Targeted degradation of membrane and extracellular proteins with LYTACs.

Targeted protein degradation technology has gained substantial momentum over the past two decades as a revolutionary strategy for eliminating pathogenic proteins that are otherwise refractory to treatment. Among the various approaches developed to harness the body's innate protein homeostasis mechanisms for this purpose, lysosome targeting chimeras (LYTACs) that exploit the lysosomal degradation pathway by coupling the target proteins with lysosome-trafficking receptors represent the latest innovation. These chimeras are uniquely tailored to degrade proteins that are membrane-bound and extracellular, encompassing approximately 40% of all proteome. Several novel LYTAC formulas have been developed recently, providing valuable insights for the design and development of therapeutic degraders. This review delineates the recent progresses of LYTAC technology, its practical applications, and the factors that dictate target degradation efficiency. The potential and emerging trends of this technology are discussed as well. LYTAC technology offers a promising avenue for targeted protein degradation, potentially revolutionizing the therapeutic landscape for numerous diseases.

Visualizing lysosomes hypochlorous acid in Parkinson's disease models by a novel fluorescent probe.

Heightened oxidative stress is the principal driver behind the altered metabolism of neurotransmitters within the brains of Parkinson's disease (PD). Hypochlorous acid (HClO), a variant of reactive oxygen species (ROS), plays a crucial role in several lysosomal activities. An irregular concentration of HClO may result in significant molecular damage and contribute to the onset of neurodegenerative disorders. Despite this, the precise role of lysosomal HClO in PD remains unclear, due to its fast reactivity and low levels. This is further complicated by the lack of effective in situ imaging techniques for accurately tracking its dynamics. Therefore, it is of great significance to use effective tools to map the lysosomal HClO during the pathological process of PD. In this study, we propose a fluorogenic probe named Lys-PTZ-HClO for the specific and sensitive detection of HClO. Lys-PTZ-HClO exhibits features like a fast response time (10 s) and a low detection limit (0.72 µM). Benefiting from its superior properties, the probe was used to visualize the basal HClO levels, and the variation of HClO levels in lysosomal of living cells. More importantly, this probe was successfully applied for the first time to reveal increased lysosomal HClO in a cellular model of PD.

NIR-Absorbing Tetraphenylethene-Containing bisBODIPY Nanoplatforms Demonstrate Effective Lysosome-Targeting and Combinational Phototherapy.

Photosensitizer-based phototherapies, including photodynamic therapy (PDT) and photothermal therapy (PTT), offer safe treatment modalities for tumor ablation with spatiotemporal precision. After photons are absorbed, PDT creates localized chemical damage by generating reactive oxygen species (ROS), while PTT induces localized thermal damage. However, PDT still faces hypoxic tumor challenges, while PTT encounters issues related to heat resistance and potential overheating. The combination of PDT and PTT shows great potential as an effective anticancer strategy. By targeting lysosomes with carefully designed phototherapeutic reagents for combined phototherapy, rapid dysfunction and cell death in cancer cells can be induced, showing promise for cancer treatment. Herein, two α-α-linked bisBODIPYs with tetraphenylethene (TPE) moieties are designed and synthesized. These TPE-substituted bisBODIPYs expand the absorption into NIR range (λmaxabs/λmaxem ∼ 740/810 nm) and confer aggregation-induced emission (AIE) activity (λmaxem ∼ 912 nm). Moreover, these bisBODIPYs self-assemble with surfactant F-127 into nanoparticles (NPs), which efficiently generate ROS (1O2 and •OH) in both solution and cellular environments and demonstrate superior photothermal conversion efficiencies (η ∼ 68.3%) along with exceptional photothermal stability. More importantly, these NPs showed lysosomal targeting and remarkable tumor ablation in cellular and murine models, indicating their potential in precision tumor therapy.

A lysosomal-targeted and viscosity-ultrasensitive near-infrared fluorescent probe for sensing viscosity in cells and a diabetic mice model.

Diabetes, as a metabolic disorder, has been implicated in organ dysfunction, often correlated with aberrant changes in viscosity. Lysosomal viscosity serves as an indicator of the lysosome's condition and activity, as it always varies synchronously with the change of lysosome's positioning, structure, and internal constituents. Diabetes, a condition within the metabolic disease category, has the potential to disrupt organ function due to irregular changes in viscosity. Therefore, early and precise diagnosis of diabetes is crucial for the prevention and management of diabetic conditions. Understanding the correlation between viscosity variations and lysosomal changes in vivo is vitally important for researching associated diseases. In this study, we developed Lyso-V, a near-infrared (NIR) fluorescent probe targeting lysosomes, with ultrasensitivity to viscosity changes. This probe, designed with a donor-π-bridge-acceptor (D-π-A) structure, exhibits a significant increase in NIR fluorescence intensity (approximately 690 times) when responding to viscosity, due to a twisted intramolecular charge transfer (TICT) mechanism. Furthermore, the probe designed specifically for lysosomes, enables the detection of changes in lysosomal viscosity as well as autophagy processes. Notably, through the application of this probe, we have detected an increased viscosity within the pathological model of the diabetic mouse. Moreover, Lyso-V was employed to measure the viscosity in diabetic mice. Owing to the multifaceted nature of the Lyso-V probe, it is anticipated to act as a practical and potent resource for deepening our understanding of the pathophysiological aspects of diabetes and aiding in its early detection.

Modular synthesis of pentacyclic-fused pyranoquinoliziniums as organelle-selective fluorescent probes.

On basis of their unique chemical and photophysical properties, and excellent biological activities, quinoliziniums have been widely used in various research fields. Herein, modular synthetic strategies for efficient synthesis of novel fluorescent quinoliziniums by using one-pot and stepwise rhodium(III)-catalyzed C-H annulations were developed. In the one-pot synthesis, the reaction between 2-aryl-4-quinolones (1) and 1,2-diarylalkynes (2) proceeded in a chemo- and regioselective manner to give quinolinone-fused isoquinolines (3) and pentacyclic-fused pyranoquinoliziniums (4). The structural diversity of pentacyclic-fused pyranoquinoliziniums (4) was expanded by the stepwise synthesis from 3 and 2, allowing the strategic incorporation of electron-donating (OMe and OH) and electron-withdrawing (Cl) substituents on the top and bottom parts of the pyranoquinoliziniums (4). These newly synthesized pyranoquinoliziniums (4) exhibited tunable absorptions (455-532 nm), emissions (520-610 nm), fluorescence lifetime (0.3-5.6 ns), large Stokes shifts (up to 120 nm), and excellent fluorescence quantum yields (up to 0.73) upon adjusting the different substituents. The the unique arrangement of N and O atoms and extended π-conjugation of 4 could cause the relocation of HOMO comparing with our previous quinoliziniums. Importantly, pyranoquinoliziniums (4a-4g and 4i) targeted the mitochondria, while 4h was localized in lysosome. Due to the remarkable photophysical properties and the potential for organelle targeting of the novel class of quinoliziniums, they could be further applied for biological, chemical and material applications.

Lysosome targeting fluorescent probe for NAAA imaging and its applications in the drug development for anti-inflammatory.

N-acylethanolamine acid amidase (NAAA) is a nucleophilic lysosomal cysteine hydrolase, which primarily mediates the hydrolytic inactivation of endogenous palmitoylethanolamide (PEA), which further influences the inflammatory process by regulating peroxisome proliferator-activated receptor-α (PPAR-α). Herein, a novel lysosome (Lyso)-targeting fluorescent probe (i.e., PMBD) was designed and synthesized for detecting endogenous NAAA selectively and sensitively, allowing real-time visual monitoring of endogenous NAAA in living cells. Moreover, PMBD can target Lyso with a high colocalization in Lyso Tracker. Finally, a high-throughput assay method for NAAA inhibitor screening was established using PMBD, and the NAAA-inhibitory effects of 42 anti-inflammatory Traditional Chinese medicines were evaluated. A novel potent inhibitor of NAAA, ellagic acid, was isolated from Cornus officinalis, which can suppress LPS-induced iNOS upregulation and NO production in RAW264.7 cells that display anti-inflammatory activities. PMBD, a novel Lyso-targeting fluorescent probe for visually imaging NAAA, could serve as a useful molecular tool for exploring the physiological functions of NAAA and drug development based on NAAA-related diseases.

A novel lysosomal targeted near-infrared probe for ratio detection of carbon monoxide in cells and in vivo.

Carbon monoxide (CO) as an endogenous gas signaling molecule possesses important physiological functions and is of great significance in the treatment of various diseases. Real-time tracking of CO in living organisms has become a research hotspot in recent years. This article presents a lysosomal targeted near-infrared ratio fluorescence probe (TBM-CO) for selective detection of CO based on the dicyanoisophorone skeleton and morpholine fragment. The probe TBM-CO with weak ICT effect can be transformed to precursor TBM-NH2 with strong ICT effect by the traditional Tsuji-Trost reaction procession in the presence of Pd2+ ions. The mechanism was proved by DFT calculation or the MS and HPLC results respectively. In the near-infrared region an obvious ratio fluorescence intensity change (F686 / F616) is observed in vitro spectral experiments. The concentration titration experiments indicate that there is a good liner relationship between the ratio fluorescence intensity and the concentration in the range of 0 to 50 µM (R2 = 0.996) and the detection limit is calculated as 0.38 µM. The cell fluorescence imaging and co-localization experiments further demonstrate that TBM-CO is able to detect the exogenous and endogenous CO in lysosomal subcellular organelle. Finally, it was used to detect the changes of CO concentration in living mice successfully. In short, a probe with three advantages of near-infrared emission, ratiometric fluorescence and organelle targeting was reported and used to detect CO successfully in cells and in living mice.

Revolutionizing viral disease treatment: Phase separation and lysosome/exosome targeting as new areas and new paradigms for antiviral drug research.

With the advancement of globalization, our world is becoming increasingly interconnected. However, this interconnection means that once an infectious disease emerges, it can rapidly spread worldwide. Specifically, viral diseases pose a growing threat to human health. The COVID-19 pandemic has underscored the pressing need for expedited drug development to combat emerging viral diseases. Traditional drug discovery methods primarily rely on random screening and structure-based optimization, and new approaches are required to address more complex scenarios in drug discovery. Emerging antiviral strategies include phase separation and lysosome/exosome targeting. The widespread implementation of these innovative drug design strategies will contribute towards tackling existing viral infections and future outbreaks.

Comparative study of the steady-state subcellular distribution of lysosome-associated membrane glycoprotein-2 (LAMP-2) isoforms with GYXXΦ-type tyrosine-based motifs that interact differently with four adaptor protein (AP) complexes.

Lysosome-associated membrane protein-1 and -2 (LAMP-1 and LAMP-2, respectively) are type I transmembrane proteins. LAMP-2 comprises three splice isoforms (LAMP-2A, -B and-C) with different cytoplasmic tails (CTs). These three CTs possess different tyrosine-based motifs (GYXXΦ, where Φ is a bulky hydrophobic amino acid) at their C-termini. Interactions between tyrosine-based motifs and µ-subunits of four tetrameric adaptor protein (AP) complexes are necessary for their vesicular transport to lysosomes. Little is known about how the interaction strengths of these tyrosine motifs with µ-subunits affect the localization of isoforms to lysosomes. The interactions were first investigated using a yeast two-hybrid system to address this question. LAMP-2A-CT interacted with all four µ-subunits (µ1, µ2, µ3A and µ4 of AP-1, AP-2, AP-3 and AP-4, respectively). The interaction with µ3A was more robust than that with other µ-subunits. LAMP-2B-CT interacted exclusively and moderately with µ3A. LAMP-2C-CT did not detectably interact with any of the four µ-subunits. Immunofluorescence microscopy showed that all isoforms were localized in late endosomes and lysosomes. LAMP-2C was present in the plasma membrane and early endosomes; however, LAMP-2A and -2B were barely detectable in these organelles. In cell fractionation, LAMP-2A was the most abundant in the dense lysosomes, whereas LAMP-2C was significantly present in the low-density fraction containing the plasma membrane and early endosomes, in addition to the dense lysosomes. LAMP-2B considerably existed in the low-density late endosomal fraction. These data strongly suggest that the LAMP-2 isoforms are distributed differently in endocytic organelles depending on their interaction strengths with AP-3.

A near-infrared fluorescent probe with viscosity sensitivity in lysosome for cancer visualization.

A viscosity-sensitive, lysosome-targeted near-infrared fluorescent probe (PYATT) was reported in this paper. The fluorescent spectra of PYATT are strongly dependent on viscosity, resulting in a Stokes shift of about 190 nm. Given its photostability, low cytotoxicity, and high fluorescence quantum yield, PYATT is expected to be used in cell imaging. Due to the higher viscosity of tumor cells than normal cells, the fluorescence intensity of PYATT in tumor cells is higher than normal cells, which can realize the visualization of tumors. The near-infrared probe (PYATT) is viscosity-dependent in lysosomes, which is valuable in early diagnosis and treatment of tumor.

Targeted protein degradation in drug development: Recent advances and future challenges.

Targeted protein degradation (TPD) has emerged as a promising therapeutic approach with potential advantages over traditional occupancy-based inhibitors in terms of dosing, side effects and targeting "undruggable" proteins. Targeted degraders can theoretically bind any nook or cranny of targeted proteins to drive degradation. This offers convenience versus the small-molecule inhibitors that must function in a well-defined pocket. The degradation process depends mainly on two cell self-destruction mechanisms, namely the ubiquitin-proteasome system and the lysosomal degradation pathway. Various TPD strategies (e.g., proteolytic-targeting chimeras, molecular glues, lysosome-targeting chimeras, and autophagy-targeting chimeras) have been developed. These approaches hold great potential for targeting dysregulated proteins, potentially offering therapeutic benefits. In this article, we systematically review the mechanisms of various TPD strategies, potential applications to drug discovery, and recent advances. We also discuss the benefits and challenges associated with these TPD strategies, aiming to provide insight into the targeting of dysregulated proteins and facilitate their clinical applications.

Ratiometric lysosome-targeting luminescent probe based on a coumarin-ruthenium(II) complex for formaldehyde detection and imaging in living cells and mouse brain tissues.

Ratiometric luminescence probes have attracted widespread attention because of their self-calibration capability. However, some defects, such as small emission shift, severe spectral overlap and poor water solubility, limit their application in the field of biological imaging. In this study, a unique luminescence probe, Ru-COU, has been developed by combining tris(bipyridine)ruthenium(II) complex with coumarin derivative through a formaldehyde-responsive linker. The probe exhibited a large emission shift (Δλ > 100 nm) and good water solubility, achieving ratiometric emission responses at 505 nm and 610 nm toward formaldehyde under acidic conditions. Besides, ratiometric luminescence imaging of formaldehyde in living cells and Alzheimer disease mouse's brain slices demonstrates the potential value of Ru-COU for the diagnosis and treatment of formaldehyde related diseases.

Enantioselective Degrader for Elimination of Extracellular Aggregation-Prone Proteins hIAPP Associated with Type 2 Diabetes.

Targeted protein degradation has demonstrated the power to modulate protein homeostasis. For overcoming the limitation to intracellular protein degradation, lysosome targeting chimeras have been recently developed and successfully utilized to degrade a range of disease-relevant extracellular and membrane proteins. Inspired by this strategy, here we describe our proof-of-concept studies using metallohelix-based degraders to deliver the extracellular human islet amyloid polypeptide (hIAPP) into the lysosomes for degradation. Our designed metallohelix can bind and inhibit hIAPP aggregation, and the conjugated tri-GalNAc motif can target macrophage galactose-type lectin 1 (MGL1), yielding chimeric molecules that can both inhibit hIAPP aggregation and direct the bound hIAPP for lysosomal degradation in macrophages. Further studies demonstrate that the enhanced hIAPP clearance has been through the endolysosomal system and depends on MGL1-mediated endocytosis. Intriguingly, Λ enantiomers show even better efficiency in preventing hIAPP aggregation and promoting internalization and degradation of hIAPP than Δ enantiomers. Moreover, metallohelix-based degraders also faciltate the clearance of hIAPP through asialoglycoprotein receptor in liver cells. Overall, our studies demonstrate that chiral metallohelix can be employed for targeted degradation of extracellular misfolded proteins and possess enantioselectivity.

A novel AIE fluorescence probe featuring with high quantum yield for high-fidelity lysosomal targeting and tracking.

High-fidelity imaging and long-term visualization of lysosomes are pivotal factors in the functional assessment of lysosomes, which perform an instrumental role in the physiological activity of cells. However, commercial probes have great limitations in lysosome exploration resulting from the aggregation-caused quenching effect as well as photobleaching instability and small Stokes shift. Therefore, we constructed a novel probe named TTAM with triphenylamine as the matrix and morpholine ring as the targeting group. In contrast with commonly accessible Lyso-tracker Red, TTAM has the merits of aggregation-induced emission effect, extremely high quantum yields (51.57 % solid-state) as well as fluorescence intensity, significant photostability, and high resolution. These properties make it ideal for imaging and activity monitoring lysosomes, which provides a powerful condition for bio-imaging.

Novel approaches to targeted protein degradation technologies in drug discovery.

INTRODUCTION: Target protein degradation (TPD) provides a novel therapeutic modality, other than inhibition, through the direct depletion of target proteins. Two primary human protein homeostasis mechanisms are exploited: the ubiquitin-proteasome system (UPS) and the lysosomal system. TPD technologies based on these two systems are progressing at an impressive pace. AREAS COVERED: This review focuses on the TPD strategies based on UPS and lysosomal system, mainly classified into three types: Molecular Glue (MG), PROteolysis Targeting Chimera (PROTAC), and lysosome-mediated TPD. Starting with a brief background introduction of each strategy, exciting examples and perspectives on these novel approaches are provided. EXPERT OPINION: MGs and PROTACs are two major UPS-based TPD strategies that have been extensively investigated in the past decade. Despite some clinical trials, several critical issues remain, among which is emphasized by the limitation of targets. Recently developed lysosomal system-based approaches provide alternative solutions for TPD beyond UPS' capability. The newly emerging novel approaches may partially address issues that have long plagued researchers, such as low potency, poor cell permeability, on-/off-target toxicity, and delivery efficiency. Comprehensive considerations for the rational design of protein degraders and continuous efforts to seek effective solutions are imperative to advance these strategies into clinical medications.

De novo design of a novel AIE fluorescent probe tailored to autophagy visualization via pH manipulation.

BACKGROUND: Macroautophagy is an essential cellular self-protection mechanism, and defective autophagy has been considered to contribute to a variety of diseases. During the process, cytoplasmic components are transported via autophagosomes to acidic lysosomes for metabolism and recycling, which represents application niches for lysosome-targeted fluorescent probes. Additionally, in view of the complexity of the autophagy pathway, it entails more stringent requirements for probes suitable for monitoring autophagy. Meanwhile, aggregation-induced emission (AIE) fluorescent probes have been impressively demonstrated in the biomedical field, which bring fascinating possibilities to the autophagy visualization. METHODS: We reported a generalizable de novo design of a novel pH-sensitive AIE probe ASMP-AP tailored to lysosome targeting for the interpretation of autophagy. Firstly, the theoretical calculation was carried out followed by the investigation of optical properties. Then, the performance of ASMP-AP in visualizing autophagy was corroborated by starvation or drugs treatments. Furthermore, the capability of ASMP-AP to monitor autophagy was demonstrated in ex vivo liver tissue and zebrafish in vivo. RESULTS: ASMP-AP displays a large stokes shift, great cell permeability and good biocompatibility. More importantly, ASMP-AP enables a good linear response to pH, which derives from the fact that its aggregation state can be manipulated by the acidity. It was successfully applied for imaging autophagy in living cells and was proved capable of monitoring mitophagy. Moreover, this novel molecular tool was validated by ex vivo visualization of activated autophagy in drug-induced liver injury model. Interestingly, it provided a meaningful pharmacological insight that the melanin inhibitor 1-phenyl-2-thiourea (PTU)-induced autophagy was clearly presented in wild-type zebrafish. CONCLUSIONS: ASMP-AP offers a simple yet effective tool for studying lysosome and autophagy. This is the first instance to visualize autophagy in zebrafish using a small-molecule probe with AIE characters, accurate lysosome targeting and simultaneous pH sensitivity. Ultimately, this novel fluorescent system has great potential for in vivo translation to fuel autophagy research.

Clearance of plasma PCSK9 via the asialoglycoprotein receptor mediated by heterobifunctional ligands.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates plasma low-density lipoprotein cholesterol (LDL-C) levels by promoting the degradation of hepatic LDL receptors (LDLRs). Current therapeutic approaches use antibodies that disrupt PCSK9 binding to LDLR to reduce circulating LDL-C concentrations or siRNA that reduces PCSK9 synthesis and thereby levels in circulation. Recent reports describe small molecules that, like therapeutic antibodies, interfere with PCSK9 binding to LDLR. We report an alternative approach to decrease circulating PCSK9 levels by accelerating PCSK9 clearance and degradation using heterobifunctional molecules that simultaneously bind to PCSK9 and the asialoglycoprotein receptor (ASGPR). Various formats, including bispecific antibodies, antibody-small molecule conjugates, and heterobifunctional small molecules, demonstrate binding in vitro and accelerated PCSK9 clearance in vivo. These molecules showcase a new approach to PCSK9 inhibition, targeted plasma protein degradation (TPPD), and demonstrate the feasibility of heterobifunctional small molecule ligands to accelerate the clearance and degradation of pathogenic proteins in circulation.

Aggregation-Induced emission photosensitizer with lysosomal response for photodynamic therapy against cancer.

Photosensitizers play a key role in bioimaging and photodynamic therapy (PDT) of cancer. However, conventional photosensitizers usually do not achieve the desired efficacy in PDT due to their poor photostability, targeting ability, and responsiveness. Herein, we designed a series of photosensitizers with aggregation-induced emission (AIE) effect using benzothiazole- triphenylamine (BZT-triphenylamine) as the parent nucleus. The synthesized compound SIN ((E)-2-(4-(diphenylamino)styryl)-3-(4-iodobutyl)benzo[d]thiazol-3-ium) exhibits good biocompatibility, photostability, and bright emission in the near-infrared range (600-800 nm). The fluorescence emission intensity is responsive to viscosity, with significant fluorescence enhancement (48 times) and high fluorescence quantum yield (4.45 %) at high viscosity. Moreover, SIN has particular lysosome targeting properties with a Pearson correlation coefficient (PCC) of 0.97 and has good 1O2 generation ability under white light irradiation, especially in a weak acidic environment. Thus, SIN can realize good bioimaging ability and photodynamic therapeutic efficacy under the highly viscous and weakly acidic environment of lysosomes in the tumor cells. This study indicates that SIN has potential as a multifunctional organic photosensitizer for bioimaging and PDT of tumor.

Photo-Activable Organosilver Nanosystem Facilitates Synergistic Cancer Theranostics.

Anticancer drug development is important for human health, yet it remains a tremendous challenge. Photodynamic therapy (PDT), which induces cancer cell apoptosis via light-triggered production of reactive oxygen species, is a promising method. However, it has minimal efficacy in subcellular targeting, hypoxic microenvironments, and deep-seated malignancies. Here, we constructed a breast cancer photo-activable theranostic nanosystem through the rational design of a synthetic lysosomal-targeted molecule with multifunctions as aggregation-induced near-infrared (NIR) emission, a photosensitizer (PDT), and organosilver (chemotherapy) for NIR imaging and synergistic cancer therapy. The synthetic molecule could self-assemble into nanoparticles (TPIMBS NPs) and be stabilized with amphiphilic block copolymers for enhanced accumulation in tumor sites through passive targeting while reducing the leakage in normal tissues. Through photochemical internalization, TPIMBS NPs preferentially concentrated in the lysosomes of cancer cells and generated reactive oxygen species (ROS) upon light irradiation, resulting in lysosomal rupture and release of PSs to the cytosol, which led to cell apoptosis. Further, the photoinduced release of Ag+ from TPIMBS NPs could act as chemotherapy, significantly improving the overall therapeutic efficacy by synergistic effects with PDT. This research sheds fresh light on the creation of effective cancer treatments.

Three near-infrared and lysosome-targeting probes for photodynamic therapy (PDT).

Lysosome, an organelle which contains a number of hydrolases and hydrogen ions, plays a crucial role in cellular survival and apoptosis. If selectively destroy lysosomes membrane, inner hydrolases and hydrogen ions will leak and induce cell death. In this work, three lysosome-targeting fluorescent probes (HCL 1-3, heptamethine cyanine lysosomal-targeting probe) were designed, synthesized and developed for photodynamic therapy. Piperazine and N, N-dimethyl structures made HCL 1-3 have good lysosome targeting ability while Pearson's correlation coefficients reached 0.85, 0.87 and 0.78. It can be concluded from MTT test, HCL 1-3 have high photo cytotoxicity and low dark cytotoxicity from MTT test. Calcein/PI staining assays also supported cytotoxicity of HCL 1-3 under light conditions. In vivo experiments, HCL 2 accumulated in tumor and a strong fluorescence signal was observed at 12 h post injection. All results showed that our experiments provide help and new ideas for cyanine dyes in cancer treatment.