Nanobubbles from gas-generating polymeric nanoparticles: ultrasound imaging of living subjects.

Ultrasound (US) is a noninvasive biomedical imaging modality that is widely available, inexpensive, safe, and provides real-time imaging and diagnosis. US contrast enhancement improves the image quality because of acoustic impedance mismatch, which can occur when a liquid or gas is encapsulated within shell materials or when liquid or solid particles with a high density difference are brought together. Microbubbles were primarily used as US contrast agents for blood flow imaging, however, development of metabolic or molecular US imaging that employs microbubbles has been limited because sub-microbubbles or nanoparticles that are appropriate for tissue penetration have poor echogenic sensitivity, while nanoparticles can not be visualized at the resolution of US imaging instruments (typically 50 to 100 mm). Recently, nanoparticles have been employed in numerous biomedical applications. Nanoparticles bypass the reticuloendothelial system, and allow prolonged circulation and passive localization within tumor vasculature by tissue extravasation and enhanced permeation and retention (EPR). Moreover, nanoparticles can be modified by addition of ligands that increase nanoparticle affinity toward specific target sites, such as tumors. In addition, the insertion of protease cleavage sites can provide activatable imaging probes, thus allowing simultaneous molecular imaging and therapeutics. The need to use nanosized carriers for in vivo imaging and therapy has led to the development of laser-induced photoacoustic US and to the exploration of materials-based approaches that employ perfluorocarbonencapsulated lipid-based nanoparticles and polymeric micelles. Herein, we describe the generation of nanobubbles, which can be imaged by US and are derived from gas-generating polymeric nanoparticles (GGPNPs). The proposed mechanism involves localization of echogenic GGPNPs in a tumor and coalescence of generated nanobubbles, followed by fusion of nanobubbles into microbubbles. Significantly, we produced GGPNP nanobubbles without encapsulation of a gas precursor (perfluorocarbon). Instead, in our system, the GGPNP carbonate side chain is degraded to form carbon dioxide. The mechanism appears to involve diffusion of water into the GGPNPs, cleavage of the carbonate side chains by hydrolysis, and formation of carbon dioxide nanobubbles on the GGPNP surface, followed by expansion or coalescence of nanobubbles into microbubbles. The resulting microbubbles exhibit resonance under a US field (Figure 1a). Polyesters with carbonate side chains (poly(BL-PO)) were synthesized by ring-opening copolymerization of gbutyrolactone and propylene oxide using samarium diiodide as initiator (Figure S1b in the Supporting Information). The carbonate side chains were created by conjugation with hydroxy groups of hydroxy g-butyrolactone and cholesteryl chloroformate or ethyl chloroformate (Figure S1–S3 in the Supporting Information). The copolymerization ratios of cholesteryl carbonate g-butyrolactone and ethyl carbonate g-butyrolactone were varied with respect to that of propylene oxide. The resulting chemical structures are shown in Figure 1b. The initial feed ratios of three monomers were 1:2:1 for poly(BL-PO). The actual ratio of three monomer units in the poly(BL-PO) was confirmed as 1:1.74:0.81 by H NMR spectroscopy (see Figure S4 in the Supporting Information). This result shows that the compositions of the copolymers were well-matched to the initial feed ratio. The presence of the cholesteryl group means that the relative proportions of cholesteryl carbonate and ethyl carbonate determine the physical stability of the polymer. The hydrolyzed particles produce biocompatible water, carbon dioxide, cholesterol, and ethanol. To prove the biocompatibility of our particles, we examined their effect on cell viability. At a concentration of up to 1 mgmL , our particles were not cytotoxic (see Figure S8 in the Supporting Information). Nanoparticles generated from poly(BL-PO) (1:2:1) were termed GGPNPs. Dynamic light scattering (DLS) indicated that the average GGPNP diameter was 581 nm (Figure S6 in the Supporting Information), and transmission electron microscopy (TEM) indicated that the GGPNP morphology was spherical and the diameter was less than 500 nm (Figure 1c). The size appeared larger by DLS probably [*] Dr. E. Kang, H. S. Min, Dr. H. J. Ahn, Dr. I. C. Yoon, Dr. K. Choi, Dr. K. Kim, Dr. I. C. Kwon Biomedical Research Center Korea Institute of Science and Technology 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136 791 (Korea) Fax: (+82)2-958-5909 E-mail: ikwon@kist.re. kr