Distinct ON/OFF fluorescence signals from dual-responsive activatable nanoprobes allows detection of inflammation with improved contrast
Graphical abstract
Introduction
Visualizing molecular features of pathological tissue micro-environments rather than the structural consequences of these abnormalities has the potential to facilitate detection of lesions at a time when treatment is most effective [1], [2]. For example, detecting cancer at an early stage is one of the most important strategies to reduce morbidity and mortality [3]. Near-infrared (NIR) optical imaging, being non-invasive, safe, highly sensitive and able to provide real-time image guidance, is emerging as a promising tool to help meet this need [4]. It is admitted that the diagnostic performance (i.e., sensitivity, accuracy and limit of detection) of a molecular imaging platform depends on its capacity to provide high specific signal relative to background signal originating from normal tissues (target-to-background ratios, T/B) [5]. Recently, nanomaterial-based optical probes have been developed to enhance detection sensitivity and extend the period available for imaging as they carry a large number of reporters, offer high surface to volume ratio causing higher binding capacity and are cleared from circulation slowly because of their larger size [6], [7]. Although this strategy improves physical detection (i.e., signal strength), achieving sufficient specificity remains a significant challenge because of the predominance of accumulation in liver and other blood-filtering organs (e.g., kidneys, and spleen) which can generate background signal, obscure weaker details, cause false positives, and decrease confidence in true positives [8]. A number of approaches (e.g., reconstruction algorithms, image subtraction) have been designed to mitigate the detrimental effect of background signal [9], [10]. While some of these methodologies work well for autofluorescence removal, they are not ideal when the noise originates from nonspecific accumulation of imaging agents.
Optical molecular probes designed to be activatable, that is, silent (‘OFF’ state) until they interact with the biological target and turn ‘ON’, can provide a decrease in background signal, hence superior accuracy and limits of detection [11], [12], [13], [14]. However, the nature of the activatable signal underlying optical probes can have a tremendous influence on their performance in vivo. For instance, in most intensity-based approaches, the inevitable residual ‘OFF’ state signal, which shares the same emission signature as the ‘ON’ state signal, can be picked up by the detector and contribute to increasing the noise level, later decreasing the detection threshold [15], [16], [17], [18]. Due to the limited amount of biomarkers and the predominance of nonspecific accumulation, the seemingly low basal signal of the ‘OFF’ state can become substantial compared to the specific signal originating from the activated probes. This is especially problematic shortly after injection when the level of activation is not yet high enough to yield good contrast. On the other hand, spectrally-resolved detection systems [19], [20], [21], [22], characterized by emission peak shifts, offer improved contrast and detection accuracy due to lower crosstalk and easier discrimination between target and non-specific background signals [23], [24]. Owing to the ability to perform ratiometric measurements, spectral-based probes can also provide quantitative information [25], [26].
Herein, we developed a novel class of activatable nanoprobes (NPs, Fig. 1), leveraging on the advantages of nanomaterial-based and spectrally-resolved detection schemes, which enabled fluorescence detection of inflamed areas with large T/B reaching as high as 22, only 3 h post-injection. In this strategy, stimuli-responsive dextran-based materials (Fig. S1, see Experimental Section, Materials) [27], [28], whose water solubility changes upon exposure to biomarkers of inflammation (i.e., acidosis and oxidative stress) [29], [30], are used to effectively control the fluorescent intensity and the spectral profile of multiple NIR-emitting IR-780 dye molecules at once (Fig. 1). To maximize the signal strength, we rely on a ‘dual responsivity’ approach to build NPs capable of simultaneous synergistic response to acidic pH and elevated levels of hydrogen peroxide (H2O2), two biomarkers intrinsically associated with inflammation processes [31], [32]. Upon intravenous injection, the nanoprobes circulate in the blood stream and through healthy tissues where they remain in their ‘OFF’ state (Fig. 1B, healthy tissue). In pathological microenvironments, the incidence of vessel leakiness and compromised blood flow results in transvascular transport of NPs into the inflamed tissues and/or retention in blood capillaries where they are selectively turned ‘ON’ by the acidic and oxidative environments (Fig. 1B, pathological tissue) [33], [34], [35]. To minimize the contribution of nonspecific accumulation, we take advantage of the distinct emission signatures of the NPs' states (silenced vs. activated) to spectrally resolve the detected signal and isolate exclusively the activated signal in inflamed areas.
Section snippets
Materials
Dextran (9–11 kDa), poly(lactic-co-glycolic acid) (PLGA, ratio: 50:50; Mw: 7–17 kDa; alkyl ester terminated), IR-780 iodide (98%), indocyanine green (ICG, USP reference standard), 2-methoxypropene (97%), pyridinium p-toluenesulfonate (99%), 4-(Hydroxymethyl)phenylboronic acid pinacol ester (97%), sodium sulfate (99%), 4-(dimethylamino)pyridine (DMAP), carbonyldiimidazole (≥97%), magnesium sulfate (≥99.5%), and triethylamine (≥99%), were purchased from Sigma Aldrich. Pluronic® F-127 (poloxamer
Preparation and characterization of activatable NIR fluorescent nanoprobes
In our nanoprobe design, the responsive polymeric material is of central importance as the speed, sensitivity, and selectivity of signal activation depends on the effectiveness of its interaction with the biological target. Naturally occurring polysaccharides possess a number of appealing characteristics (e.g. biocompatibility, biodegradability, low toxicity, high abundance and low cost) and thus have been increasingly incorporated into a variety of nanoparticle formulations [36]. Because of
Discussion
Combining fast responsive polymeric systems with NIR fluorescent dyes to create robust activatable contrast agents offers a number of advantages. First, these biosensors are highly versatile, as all their constituting elements are interchangeable. A variety of fluorescent dyes and stimuli-responsive polymers can be used to tune NPs' emission and the chemical species to which they selectively respond. As demonstrated in this study, combining materials responsive to complementary biomarkers (e.g.
Conclusion
We have developed a system capable of detecting inflammation-associated chemical species with high sensitivity and specificity. Since molecular changes occur before structural, functional, or anatomical changes, our imaging methodology has the potential to facilitate earlier identification of small inflammatory foci and timely clinical intervention. The impact in oncology could be crucial, as early detection greatly enhances survival. Also, high-resolution delineation of primary tumor sites
Acknowledgments
This research was made possible by an NIH New Innovator Award (DP 2OD006499), King Abdulaziz City for Science and Technology (through the KACST-UCSD Center of Excellence in Nanomedicine and Engineering), National Institute of Arthritis and Musculoskeletal and Skin Diseases (MG: 1K08AR064834), NCRR (S10 RR027970), and NIH/NHLBI (P01 HL091830). NMR data was acquired at the UCSD Skaggs School of Pharmacy and Pharmaceutical Sciences NMR Facility. Serum biochemistry was undertaken and analyzed by
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