas with a low degree of dispersion, tetradecafluorohexane (C6F14), in step B.2 According to our design, the resultant hybrid microbubbles would possess MRI/US dual-imaging enhancement capabilities due to the incorporation of the IONPs that can enhance MRI imaging and also due to the encapsulated C6F14 gas that can enhance the US imaging contrast. To explore the mechanism of the proposed self-assembly approach to fabricating these microbubbles, we observed the morphologies of the products obtained after each step or substep through a combination of transmission electron microscopy (TEM) and laser scanning confocal microscopy (LSCM). To gain insight into this mechanism, the amino groups of the poly(allylamine) chains were partially reacted with fluorescein sodium salt so that they could be readily observed via LSCM. The images in Figure 1a−c show the respective morphologies of the ICPAs, IPHAs, and microcapsules obtained from the three substeps in step A. In the first substep of step A, the complexation between poly(allylamine) and citrate yielded ICPAs with a solid spherical morphology (Figure 1a). The poly(allylamine) chains appeared green and were located homogeneously throughout the spheres. The addition of the IONPs during the second substep of step A yielded IPHAs with a similar morphology and size to those of the ICPAs (Figure 1b), and the TEM indicated that the IONPs, which exhibited a strong contrast, were located at the periphery of the microspheres. Furthermore, after the third substep of step A, the products underwent a significant morphological transition from spherical IPHAs to microcapsules with a hollow inner cavity, as shown in Figure 1c. Besides, it was clearly evident that both the IONPs and the poly(allylamine) chains occupied the shells of the microcapsules (Figure S5 and Table S1 in SI). On this basis, the microcapsules were filled with C6F14 through a gas supersaturation method as illustrated in step B of Scheme 1 to produce gas-filled microbubbles. As is evident from the TEM images of the resultant microbubbles shown in Figure 1d, step B did not cause any morphological changes, thus indicating that the self-assembled microcapsules were structurally robust and could withstand the gas-filling process. The resultant microbubbles were anticipated to exhibit MRI/US dual-imaging enhancement capabilities.

attract a negatively charged shell precursor to produce the targeted microcapsules. Although there are currently only a limited range of inorganic nanoparticles that have proven suitable for this PSA self-assembly approach, this strategy has provided valuable insight that may be applied toward designing an approach that yields self-assembled microcapsules or even gas-filled microbubbles for imaging contrast applications.

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RESULTS AND DISCUSSION Herein, we report a facile self-assembly approach for fabricating IONP/polymer hybrid microbubbles for MRI/US dual imaging applications. As shown in Scheme 1, this approach involves two Scheme 1. Schematic Diagram Depicting the Formation of Self-Assembled Microcapsules and Microbubbles

steps, including the initial fabrication of the IONPs/polymer hybrid microcapsules via self-assembly (step A) and a subsequent gas-filling process to yield the final microbubbles (step B). Between the two steps, the gas-filling process in step B involved a single stage (the detailed procedure is illustrated in the Experimental Section of the Supporting Information (SI)), whereas step A involves three substeps. In the first substep of step A, an amino-group-bearing cationic polymer, poly(allylamine), and a carboxyl-group-containing multivalent salt (trisodium citrate dehydrate, abbreviated as Cit) undergo complexation together in aqueous media via ionic interactions to yield ionically cross-linked polymer aggregates (ICPAs). During the second substep, citric acid (CA)-capped Fe3O4 IONPs (CA-capped IONPs, characterized in Figure S1−S4 in the SI) are added to the solution to form IONP/polymer

Figure 1. TEM images of ICPAs (a), IPHAs (b), and microcapsules observed before (c) and after (d) the gas-filling process. The insets in panels a− c show the corresponding confocal microscopy images of samples in which the poly(allylamine) chains had been labeled using fluorescein groups, and they thus show the location of poly(allylamine) chains during the self-assembly process. 10558

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The MRI enhancement provided by both the self-assembled hybrid microcapsules and the gas-filled microbubbles was first evaluated in vitro. IONPs have been shown previously to enhance the transverse relaxivity (r2, the reciprocal of the proton spin−spin relaxation time that corresponds to the slope of the plot of the inverse of T2 versus the iron concentration) as MRI T2 contrast enhancement agents.21,22 The term r2 is typically used as a standard for evaluating the relaxivity of an MRI T2 contrast agent, and a larger slope indicates a higher relaxivity.23 With this in mind, the T2 enhancement capabilities of the IONPs, the IONPs/polymer hybrid microcapsules, and the microbubbles were investigated, and these results are summarized in Figure 2. It was apparent that all three samples

Figure 3. In vitro US images of degassed water, microcapsules, and microbubbles with gas interiors and Sono Vue with emission frequencies of 4 and 11 MHz. The concentration of the microcapsules and the microbubbles was 0.25 mg/mL.

the higher sound attenuation that is provided by C6F14 within the gas-filled microbubbles. The US signal intensities of degassed water, microcapsules, gas-filled microbubbles, and commercial US contrast agent Sono Vue were measured by the mean gray scale of their US images, and the results are shown in Figure 4. With US

Figure 2. Spin-spin relaxivity (r2) of IONPs, microcapsules, and microbubbles. The insets show the in vitro T2-weighted images of IONPs, microcapsules, and microbubbles.

resulted in a significant signal reduction. The IONPs possess an r2 value of 16 mM−1 s−1, and the IONP-loaded vesicles have an r2 relaxivity of 122 mM−1 s−1. As reported in the literature, the value of r2 is strongly dependent on the local field inhomogeneity that correlates with the relative volume of the IONPs and the difference in the magnetic susceptibility between the particle and the suspension medium.23,24 The r2 relaxivity increased when the IONPs assembled to form vesicles because the resultant aggregates provided the IONPs with both a high relative volume fraction and magnetic susceptibility. After the C6F14 gas was introduced into the interior of the microcapsules, the r2 relaxation of the microbubbles decreased to 12 mM−1 s−1. This r2 value was still comparable to those of commercial US contrast agents, which are typically in the range of 7−17 mM−1 s−1.25,26 The reason for the decrease in r2 exhibited by the gas-filled microbubbles may be attributed to the IONPs within the shell undergoing fewer interactions with water protons after the microcapsule cavities had been filled with the gas, thus resulting in a decreased local field inhomogeneity. To investigate the US imaging contrast efficiency of the microcapsules and the gas-filled microbubbles, in vitro ultrasonography was performed with emission frequencies of 4 and 11 MHz, which are two commonly used frequencies27 in medical diagnostic applications. As shown in Figure 3, the image intensity was enhanced at both frequencies in the presence of gas-filled microbubbles, in comparison to that observed in the presence of degassed water and the gas-free microcapsules. According to the mechanism of 2D imaging, the image intensity is directly proportional to the strength of the sound attenuation.28 Therefore, the enhancement of the intensity of the in vitro ultrasonography images results from

Figure 4. US signal intensities of degassed water, microcapsules, gasfilled microbubbles, and Sono Vue with emission frequencies of 11 and 4 MHz.

emission at frequencies of 4 and 11 MHz, degassed water and microcapsules have quite low US signal intensities, which are 1 at 4 MHz and 7 at 11 MHz US sound emissions, respectively. However, both the as-prepared microbubbles and Sono Vue microbubbles possess greatly enhanced US signal intensities. The US signal intensities of the self-assembled microbubbles are enhanced to 46 at 4 MHz and 64 at 11 MHz US sound emissions, and the US signal intensities of Sono Vue microbubbles are 43 and 56 at 4 and 11 MHz US sound emissions, respectively. The comparasion indicates that the US imaging capabilities of the microbubbles are comparable to the Sono Vue microbubbles. As a US contrast agent widely used in clinical applications, the Sono Vue microbubble is a sulfur hexafluororide (SF6) emulsion with a lipid shell.29 Although the US imaging property of Sono Vue microbubbles is remarkable, there is still much more room for the improvement of the property considering the instability of the lipid shells at the emission of ultrasonic pressure and the relatively wider size distribution of the microbubbles. In comparasion, the Fe3O4/ polymer hybrid microbubbles possess the advantages of tunable size and good shell stability under US exposure. The in vitro ultrasonography study indicated that the Fe3O4/polymer hybrid microbubble has significant US signal enhancement capability as a novel US contrast agent. 10559

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In consideration of the flexibility of the polymer-mediated IONP self-assembly approach12,30 in controlling the diameters and the properties of the resultant microcapsules, the relationship between the diameters of the products and the ratios of the precursors was investigated, and the results are shown in Figures S6 and S7 and Tables S2 and S3 in the SI. The related results demonstrated that with the same Fe concentration, the larger microbubbles provided better ultrasonic contrast. Besides, the cytotoxicity of the IONP/polymer hybrid microcapsules was investigated through various approaches, and the results shown in Figures S8 and S9 of the SI indicated that the microcapsules exhibited a low cellular cytotoxicity. We also evaluated the effectiveness of the microbubbles as contrast agents for the in vivo MRI and US imaging of a rat’s liver (Figures S10 and S11 in the SI). The studies performed using rats as models suggested that these self-assembled gas-filled microbubbles hold great potential as MRI/US dual-imaging contrast agents.

(5) Laurent, S.; Forge, D.; Port, M.; Roch, A.; Robic, C.; Vander Elst, L.; Muller, R. N. Magnetic Iron Oxide Nanoparticles: Synthesis, Stabilization, Vectorization, Physicochemical Characterizations, and Biological Applications. Chem. Rev. 2008, 108, 2064−2110. (6) Kiessling, F.; Schmitz, G.; Gatjens, J. Chapter 7.3 Design and Use of Contrast Agents for Ultrasound Imaging. In Biomedical Imaging: The Chemistry of Labels, Probes and Contrast Agents; The Royal Society of Chemistry: Cambridge, 2012; pp 391−410. (7) Yang, F.; Li, Y.; Chen, Z.; Zhang, Y.; Wu, J.; Gu, N. Superparamagnetic iron oxide nanoparticle-embedded encapsulated microbubbles as dual contrast agents of magnetic resonance and ultrasound imaging. Biomaterials 2009, 30, 3882−3890. (8) Liu, Z.; Lammers, T.; Ehling, J.; Fokong, S.; Bornemann, J.; Kiessling, F.; Gätjens, J. Iron oxide nanoparticle-containing microbubble composites as contrast agents for MR and ultrasound dualmodality imaging. Biomaterials 2011, 32, 6155−6163. (9) Xu, B.; Dou, H.; Tao, K.; Sun, K.; Ding, J.; Shi, W.; Guo, X.; Li, J.; Zhang, D.; Sun, K. Two-in-One” Fabrication of Fe3O4/MePEG-PLA Composite Nanocapsules as a Potential Ultrasonic/MRI Dual Contrast Agent. Langmuir 2011, 27, 12134−12142. (10) Niu, C.; Wang, Z.; Lu, G.; Krupka, T. M.; Sun, Y.; You, Y.; Song, W.; Ran, H.; Li, P.; Zheng, Y. Doxorubicin loaded superparamagnetic PLGA-iron oxide multifunctional microbubbles for dual-mode US/MR imaging and therapy of metastasis in lymph nodes. Biomaterials 2013, 34, 2307−2317. (11) Huang, C.; Qu, G.; Wei, Z.; Xu, B.; Ding, J.; Dou, H.; Sun, K. Fabrication and Medical Imaging of Ultrasonic Imaging Nanocaspsules. Chem. J. Chin. Univ. 2014, 35, 191−198. (12) Shenhar, R.; Norsten, T. B.; Rotello, V. M. Polymer-Mediated Nanoparticle Assembly: Structural Control and Applications. Adv. Mater. 2005, 17, 657−669. (13) Grzelczak, M.; Vermant, J.; Furst, E. M.; Liz-Marzán, L. M. Directed Self-Assembly of Nanoparticles. ACS Nano 2010, 4, 3591− 3605. (14) Tian, Y.; Zhang, Y.-L.; Ku, J.-F.; He, Y.; Xu, B.-B.; Chen, Q.-D.; Xia, H.; Sun, H.-B. High performance magnetically controllable microturbines. Lab Chip 2010, 10, 2902−2905. (15) Rana, R. K.; Murthy, V. S.; Yu, J.; Wong, M. S. Nanoparticle Self-Assembly of Hierarchically Ordered Microcapsule Structures. Adv. Mater. 2005, 17, 1145−1150. (16) Bagaria, H. G.; Wong, M. S. Polyamine-salt aggregate assembly of capsules as responsive drug delivery vehicles. J. Mater. Chem. 2011, 21, 9454−9466. (17) Nguyen, Q. X.; Belgard, T. G.; Taylor, J. J.; Murthy, V. S.; Halas, N. J.; Wong, M. S. Water-Phase Synthesis of Cationic Silica/Polyamine Nanoparticles. Chem. Mater. 2012, 24, 1426−1433. (18) Yu, J.; Murthy, V. S.; Rana, R. K.; Wong, M. S. Synthesis of nanoparticle-assembled tin oxide/polymer microcapsules. Chem. Commun. 2006, 10, 1097−1099. (19) Amali, A. J.; Rana, R. K. Trapping Pd(0) in nanoparticleassembled microcapsules: an efficient and reusable catalyst. Chem. Commun. 2008, 35, 4165−4167. (20) Song, S.; Dou, H.; Sun, K. Properties of Hybrid Capsules Fabricated from Template Free Self assembly. J. Funct. Polym. 2011, 24, 438−446. (21) Jaganathan, H.; Gieseck, R. L.; Ivanisevic, A. Transverse Relaxivity Changes after Layer-by-Layer Encapsulation of Multicomponent DNA Templated Nanostructures. J. Phys. Chem. C 2010, 114, 22508−22513. (22) Fan, H.-M.; Olivo, M.; Shuter, B.; Yi, J.-B.; Bhuvaneswari, R.; Tan, H.-R.; Xing, G.-C.; Ng, C.-T.; Liu, L.; Lucky, S. S.; Bay, B.-H.; Ding, J. Quantum Dot Capped Magnetite Nanorings as High Performance Nanoprobe for Multiphoton Fluorescence and Magnetic Resonance Imaging. J. Am. Chem. Soc. 2010, 132, 14803−14811. (23) Duan, H.; Kuang, M.; Wang, X.; Wang, Y. A.; Mao, H.; Nie, S. Reexamining the Effects of Particle Size and Surface Chemistry on the Magnetic Properties of Iron Oxide Nanocrystals: New Insights into Spin Disorder and Proton Relaxivity. J. Phys. Chem. C 2008, 112, 8127−8131.

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CONCLUSIONS A hybrid microbubble as a MRI/US contrast agent was developed through an Fe3O4 nanoparticle/polymer selfassembly approach. Both in vitro and in vivo experiments demonstrated that the resultant gas-filled microbubbles exhibit excellent MRI/ultrasonic dual-imaging enhancement capabilities. In addition, this flexible approach allows the facile control of the size and thus the imaging capabilities of the microbubbles through the tuning of the molar ratio between the precursors.

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ASSOCIATED CONTENT

S Supporting Information *

More details of the experimental methods and characterization of IONPs, microcapsules, and microbubbles. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

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

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS H.D. is grateful for the financial support of the National Natural Science Foundation of China (nos. 21174082 and 21374061), the SJTU SMC-Chen Xing Young Scholars Award, the Program of New Century Excellent Talent in University (NCET-13-0360), and the Instrumental Analysis Center of the SJTU.

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