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.

Bioorthogonal Cu Single-Atom Nanozyme for Synergistic Nanocatalytic Therapy, Photothermal Therapy, Cuproptosis and Immunotherapy.

Single-atom nanozymes (SAzymes) with atomically dispersed active sites are potential substitutes for natural enzymes. A systematic study of its multiple functions can in-depth understand SAzymes's nature, which remains elusive. Here, we develop a novel ultrafast synthesis of sputtered SAzymes by in situ bombarding-embedding technique. Using this method, sputtered copper (Cu) SAzymes (CuSA) is developed with unreported unique planar Cu-C3 coordinated configuration. To enhance the tumor-specific targeting, we employ a bioorthogonal approach to engineer CuSA, denoted as CuSACO. CuSACO not only exhibits minimal off-target toxicity but also possesses exceptional ultrahigh catalase-, oxidase-, peroxidase-like multienzyme activities, resulting in reactive oxygen species (ROS) storm generation for effective tumor destruction. Surprisingly, CuSACO can release Cu ions in the presence of glutathione (GSH) to induce cuproptosis, enhancing the tumor treatment efficacy. Notably, CuSACO's remarkable photothermal properties enables precise photothermal therapy (PTT) on tumors. This, combined with nanozyme catalytic activities, cuproptosis and immunotherapy, efficiently inhibiting the growth of orthotopic breast tumors and gliomas, and lung metastasis. Our research highlights the potential of CuSACO as an innovative strategy to utilize multiple mechanism to enhance tumor therapeutic efficacy, broadening the exploration and development of enzyme-like behavior and physiological mechanism of action of SAzymes.

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.

A Fast and Reversible Responsive Bionic Transmembrane Nanochannel for Dynamic Single-Cell Quantification of Glutathione.

Biological channels can rapidly and continuously modulate ion transport behaviors in response to external stimuli, which play essential roles in manipulating physiological and pathological processes in cells. Here, to mimic the biological channels, a bionic nanochannel is developed by synergizing a cationic silicon-substituted rhodamine (SiRh) with a glass nanopipette for transmembrane single-cell quantification. Taking the fast and reversible nucleophilic addition reaction between glutathione (GSH) and SiRh, the bionic nanochannel shows a fast and reversible response to GSH, with its inner-surface charges changing between positive and negative charges, leading to a distinct and reversible switch in ionic current rectification (ICR). With the bionic nanochannel, spatiotemporal-resolved operation is performed to quantify endogenous GSH in a single cell, allowing for monitoring of intracellular GSH fluctuation in tumor cells upon photodynamic therapy and ferroptosis. Our results demonstrate that it is a feasible tool for in situ quantification of the endogenous GSH in single cells, which may be adapted to addressing other endogenous biomolecules in single cells by usage of other stimuli-responsive probes.

Ratiometric Near-Infrared Fluorescence Liposome Nanoprobe for H2S Detection In Vivo.

Accurate detection of H2S is crucial to understanding the occurrence and development of H2S-related diseases. However, the accurate and sensitive detection of H2S in vivo still faces great challenges due to the characteristics of H2S diffusion and short half-life. Herein, we report a H2S-activatable ratiometric near-infrared (NIR) fluorescence liposome nanoprobe HS-CG by the thin-film hydration method. HS-CG shows "always on" fluorescence signal at 816 nm and low fluorescence signal at 728 nm; the NIR fluorescence ratio between 728 and 816 nm (F728/F816) is low. Upon reaction with H2S, the fluorescence at 728 nm could be more rapidly turned on due to strong electrostatic interaction between enriched HS- and positively charged 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC) doped in the liposome nanoprobe HS-CG, resulting in a large enhancement of F728/F816, which allows for sensitive visualization of the tumor H2S levels in vivo. This study demonstrates that this strategy of electrostatic adsorption between HS- and positively charged molecules provides a new way to enhance the reaction rate of the probe and H2S, thus serving as an effective platform for improving the sensitivity of imaging.

Smart Nanosensitizers for Activatable Sono-Photodynamic Immunotherapy of Tumors by Redox-Controlled Disassembly.

Tumor-targeted and stimuli-activatable nanosensitizers are highly desirable for cancer theranostics. However, designing smart nanosensitizers with multiple imaging signals and synergistic therapeutic activities switched on is challenging. Herein, we report tumor-targeted and redox-activatable nanosensitizers (1-NPs) for sono-photodynamic immunotherapy of tumors by molecular co-assembly and redox-controlled disassembly. 1-NPs show a high longitudinal relaxivity (r1 =18.7±0.3 mM-1 s-1 ), but "off" dual fluorescence (FL) emission (at 547 and 672 nm), "off" sono-photodynamic therapy and indoleamine 2,3-dioxygenase 1 (IDO1) inhibition activities. Upon reduction by glutathione (GSH), 1-NPs rapidly disassemble and remotely release small molecules 2-Gd, Zn-PPA-SH and NLG919, concurrently switching on (1) dual FL emission, (2) sono-photodynamic therapy and (3) IDO1 inhibition activities. After systemic injection, 1-NPs are effective for bimodal FL and magnetic resonance (MR) imaging-guided sono-photodynamic immunotherapy of orthotropic breast and brain tumors in mice under combined ultrasound (US) and 671-nm laser irradiation.

Tailoring a Near-Infrared Macrocyclization Scaffold Allows the Control of In Situ Self-Assembly for Photoacoustic/PET Bimodal Imaging.

Enzyme-triggered macrocyclization and in situ self-assembly of small molecules into nanoparticles has shown promise to design activatable probes for molecular imaging. However, controlling macrocyclization and self-assembly to concurrently augment positron emission tomography (PET) and photoacoustic (PA) signals for bimodality imaging is challenging. Herein, we report the engineering of a triazole-IR780 fluorophore as a versatile macrocyclization scaffold for controlling in situ self-assembly and design a caspase-3-activatable PA/PET bimodal probe ([18 F]-IR780-1) for in vivo imaging of tumor apoptosis. By leveraging the high-sensitivity whole-body imaging signals offered by PET with the high-resolution imaging signals offered by PA, [18 F]-IR780-1 can provide a promising tool for the early evaluation of antitumor efficacy, helpful for optimizing the therapeutic protocol for patients. This scaffold may be adopted to design other activatable bimodal probes for in vivo imaging.

Alkaline Phosphatase Enabled Fluorogenic Reaction and in situ Coassembly of Near-Infrared and Radioactive Nanoparticles for in vivo Imaging.

Smart near-infrared (NIR) fluorescence (FL) and positron emission tomography (PET) bimodal probes have shown promise for preoperative and intraoperative imaging of tumors. In this paper, we report an enzyme-activatable probe (P-CyFF-68Ga) and its cold probe (P-CyFF-Ga) using an enzyme-induced fluorogenic reaction and in situ coassembly strategy and demonstrate the utility for NIR FL/PET bimodality imaging of enzymatic activity. P-CyFF-68Ga and P-CyFF-Ga can be converted into dephosphorylated CyFF-68Ga and CyFF-Ga in response to alkaline phosphatase (ALP) and subsequently coassemble into fluorescent and radioactive nanoparticles (NP-68Ga). The ALP-triggered in situ formed NP-68Ga is prone to anchoring on the ALP-positive HeLa cell membrane, permitting the concurrent enrichment of NIR FL and radioactivity. The enhancements in NIR FL and radioactivity enables high sensitivity and deep-tissue imaging of ALP activity, consequently facilitating the delineation of HeLa tumor foci from the normal tissues in vivo.

Promising potential of a 18F-labelled small-molecular radiotracer to evaluate PD-L1 expression in tumors by PET imaging.

Programmed death ligand 1 (PD-L1) expression level is a reproducible biomarker for guiding stratification of patients to immunotherapy. However, the most widely used immunohistochemistry method is incompetent to fully understand the PD-L1 expression level in the whole body because of the highly complex PD-L1 expression in the tumor microenvironment. In this work, a novel small-molecular radiotracer [18F]LG-1 based on the biphenyl active structure was developed to evaluate PD-L1 expression in tumors. [18F]LG-1 was obtained by conjugating and radiolabeling with [18F]FDG with high radiochemical purity (>98.0%) and high molar activity (37.2 ± 2.9 MBq/nmol). In vitro experimental results showed that [18F]LG-1 could target PD-L1 in tumor cells and the cellular uptake in A375-hPD-L1 cells (PD-L1 + ) was clearly higher than that in A375 cells (PD-L1-). In vivo dynamic PET images of [18F]LG-1 provided clear visualization of A375-hPD-L1 tumor with high tumor-to-background contrast, and the tumor uptake was determined to be 3.98 ± 0.21 %ID/g at 60 min, which was 2.6-fold higher than that of A375 tumor. These results suggested that [18F]LG-1 had great potential as a promising PD-L1 radiotracer.

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.

Development of biotin-tagged near-infrared fluorescence probes for tumor-specific imaging.

Near-infrared (NIR) probes are applicable for tumor imaging due to deep tissue penetration and low background signal. And cyanine dyes with long emission wavelength are excellent fluorophores to develop NIR probes. However, the aggregation of cyanine dyes in aqueous solution reduces the utilization of light. To solve this problem, polyethylene glycol (PEG) was introduced into the probes to reduce their aggregation. In our work, two new NIR probes G1 and G2 were designed and prepared by conjugating the cyanine dye G0 with Biotin-PEG5-Azide. The conjugated biotin could enhance the target specificity of probes. And the photophysical and photochemical parameters demonstrated that G1 and G2 had a reduced aggregation tendency. In vitro fluorescence imaging proved that these two probes could be specifically taken up by HeLa cells, and in vivo imaging demonstrated that these two probes could specifically target tumors with large tumor-to-muscle (T/M) ratios. All these results indicated that G1 and G2 are promising NIR fluorescent contrast agents for tumor-specific imaging.

Dual-targeted 5-aminolevulinic acid derivatives with glutathione depletion function for enhanced photodynamic therapy.

Photodynamic therapy (PDT) is a promising tumor therapy which utilizes reactive oxygen species (ROSs) to cause tumor cells death. 5-aminolevulinic acid (ALA) and two of its esters are FDA-approved photosensitizers. However, their clinical application suffers from their instability and lack of tumor selectivity. In addition, the overexpression of glutathione (GSH) in some tumor cells reduces the PDT efficiency due to the ROS-scavenging ability of GSH. In this work, we present three multifunctional ALA derivates with the characteristics of dual-targeting and GSH depletion to improve the therapeutic effect of ALA-based PDT. The general structure of these compounds consists of an ALA methyl ester (ALA-OMe) moiety that can metabolize to photosensitive protoporphyin IX (PpIX) inside the cells, a biotin group for targeting biotin receptor-positive tumor cells and a disulfide bond-based self-immolative linker which can be activated by GSH to liberate ALA-OMe. Simultaneously, the reaction between the disulfide bond and GSH also depletes intracellular GSH, causing tumor cells more vulnerable to ROSs. All three compounds exhibited high stability under physiological conditions. In vitro experiments demonstrated that the more lipophilic compounds 1 and 2 were much more efficient in inducing PpIX production in biotin receptor-overexpressed HeLa cells as compared with their parent compound (ALA-OMe). And the PpIX generation induced by compounds 1 and 2 was positively correlated with the overexpression of biotin receptor and GSH level in tumor cells. More importantly, the GSH depletion ability of them significantly increased their phototoxicity. Furthermore, in comparison with ALA-OMe, compound 2 showed much higher in vivo efficiency in PpIX production. All the results demonstrate that the combination strategy of dual-targeting and GSH depletion can be used to concurrently enhance the tumor-specificity and anti-tumor efficiency of ALA-based PDT. And this strategy may be used for designing other ALA-based photosensitizers with higher tumor-specificity and better therapeutic effects.

A fluorine-18 labeled radiotracer for PET imaging of γ-glutamyltranspeptidase in living subjects.

The expression level of γ-glutamyltranspeptidase (GGT) in some malignant tumors is often abnormally high, while its expression is low in normal tissues. Therefore, GGT is considered as a key biomarker for cancer diagnosis. Several GGT-targeting fluorescence probes have been designed and prepared, but their clinical applications are limited due to their shallow tissue penetration. Considering the advantages of positron emission tomography (PET) such as high sensitivity and deep tissue penetration, we designed a novel PET imaging probe for targeted monitoring of the expression of GGT in living subjects, ([18F]γ-Glu-Cys-PPG(CBT)-AmBF3)2, hereinafter referred to as ([18F]GCPA)2. The non-radioactive probe (GCPA)2 was synthesized successfully and [18F]fluorinated rapidly via the isotope exchange method. The radiotracer ([18F]GCPA)2 could be obtained within 0.5 h with the radiochemical purity over 98% and the molar activity of 10.64 ± 0.89 GBq µmol-1. It showed significant difference in cellular uptake between GGT-positive HCT116 cells and GGT-negative L929 cells (2.90 ± 0.12% vs. 1.44 ± 0.15% at 4 h, respectively). In vivo PET imaging showed that ([18F]GCPA)2 could quickly reach the maximum uptake in tumor (4.66 ± 0.79% ID g-1) within 5 min and the tumor-to-muscle uptake ratio was higher than 2.25 ± 0.08 within 30 min. Moreover, the maximum tumor uptake of the control group co-injected with the non-radioactive probe (GCPA)2 or pre-treated with the inhibitor GGsTop decreased to 3.29 ± 0.24% ID g-1 and 2.78 ± 0.32% ID g-1 at 10 min, respectively. In vitro and in vivo results demonstrate that ([18F]GCPA)2 is a potential PET probe for sensitively and specifically detecting the expression level of GGT.

One-step radiosynthesis and initial evaluation of a small molecule PET tracer for PD-L1 imaging.

Programmed cell death protein-ligand 1 (PD-L1) is a crucial biomarker in immunotherapy and its expression level plays a key role in the guidance of anti-PD-L1 therapy. It had been reported that PD-L1 was quantified by noninvasive imaging with more developed radiotracers. In our study, a novel [18F]fluoride labeled small molecule inhibitor, [18F]LN was designed for positron emission tomography (PET) imaging in both PD-L1 transfected (A375-hPD-L1) and non-transfected (A375) melanoma-bearing mice. LN showed the specificity (IC50 = 50.39 ± 2.65 nM) to PD-L1 confirmed by competitive combination and cell flow cytometry (FACS) analysis. The radiotracer [18F]LN was obtained via 18F-19F isotope exchange from precursor LN. After radiosynthesis, [18F]LN was achieved with a high radiochemical purity (RCP) above 95% and got a favorable molar activity of 36.34 ± 5.73 GBq/µmol. [18F]LN displayed the moderate affinity (Kd = 65.27 ± 3.47 nM) to PD-L1 by specific binding assay. And it showed 1.3-fold higher uptake in A375-hPD-L1 cells than that in A375 cells. PET imaging revealed that [18F]LN could enter into PD-L1 expressing tumor site and visualize the outline of tumor. And tumor uptake (1.96 ± 0.27 %ID/g) reached the maximum at 15 min in the positive group, showed 2.2-fold higher than the negative (0.89 ± 0.31 %ID/g) or the blocked (1.07 ± 0.26 %ID/g) groups. Meanwhile, biodistribution could slightly distinguish the positive from the negative. The results indicated [18F]LN would become an efficient tool for evaluating PD-L1 expression with further optimization.

Structure-based virtual screening and biological evaluation of novel non-bisphosphonate farnesyl pyrophosphate synthase inhibitors.

Farnesyl pyrophosphate synthase (FPPS) is known to participate in a variety of disease-related cell signaling pathway and bisphosphonates (BPs) are served as FPPS inhibitors. However, the high polarity of BPs often induces a series of side effects, limiting their applications. In the present study, novel non-BP FPPS inhibitors were discovered by in silico screening and experimental validation. From the structure-based virtual screening (SBVS) strategy combining molecular docking, pharmacophore and binding affinity prediction, 10 hits with novel scaffolds were filtered. The inhibition activity of hits against FPPS was identified and 7 hits showed comparable or higher inhibition activity than Zoledronate. The hit VS-4 with higher lipophilicity (XlogP = 1.81) and binding affinity (KD = 14.3 ± 2.63 µM) to FPPS was selected for further study on cancer cells with different FPPS expression level. Experimental results revealed that VS-4 could better target the FPPS high-expressing colon LoVo and HCT116 cancer cell lines with IC50 of 51.772 ± 0.473 and 43.553 ± 1.027 µM, respectively, whereas the IC50 value against FPPS low expressing MDA-MB-231 cells was >100 µM. The mechanism of VS-4 against colon cancer cells was investigated by flow cytometry and the results indicated that VS-4 induced cell apoptosis by increasing the intracellular reactive oxygen species (ROS) level. Taken together, the SBVS strategy could be used to discover promising non-BP FPPS inhibitors and the lead compound VS-4 might shed a light on designing more potent inhibitors as novel anticancer drugs.