Stimuli-responsive nanomaterials are increasingly important in a variety of applications such as biosensing, molecular imaging, drug delivery and tissue engineering. For cancer detection, a paramount challenge still exists in search of methods that can illuminate tumors universally regardless of their genotypes and phenotypes. Here we capitalized on the acidic, angiogenic tumor microenvironment to achieve broad detection of tumor tissues in a wide variety of mouse cancer models. This was accomplished using ultra-pH sensitive fluorescent nanoprobes that have tunable, exponential fluorescence activation upon encountering subtle, physiologically relevant pH transitions. These nanoprobes were silent in the circulation, then dramatically activated (>300 fold) in response to neovasculature or to the low extracellular pH in tumors. Thus, we have established non-toxic, fluorescent nanoreporters that can non-linearly amplify tumor microenvironmental signals, permitting identification of tumor tissue independently of histological type or driver mutation, and detection of acute treatment responses much more rapidly than conventional imaging approaches.

Switch it up: Tunable, pH‐responsive nanoparticles can be selectively activated in different endocytic compartments. At high pH values, micelle formation (see picture, left) quenches fluorescence by Förster resonance energy transfer. The micelles disassemble at low pH values, leading to fluorescence emission. This nonlinear on/off nanoplatform offers many exciting opportunities in diagnostic imaging and drug‐delivery applications.

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Tunable, ultra-pH responsive fluorescent nanoparticles with multichromatic emissions are highly valuable in a variety of biological studies, such as endocytic trafficking, endosome/lysosome maturation, and pH regulation in subcellular organelles. Small differences (e.g., <1 pH unit) and yet finely regulated physiological pH inside different endocytic compartments present a huge challenge to the design of such a system. Herein, we report a general strategy to produce pH-tunable, highly activatable multicolored fluorescent nanoparticles using commonly available pH-insensitive dyes with emission wavelengths from green to near IR range. pH-induced micellization is the primary driving force of fluorescence activation between the ON (unimer) and OFF (micelle) states. Among three possible photochemical mechanisms, homo Förster resonance energy transfer (homo-FRET) was found to be the most facile strategy to render ultra-pH response over the H-dimer and photoinduced electron transfer (PeT) mechanisms. Based on this insight, we selected several fluorophores with small Stoke shifts (<40 nm) and established a panel of multicolored nanoparticles with wide emission range (500-820 nm) and different pH transitions. Each nanoparticle maintained the sharp pH response (ON/OFF <0.25 pH unit) with corresponding pH transition point at pH 5.2, 6.4, 6.9 and 7.2. Incubation of a mixture of multicolored nanoparticles with human H2009 lung cancer cells demonstrated sequential activation of the nanoparticles inside endocytic compartments directly correlating with their pH transitions. This multicolored, pH-tunable nanoplatform offers many exciting opportunities for the study of many important cell physiological processes such as pH regulation and endocytic trafficking of subcellular organelles.

The endosomal barrier is a major bottleneck for the effective intracellular delivery of siRNA by nonviral nanocarriers. Here, we report a novel amphotericin B (AmB)-loaded, dual pH-responsive micelleplex platform for siRNA delivery. Micelles were self-assembled from poly(2-(dimethylamino)ethyl methacrylate)-block-poly(2-(diisopropylamino)ethyl methacrylate) (PDMAb-PDPA) diblock copolymers. At pH 7.4, AmB was loaded into the hydrophobic PDPA core, and siRNA was complexed with a positively charged PDMA shell to form the micelleplexes. After cellular uptake, the PDMA-b-PDPA/siRNA micelleplexes dissociated in early endosomes to release AmB. Live cell imaging studies demonstrated that released AmB significantly increased the ability of siRNA to overcome the endosomal barrier. Transfection studies showed that AmBloaded micelleplexes resulted in significant increase in luciferase (Luc) knockdown efficiency over the AmB-free control. The enhanced Luc knockdown efficiency was abolished by bafilomycin A1, a vacuolar ATPase inhibitor that inhibits the acidification of the endocytic organelles. These data support the central hypothesis that membrane poration by AmB and increased endosomal swelling and membrane tension by a "proton sponge" polymer provided a synergistic strategy to disrupt endosomes for improved intracellular delivery of siRNA. Graphical Abstract*

pH is an important physiological parameter that plays a critical role in cellular and tissue homeostasis. Conventional small molecular pH sensors (e.g., fluorescein, Lysosensor) are limited by broad pH response and restricted fluorescent emissions. Previously, we reported the development of ultra-pH-sensitive (UPS) nanoprobes with sharp pH response using fluorophores with small Stokes shifts (<40 nm). In this study, we expand the UPS design to a library of nanoprobes with operator-predetermined pH transitions and wide fluorescent emissions (400–820 nm). A copolymer strategy was employed to fine tune the hydrophobicity of the ionizable hydrophobic block, which led to a desired transition pH based on standard curves. Interestingly, matching the hydrophobicity of the monomers was critical to achieve a sharp pH transition. To overcome the fluorophore limitations, we introduced copolymers conjugated with fluorescence quenchers (FQs). In the micelle state, the FQs effectively suppressed the emission of fluorophores regardless of their Stokes shifts and further increased the fluorescence activation ratios. As a proof of concept, we generated a library of 10 nanoprobes each encoded with a unique fluorophore. The nanoprobes cover the entire physiologic range of pH (4–7.4) with 0.3 pH increments. Each nanoprobe maintained a sharp pH transition (on/off < 0.25 pH) and high fluorescence activation ratio (>50-fold between on and off states). The UPS library provides a useful toolkit to study pH regulation in many pathophysiological indications (e.g., cancer, lysosome catabolism) as well as establishing tumor-activatable systems for cancer imaging and drug delivery.

Polymeric micelles are supramolecular, core-shell nanoparticles that offer considerable advantages for cancer diagnosis and therapy. Their relatively small size (10-100 nm), ability to solubilize hydrophobic drugs as well as imaging agents, and improved pharmacokinetics provide a useful bioengineering platform for cancer applications. Several polymeric micelle formulations are currently undergoing phase I/II clinical trials, which have shown improved antitumor efficacy and reduced systemic toxicity. This minireview will focus on recent advancements in the multifunctional design of micellar nanomedicine with tumor targeting, stimulated drug release, and cancer imaging capabilities. Such functionalization strategies result in enhanced micellar accumulation at tumor sites, higher drug bioavailability, as well as improved tumor diagnosis and visualization of therapy. Ultimately, integrated nanotherapeutic systems (e.g., theranostic nanomedicine) may prove essential to address the challenges of tumor heterogeneity and adaptive resistance to achieve efficacious treatment of cancer.

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