Biodegradable pH-sensing dendritic nanoprobes for near-infrared fluorescence lifetime and intensity imaging.

The overproduction of acidic byproducts is implicated in the development of numerous diseases, and there is currently great interest in developing contrast agents that can image acidic tissue in vivo.1 Here we report a pH-sensing biodegradable near-infrared (NIR) nanoprobe capable of providing complementary information through both fluorescence lifetime measurements and signal amplification in acidic environments in vivo (Figure 1). In the “off” state, the NIR fluorescence of this sensing nanoprobe is deactivated (Figure 2) by exploiting the polyvalent periphery of a compact biodegradable aliphatic polyester dendrimer (in blue Figure 1 and Scheme 1).2 NIR dyes (in red) are attached via acidsensitive linkages to the periphery of the dendrimer. In such close proximity, the dyes form H-type homoaggregates via face-to-face stacking (Figure 2a). This agglomeration suppresses both their fluorescence lifetime and intensity through nonradiative decay3 and thereby minimizes background noise.4 In acidic environments, this latent NIR nanoprobe is activated (Figure 2b) by the release of multiple copies of NIR fluorophores. As the fluorophores drift apart, their fluorescence lifetime begins to reflect the new environment which they are in, with simultaneous amplification of the fluorescence intensity signal (Figure 1). The dendrimer is PEGylated (in green Figure 1 and Scheme 1) for improved biocompatibility and to impart biological stealth.5 A number of small-molecule NIR dyes that operate in the tissue transparency window (750-900 nm)6 respond to changes in proton concentrations at physiologically relevant pH, with a change in fluorescence intensity.7,8 However, upon intravenous injection, these small-molecule NIR imaging probes immediately bind to serum proteins.9 This opsonization process may lead to poor bioavailability and more importantly dominates the excited-state fluorescent properties of the fluorophores.10As a result, limited functional information on the in vivo environment can be extracted through lifetime imaging.10 NIR probes that can be shielded from immediate opsonization and activated in desired target tissue are a promising avenue to functional fluorescence lifetime and intensity imaging. Nanoscale probes based on polymer-dendrimer hybrids offer an ideal and modular platform for biomedical applications.11 The level of branching and hydrodynamic volume of the polymeric carrier can be tuned12 to suit the bioavailability needs of the application.5 Furthermore, deactivation/activation of fluorescence intensity can be achieved by using a variety of environmentally sensitive linkages.8,13 In heterogeneous media, such as tissue and cells, quantitative fluorescence intensity measurements require the modeling of photon transport in the media which depends on fluorophore concentration, path length, photobleaching, and scattering.14 In contrast, the excited states of NIR fluorophores are less dependent on these parameters and respond to their local environment with a sensitivity comparable to radioactive probes without the inherent danger of radioactivity.14 We show here that shielding the dyes within the PEGylated carrier can control the influence of serum proteins on the fluorescence lifetime properties of the fluorophores. Pentaerythritol was chosen as the dendrimer core for its compactness that is thought to promote aggregation. It can be divergently dendronized in one step to afford eight branching points.15 Aliphatic polyester dendrimers based on the monomer 2,2bis(hydroxymethyl) propanoic acid are especially promising for imaging applications because of their low toxicity, low immunogenicity, and biodegradable structure.2,16 We introduced heterobifunctionality by coupling Boc-4-acetylphenylalanine (Scheme 1) to the pentaerythritol polyester dendrimer.17 The amino group was deprotected, and eight PEG chains (5000 Da) were attached through † University of California, Berkeley. ‡ Washington University, St. Louis. Figure 1. (a) “OFF: state: in neutral pH, found in healthy tissue, the NIR fluorescence intensity of the nanoprobe is silenced and its fluorescence lifetime properties are insensitive to the presence of in vivo proteins. (b) “ON” state: in acidic pH found in diseased tissue, the NIR fluorescence intensity of the nanoprobe increases as the dyes (red) are released and their fluorescence lifetime is sensitive to the presence of in vivo proteins.