Controlled In Situ Self-Assembly of Biotinylated Trans-Cyclooctene Nanoparticles for Orthogonal Dual-Pretargeted Near-Infrared Fluorescence and Magnetic Resonance Imaging.

A pretargeted strategy that decouples targeting vectors from radionuclides has shown promise for nuclear imaging and/or therapy in vivo. However, the current pretargeted approach relies on the use of antibodies or nanoparticles as the targeting vectors, which may be compromised by poor tissue penetration and limited accumulation of targeting vectors in the tumor tissues. Herein, we present an orthogonal dual-pretargeted approach by combining stimuli-triggered in situ self-assembly strategy with fast inverse electron demand Diels-Alder (IEDDA) reaction and strong biotin-streptavidin (SA) interaction for near-infrared fluorescence (NIR FL) and magnetic resonance (MR) imaging of tumors. This approach uses a small-molecule probe (P-Cy-TCO&Bio) containing both biotin and trans-cyclooctene (TCO) as a tumor-targeting vector. P-Cy-TCO&Bio can efficiently penetrate subcutaneous HeLa tumors through biotin-assisted targeted delivery and undergo in situ self-assembly to form biotinylated TCO-bearing nanoparticles (Cy-TCO&Bio NPs) on tumor cell membranes. Cy-TCO&Bio NPs exhibited an "off-on" NIR FL and retained in the tumors, offering a high density of TCO and biotin groups for the concurrent capture of Gd-chelate-labeled tetrazine (Tz-Gd) and IR780-labeled SA (SA-780) via the orthogonal IEDDA reaction and SA-biotin interaction. Moreover, Cy-TCO&Bio NPs offered multiple-valent binding modes toward SA, which additionally regulated the cross-linking of Cy-Gd&Bio NPs into microparticles (Cy-Gd&Bio/SA MPs). This process could significantly (1) increase r1 relaxivity and (2) enhance the accumulation of Tz-Gd and SA-780 in the tumors, resulting in strong NIR FL, bright MR contrast, and an extended time window for the clear and precise imaging of HeLa tumors.

Cascade In Situ Self-Assembly and Bioorthogonal Reaction Enable the Enrichment of Photosensitizers and Carbonic Anhydrase Inhibitors for Pretargeted Cancer Theranostics.

We report here a tumor-pretargted theranostic approach for multimodality imaging-guided synergistic cancer PDT by cascade alkaline phosphatase (ALP)-mediated in situ self-assembly and bioorthogonal inverse electron demand Diels-Alder (IEDDA) reaction. Using the enzymatic catalysis of ALP that continuously catalyses the dephosphorylation and self-assembly of trans-cyclooctene (TCO)-bearing P-FFGd-TCO, a high density of fluorescent and magnetic TCO-containing nanoparticles (FMNPs-TCO) can be synthesized and retained on the membrane of tumor cells. They can act as 'artificial antigens' amenable to concurrently capture lately administrated tetrazine (Tz)-decorated PS (775NP-Tz) and carbonic anhydrase (CA) inhibitor (SA-Tz) via the fast IEDDA reaction. This two-step pretargeting process can further induce FMNPs-TCO regrowth into microparticles (FMNPs-775/SA) directly on tumor cell membranes, which is analyzed by bio-SEM and fluorescence imaging. Thus, efficient enrichment of both SA-Tz and 775NP-Tz in tumors can be achieved, allowing to alleviate hypoxia by continuously inhibiting CA activity and improving PDT of tumors. Findings show that subcutaneous HeLa tumors could be completely eradicated and no tumor recurred after irradiation with an 808 nm laser (0.33 W cm-2 , 10 min). This pretargeted approach may be applied to enrich other therapeutic agents in tumors to improve targeted therapy.

Enzyme-Mediated In Situ Self-Assembly Promotes In Vivo Bioorthogonal Reaction for Pretargeted Multimodality Imaging.

Pretargeted imaging has emerged as a promising approach to advance nuclear imaging of malignant tumors. Herein, we combine the enzyme-mediated fluorogenic reaction and in situ self-assembly with the inverse electron demand Diels-Alder (IEDDA) reaction to develop an activatable pretargeted strategy for multimodality imaging. The trans-cyclooctene (TCO) bearing small-molecule probe, P-FFGd-TCO, can be activated by alkaline phosphatase and in situ self-assembles into nanoaggregates (FMNPs-TCO) retained on the membranes, permitting to (1) amplify near-infrared (NIR) fluorescence (FL) and magnetic resonance imaging (MRI) signals, and (2) enrich TCOs to promote IEDDA ligation. The Gallium-68 (68 Ga) labeled tetrazine can readily conjugate the tumor-retained FMNPs-TCO to enhance radioactivity uptake in tumors. Strong NIR FL, MRI, and positron emission tomography (PET) signals are concomitantly achieved, allowing for pretargeted multimodality imaging of ALP activity in HeLa tumor-bearing mice.

Visualizing Endogenous Sulfur Dioxide Derivatives in Febrile-Seizure-Induced Hippocampal Damage by a Two-Photon Energy Transfer Cassette.

Febrile seizure (FS), a frequently encountered seizure disorder in pediatric populations, can cause hippocampus damage. It has been elucidated that sulfur dioxide (SO2) content is overproduced during the development of FS and related brain injury. Thus, monitoring in situ the level of endogenous SO2 in FS-related models is helpful to estimate the pathogenesis of FS-induced brain injury, but the effect detection method remains to be explored. Herein, we developed a two-photon energy transfer cassette based on an acedan-anthocyanidin scaffold, TP-Ratio-SO2, allowing us to achieve this purpose. TP-Ratio-SO2 specifically responds to SO2 derivatives (HSO3-/SO32-) in an ultrafast fashion (less than 3 s), and HSO3-/SO32- can be sensitively determined with a detection limit of 26 nM. Moreover, it exhibits significant changes in two well-resolved fluorescence emissions (Δλ = 140 nm) by reacting with HSO3-/SO32-, behaving as a ratiometric fluorescent sensor. Importantly, ratiometric imaging of endogenous SO2 derivatives generation in hyperpyretic U251 cells and in a rat model of FS-treated hippocampus damage was successfully carried out by TP-Ratio-SO2, demonstrating that it may be a promising tool for studying the role of SO2 in FS-associated neurological diseases.

A carbonic anhydrase-targeted NIR-II fluorescent cisplatin theranostic nanoparticle for combined therapy of pancreatic tumors.

Optically active organic nanoparticles capable of emitting strong near-infrared II (NIR-II) fluorescence and eliciting tumor hyperthermia are promising for tumor imaging and photothermal therapy (PTT). However, their applications for the treatment of pancreatic tumors via mere PTT are challenging as both the nanoparticles and light are hard to enter the deeply located pancreatic tumors. Here, we report a NIR-II light excitable, carbonic anhydrase (CA)-targeting cisplatin prodrug-decorated nanoparticle (IRNPs-SBA/PtIV) for NIR-II fluorescence imaging (FLI)-guided combination PTT and chemotherapy of pancreatic tumors. IRNPs-SBA/PtIV is designed to hold a high photothermal conversion efficiency (PCE ≈ 65.17 %) under 1064 nm laser excitation, a strong affinity toward CA (Kd = 14.40 ± 5.49 nM), and a prominent cisplatin release profile in response to glutathione (GSH) and 1064 nm laser irradiation. We show that IRNPs-SBA/PtIV can be actively delivered into pancreatic tumors where the CA is upregulated, and emits NIR-II fluorescence to visualize tumors with a high sensitivity and penetration depth under 980 nm laser excitation. Moreover, the tumor-resided IRNPs-SBA/PtIV can efficiently inhibit the CA activity and consequently, relieve the acidic and hypoxic tumor microenvironment, benefiting to intensify chemotherapy. Guided by the NIR-II FLI, IRNPs-SBA/PtIV is capable of efficiently inhibiting pancreatic tumor growth via combinational PTT and chemotherapy with 1064 nm laser excitation under a low-power density (0.5 W cm-2, 10 min). This study demonstrates promise to fabricate NIR-II excitable nanoparticles for FLI-guided precise theranostics of pancreatic tumors.

Smart Molecular Imaging and Theranostic Probes by Enzymatic Molecular In Situ Self-Assembly.

Enzymatic molecular in situ self-assembly (E-MISA) that enables the synthesis of high-order nanostructures from synthetic small molecules inside a living subject has emerged as a promising strategy for molecular imaging and theranostics. This strategy leverages the catalytic activity of an enzyme to trigger probe substrate conversion and assembly in situ, permitting prolonging retention and congregating many molecules of probes in the targeted cells or tissues. Enhanced imaging signals or therapeutic functions can be achieved by responding to a specific enzyme. This E-MISA strategy has been successfully applied for the development of enzyme-activated smart molecular imaging or theranostic probes for in vivo applications. In this Perspective, we discuss the general principle of controlling in situ self-assembly of synthetic small molecules by an enzyme and then discuss the applications for the construction of "smart" imaging and theranostic probes against cancers and bacteria. Finally, we discuss the current challenges and perspectives in utilizing the E-MISA strategy for disease diagnoses and therapies, particularly for clinical translation.

A Near-infrared Fluorescence and Positron Emission Tomography Bimodal Probe for In Vivo Imaging of Amyloid-ß Species.

Noninvasive imaging of amyloid-ß (Aß) species in vivo is important for the early diagnosis of Alzheimer's disease (AD). In this paper, we report a near-infrared (NIR) fluorescence (FL) and positron emission tomography (PET) bimodal probe (NIR-[68Ga]) for in vivo imaging of both soluble and insoluble Aß species. NIR-[68Ga] holds a high binding affinity, high selectivity and high sensitivity toward Aß42 monomers, oligomers, and aggregates in vitro. In vivo imaging results show that NIR-[68Ga] can cross the blood-brain-barrier (BBB), and produce significantly higher PET and NIR FL bimodal signals in the brains of APP/PS1 transgenic AD mice relative to that of age-matched wild-type mice, which are also validated by the ex vivo autoradiography and histological staining images. Our results demonstrate that NIR-[68Ga] is an efficient NIR FL and PET bimodal probe for the sensitive imaging of soluble and insoluble Aß species in AD mice.

Controlled sequential in situ self-assembly and disassembly of a fluorogenic cisplatin prodrug for cancer theranostics.

Temporal control of delivery and release of drugs in tumors are important in improving therapeutic outcomes to patients. Here, we report a sequential stimuli-triggered in situ self-assembly and disassembly strategy to direct delivery and release of theranostic drugs in vivo. Using cisplatin as a model anticancer drug, we design a stimuli-responsive small-molecule cisplatin prodrug (P-CyPt), which undergoes extracellular alkaline phosphatase-triggered in situ self-assembly and succeeding intracellular glutathione-triggered disassembly process, allowing to enhance accumulation and elicit burst release of cisplatin in tumor cells. Compared with cisplatin, P-CyPt greatly improves antitumor efficacy while mitigates off-target toxicity in mice with subcutaneous HeLa tumors and orthotopic HepG2 liver tumors after systemic administration. Moreover, P-CyPt also produces activated near-infrared fluorescence (at 710 nm) and dual photoacoustic imaging signals (at 700 and 750 nm), permitting high sensitivity and spatial-resolution delineation of tumor foci and real-time monitoring of drug delivery and release in vivo. This strategy leverages the advantages offered by in situ self-assembly with those of intracellular disassembly, which may act as a general platform for the design of prodrugs capable of improving drug delivery for cancer theranostics.

Engineering of donor-acceptor-donor curcumin analogues as near-infrared fluorescent probes for in vivo imaging of amyloid-ß species.

Near-infrared (NIR) fluorescent imaging of both soluble and insoluble Aß species in the brain of Alzheimer's disease (AD) is crucial for the early diagnosis and intervention of AD. To date, a variety of NIR fluorescent probes have been reported for the detection of Aß species. Among these probes, CRANAD-58 was reported to have the capability to detect both soluble and insoluble Aß species, which is vital to monitor the changes of Aß species during the pathological course of the disease. Though CRANAD-58 has shown promise to noninvasively detect Aß species in transgenic AD mice, the emission wavelength (~670 nm) is still too short for further applications. Therefore, new probes with longer emission wavelength and improved physiological properties are in highly demand. Herein, we report the design and engineering of nine donor-acceptor-donor molecules as "off-on" near-infrared fluorescent probes for in vivo imaging of both soluble and insoluble Aß species in living AD mice owing to its improved in vitro properties and in vivo performance. Methods: We report a two-round strategy to develop nine "off-on" NIR fluorescence probes via structural modification of a curcumin analogue-based donor-acceptor-donor architecture. In round one, probes 1 and 2 were synthesized, and probe 2 was identified to be an optimum probe as it showed distinct "off-on" NIR fluorescence at > 690 nm upon binding to Aß monomers, oligomers and aggregates. To further improve the in vivo performance, further structural modification of probe 2 into probes 3-9 was then conducted. The fluorescence response with Aß species and histological staining in vitro and in vivo imaging of Aß species in APP/PS1 transgenic AD mice and age-matched wild-type mice were performed. Results: We demonstrate that, compared to probe 2, probe 9 with improved physiological properties hold the fastest kinetics (~10 min) to produce not only higher brain fluorescence intensity in 10-month-old APP/PS1 transgenic AD mice, but also afford a higher discrepancy in brain fluorescence to discriminate AD mice from wild-type (WT) mice. Probe 9 also hold the ability to detect soluble Aß species in 6-month-old APP/PS1 transgenic mice. Probe 9 was further applied for dynamic visualization of Aß plaques in a skull-thinning 14-month-old APP/PS1 mouse, which revealed its immediate penetration into brain parenchyma and selective labeling of both parenchymal and angiopathic Aß plaques. In addition, probe 9 possessed significantly high attenuation effect on the aggregation of Aß monomers. Conclusion: Our results demonstrate the good potential of probe 9 for longitudinal NIR fluorescence imaging of soluble and insoluble Aß species in APP/PS1 transgenic AD mice, which may act as a useful tool for early diagnosis and intervention of AD.

Recent advances in stimuli-responsive in situ self-assembly of small molecule probes for in vivo imaging of enzymatic activity.

Stimuli-responsive in situ self-assembly of small molecule probes into nanostructures has been promising for the construction of molecular probes for in vivo imaging. In the past few years, a number of intelligent molecular imaging probes with fluorescence, magnetic resonance imaging (MRI), positron electron tomography (PET) or photoacoustic imaging (PA) modality have been developed based on the in situ self-assembly strategy. In this minireview, we summarize the recent advances in the development of different modality imaging probes through controlling in situ self-assembly for in vivo imaging of enzymatic activity. This review starts from the brief introduction of two different chemical approaches amenable for in situ self-assembly, including (1) stimuli-mediated proteolysis and (2) stimuli-triggered biocompatible reaction. We then discuss their applications in the design of fluorescence, MRI, PET, PA, and bimodality imaging probes for in vivo imaging of different enzymes, such as caspase-3, furin, gelatinase and phosphatase. Finally, we discuss the current and prospective challenges in the stimuli-responsive in situ self-assembly strategy for in vivo imaging.

Responsive Trimodal Probes for In Vivo Imaging of Liver Inflammation by Coassembly and GSH-Driven Disassembly.

Noninvasive in vivo imaging of hepatic glutathione (GSH) levels is essential to early diagnosis and prognosis of acute hepatitis. Although GSH-responsive fluorescence imaging probes have been reported for evaluation of hepatitis conditions, the low penetration depth of light in liver tissue has impeded reliable GSH visualization in the human liver. We present a liver-targeted and GSH-responsive trimodal probe (GdNPs-Gal) for rapid evaluation of lipopolysaccharide- (LPS-) induced acute liver inflammation via noninvasive, real-time in vivo imaging of hepatic GSH depletion. GdNPs-Gal are formed by molecular coassembly of a GSH-responsive Gd(III)-based MRI probe (1-Gd) and a liver-targeted probe (1-Gal) at a mole ratio of 5/1 (1-Gd/1-Gal), which shows high r 1 relaxivity with low fluorescence and fluorine magnetic resonance spectroscopic (19F-MRS) signals. Upon interaction with GSH, 1-Gd and 1-Gal are cleaved and GdNPs-Gal rapidly disassemble into small molecules 2-Gd, 2-Gal, and 3, producing a substantial decline in r 1 relaxivity with compensatory enhancements in fluorescence and 19F-MRS. By combining in vivo magnetic resonance imaging (1H-MRI) with ex vivo fluorescence imaging and 19F-MRS analysis, GdNPs-Gal efficiently detect hepatic GSH using three independent modalities. We noninvasively visualized LPS-induced liver inflammation and longitudinally monitored its remediation in mice after treatment with an anti-inflammatory drug, dexamethasone (DEX). Findings highlight the potential of GdNPs-Gal for in vivo imaging of liver inflammation by integrating molecular coassembly with GSH-driven disassembly, which can be applied to other responsive molecular probes for improved in vivo imaging.