Near-Infrared Light-Responsive Size-Selective Lateral Flow Chip for Single-Cell Manipulation of Circulating Tumor Cells.

Accurately obtaining information on the heterogeneity of CTCs at the single-cell level is a very challenging task that may facilitate cancer pathogenesis research and personalized therapy. However, commonly used multicellular population capture and release assays tend to lose effective information on heterogeneity and cannot accurately assess molecular-level studies and drug resistance assessment of CTCs in different stages of tumor metastasis. Herein, we designed a near-infrared (NIR) light-responsive microfluidic chip for biocompatible single-cell manipulation and study the heterogeneity of CTCs by a combination of the lateral flow microarray (LFM) chip and photothermal response system. First, immunomagnetic labeling and a gradient magnetic field were combined to distribute CTCs in different regions of the chip according to the content of surface markers. Subsequently, the LFM chip achieves high single-cell capture efficiency and purity (even as low as 5 CTCs per milliliter of blood) under the influence of lateral fluid and magnetic fields. Due to the rapid dissolution of the gelatin capture structure at 37 °C and the photothermal properties of gold nanorods, the captured single CTC cell can be recovered in large quantities at physiological temperature or released individually at a specific point by NIR. The multifunctional NIR-responsive LFM chip demonstrates excellent performance in capture and site release of CTCs with high viability, which provides a robust and versatile means for CTCs heterogeneity study at the single-cell level.

Surfactant like peptides for targeted gene delivery to cancer cells.

Surfactant like peptides (SLPs) are a class of amphiphilic peptides widely used for drug delivery and tissue engineering. However, there are very few reports on their application for gene delivery. The current study was aimed at development of two new SLPs, named (IA)4K and (IG)4K, for selective delivery of antisense oligodeoxynucleotides (ODNs) and small interfering RNA (siRNA) to cancer cells. The peptides were synthesized by Fmoc solid phase synthesis. Their complexation with nucleic acids was studied by gel electrophoresis and DLS. The transfection efficiency of the peptides was assessed in HCT 116 colorectal cancer cells and human dermal fibroblasts (HDFs) using high content microscopy. The cytotoxicity of the peptides was assessed by standard MTT test. The interaction of the peptides with model membranes was studied using CD spectroscopy. Both SLPs delivered siRNA and ODNs to HCT 116 colorectal cancer cells with high transfection efficiency which was comparable to the commercial lipid-based transfection reagents, but with higher selectivity for HCT 116 compared to HDFs. Moreover, both peptides exhibited very low cytotoxicity even at high concentrations and long exposure time. The current study provides more insights into the structural features of SLPs required for nucleic acid complexation and delivery and can therefore serve as a guide for the rational design of new SLPs for selective gene delivery to cancer cells to minimize the adverse effects in healthy tissues.

Silk Fibroin as a Functional Biomaterial for Tissue Engineering.

Tissue engineering (TE) is the approach to combine cells with scaffold materials and appropriate growth factors to regenerate or replace damaged or degenerated tissue or organs. The scaffold material as a template for tissue formation plays the most important role in TE. Among scaffold materials, silk fibroin (SF), a natural protein with outstanding mechanical properties, biodegradability, biocompatibility, and bioresorbability has attracted significant attention for TE applications. SF is commonly dissolved into an aqueous solution and can be easily reconstructed into different material formats, including films, mats, hydrogels, and sponges via various fabrication techniques. These include spin coating, electrospinning, freeze drying, physical, and chemical crosslinking techniques. Furthermore, to facilitate fabrication of more complex SF-based scaffolds with high precision techniques including micro-patterning and bio-printing have recently been explored. This review introduces the physicochemical and mechanical properties of SF and looks into a range of SF-based scaffolds that have been recently developed. The typical TE applications of SF-based scaffolds including bone, cartilage, ligament, tendon, skin, wound healing, and tympanic membrane, will be highlighted and discussed, followed by future prospects and challenges needing to be addressed.

Recent Advances in Microfluidics for the Preparation of Drug and Gene Delivery Systems.

Drug delivery systems (DDSs) have great potential for improving the treatment of several diseases, especially microbial infections and cancers. However, the formulation procedures of DDSs remain challenging, especially at the nanoscale. Reducing batch-to-batch variation and enhancing production rate are some of the essential requirements for accelerating the translation of DDSs from a small scale to an industrial level. Microfluidic technologies have emerged as an alternative to the conventional bench methods to address these issues. By providing precise control over the fluid flows and rapid mixing, microfluidic systems can be used to fabricate and engineer different types of DDSs with specific properties for efficient delivery of a wide range of drugs and genetic materials. This review discusses the principles of controlled rapid mixing that have been employed in different microfluidic strategies for producing DDSs. Moreover, the impact of the microfluidic device design and parameters on the type and properties of DDS formulations was assessed, and recent applications in drug and gene delivery were also considered.

Interfacial Assembly Inspired by Marine Mussels and Antifouling Effects of Polypeptoids: A Neutron Reflection Study.

Polypeptoid-coated surfaces and many surface-grafted hydrophilic polymer brushes have been proven efficient in antifouling-the prevention of nonspecific biomolecular adsorption and cell attachment. Protein adsorption, in particular, is known to mediate subsequent cell-surface interactions. However, the detailed antifouling mechanism of polypeptoid and other polymer brush coatings at the molecular level is not well understood. Moreover, most adsorption studies focus only on measuring a single adsorbed mass value, and few techniques are capable of characterizing the hydrated in situ layer structure of either the antifouling coating or adsorbed proteins. In this study, interfacial assembly of polypeptoid brushes with different chain lengths has been investigated in situ using neutron reflection (NR). Consistent with past simulation results, NR revealed a common two-step structure for grafted polypeptoids consisting of a dense inner region that included a mussel adhesive-inspired oligopeptide for grafting polypeptoid chains and a highly hydrated upper region with very low polymer density (molecular brush). Protein adsorption was studied with human serum albumin (HSA) and fibrinogen (FIB), two common serum proteins of different sizes but similar isoelectric points (IEPs). In contrast to controls, we observed higher resistance by grafted polypeptoid against adsorption of the larger FIB, especially for longer chain lengths. Changing the pH to close to the IEPs of the proteins, which generally promotes adsorption, also did not significantly affect the antifouling effect against FIB, which was corroborated by atomic force microscopy imaging. Moreover, NR enabled characterization of the in situ hydrated layer structures of the polypeptoids together with proteins adsorbed under selected conditions. While adsorption on bare SiO2 controls resulted in surface-induced protein denaturation, this was not observed on polypeptoids. Our current results therefore highlight the detailed in situ view that NR may provide for characterizing protein adsorption on polymer brushes as well as the excellent antifouling behavior of polypeptoids.

Reactive Inkjet Printing of Functional Silk Stirrers for Enhanced Mixing and Sensing.

Stirring small volumes of solution can reduce immunoassay readout time, homogenize cell cultures, and increase enzyme reactivity in bioreactors. However, at present many small scale stirring methods require external actuation, which can be cumbersome. To address this, here, reactive inkjet printing is shown to be able to produce autonomously rotating biocompatible silk-based microstirrers that can enhance fluid mixing. Rotary motion is generated either by release of a surface active agent (small molecular polyethylene glycol) resulting in Marangoni effect, or by catalytically powered bubble propulsion. The Marangoni driven devices do not require any chemicals to be added to the fluid as the "fuel," while the catalytically powered devices are powered by decomposing substrate molecules in solution. A comparison of Marangoni effect and enzyme powered stirrers is made. Marangoni effect driven stirrers rotate up to 600 rpm, 75-100-fold faster than enzyme driven microstirrers, however enzyme powered stirrers show increased longevity. Further to stirring applications, the sensitivity of the motion generation mechanisms to fluid properties allows the rotating devices to also be exploited for sensing applications, for example, acting as motion sensors for water pollution.

A Review of Curcumin and Its Derivatives as Anticancer Agents.

Cancer is the second leading cause of death in the world and one of the major public health problems. Despite the great advances in cancer therapy, the incidence and mortality rates of cancer remain high. Therefore, the quest for more efficient and less toxic cancer treatment strategies is still at the forefront of current research. Curcumin, the active ingredient of the Curcuma longa plant, has received great attention over the past two decades as an antioxidant, anti-inflammatory, and anticancer agent. In this review, a summary of the medicinal chemistry and pharmacology of curcumin and its derivatives in regard to anticancer activity, their main mechanisms of action, and cellular targets has been provided based on the literature data from the experimental and clinical evaluation of curcumin in cancer cell lines, animal models, and human subjects. In addition, the recent advances in the drug delivery systems for curcumin delivery to cancer cells have been highlighted.

Toxic effect of the novel chiral insecticide IPP and its biodegradation intermediate in nematode Caenorhabditis elegans.

Caenorhabditis elegans, a kind of model organism, was used to investigate biodegradation pathway of IPP and M1 in nematodes, in vivo toxicity from IPP and M1 and the possible underlying molecular mechanism. The results showed that both IPP and M1 could decrease lifespan, locomotion behavior, reproductive ability and AChE activity. During IPP biodegradation process, three intermediates (M1-M3) were monitored and identified. Based on the identified metabolites and their biodegradation courses, a possible biodegradation pathway was proposed. IPP was probably transformed to different three metabolites in nematodes through oxidation and elimination of methyl and propyl etc. Under the same concentration, IPP had more severe toxicity than M1 on nematodes. IPP and M1 might reduce lifespan and decrease reproductive ability through influencing insulin/IGF signaling pathway and TOR signaling pathway. They could decrease expression levels of daf-16, sgk-1, aak-2, daf-15 and rict-1 genes, which involved in IGF and TOR signaling pathway.

Reactive Inkjet Printing of Biocompatible Enzyme Powered Silk Micro-Rockets.

Inkjet-printed enzyme-powered silk-based micro-rockets are able to undergo autonomous motion in a vast variety of fluidic environments including complex media such as human serum. By means of digital inkjet printing it is possible to alter the catalyst distribution simply and generate varying trajectory behavior of these micro-rockets. Made of silk scaffolds containing enzymes these micro-rockets are highly biocompatible and non-biofouling.

Interfacial Adsorption of Silk Fibroin Peptides and Their Interaction with Surfactants at the Solid-Water Interface.

Regenerated silk fibroin (RSF) is a Food and Drug Administration-approved material and has been widely used in many biomedical and cosmetic applications. Because of the amphiphilic nature of the primary repeat amino acid sequence (e.g., AGAGAS), RSF peptides can significantly reduce the water surface tension and therefore have the potential to be used as a surface active component for many applications, particularly in the biomedical, cosmetic, pharmaceutical, and food industries. In this paper, the adsorption of RSF peptides separated into molecular fractions of 5-30, 30-300, and >300 kDa has been studied at the solid-water interface by neutron reflection and spectroscopic ellipsometry to assess its surface active behavior. A stable layer of RSF was found to be irreversibly adsorbed at the hydrophilic SiO2-water interface. Changes in solution concentration, pH, and ionic strength all had an impact on the final adsorbed amount found at the interface. There were no significant differences between the final adsorbed amounts or layer structure among the three RSF molecular fractions studied; however, >300 kDa RSF was more stable to changes in solution ionic strength. Adsorption of conventional anionic and cationic surfactants, sodium dodecyl sulfate (SDS) and dodecyl trimethylammonium bromide (C12TAB), to the preadsorbed 5-30 kDa RSF revealed penetration of the surfactant into the RSF layer, at concentrations below the critical micellar concentration (CMC). SDS was found in the preadsorbed RSF layer and gradually removed RSF from the surface with an increase in SDS concentration. At concentrations above the CMC, there is near complete removal of RSF by SDS at the interface. C12TAB adsorbed into the preadsorbed RSF layer with considerably less removal of RSF from the interface compared to SDS. At concentrations above the CMC, both C12Tab and RSF were found to coexist at the interface, forming a less thick layer but with a considerable amount of RSF still present.

Synergistic effect of bioactive lipid and condition medium on cardiac differentiation of human mesenchymal stem cells from different tissues.

Human umbilical cord mesenchymal stem cells (hUCMSCs) and human adipose tissue mesenchymal stem cells (hATMSCs) have the potential to differentiate into cardiomyocytes, making them promising therapeutic candidates for treating damaged cardiac tissues. Currently, however, the differentiated cells induced from hUCMSCs or hATMSCs can hardly display functional characteristics similar to cardiomyocytes. In this study, we have investigated the effects of bioactive lipid sphingosine-1-phosphate (S1P) on cardiac differentiations of hUCMSCs and hATMSCs in condition medium composed of cardiac myocytes culture medium or 5-azacytidine. Cardiac differentiations were identified through immunofluorescence staining, and the results were observed with fluorescence microscopy and confocal microscopy. Synergistic effects of S1P and condition medium on cell viability were evaluated by MTT assays. Functional characteristics similar to cardiomyocytes were evaluated through detecting calcium transient. The differentiated hUCMSCs or hATMSCs in each group into cardiomyocytes showed positive expressions of cardiac specific proteins, including α-actin, connexin-43 and myosin heavy chain-6 (MYH-6). MTT assays showed that suitable differentiation time was 14 days and that the optimal concentration of S1P was 0.5 µM. Moreover, incorporation of S1P and cardiac myocytes culture medium gave rise to calcium transients, an important marker for displaying in vivo electrophysiological properties. This feature was not observed in the S1P-5-azacytidine group, indicating the possible lack of cellular stimuli such as transforming growth factor-beta, TGF-ß.

Co-adsorption of peptide amphiphile V(6)K and conventional surfactants SDS and C(12)TAB at the solid/water interface.

Recent research has reported many attractive benefits from short peptide amphiphiles. A practical route for them to enter the real world of applications is through formulation with conventional surfactants. This study reports the co-adsorption of the surfactant-like peptide, V6K, with conventional anionic and cationic surfactants at the solid/water interface. The time-dependant adsorption behaviour was examined using spectroscopic ellipsometry whilst adsorbed layer composition and structural distribution of the components were investigated by neutron reflection with the use of hydrogen/deuterium labelling of the surfactant molecules. Both binary (surfactant/peptide mixtures) and sequential (peptide followed by surfactant) adsorption have been studied. It was found that at the hydrophilic SiO2/water interface, the peptide was able to form a stable, flat, defected bilayer structure however both the structure and adsorbed amount were highly dependent on the initial peptide concentration. This consequently affected surfactant adsorption. In the presence of a pre-adsorbed peptide layer anionic sodium dodecyl sulfate (SDS) could readily co-adsorb at the interface; however, cationic dodecyl trimethyl ammonium bromide (C12TAB) could not co-adsorb due to the same charge character. However on a trimethoxy octyl silane (C8) coated hydrophobic surface, V6K formed a monolayer, and subsequent exposure to cationic and anionic surfactants both led to some co-adsorption at the interface. In binary surfactant/peptide mixtures, it was found that adsorption was dependent on the molar ratio of the surfactant and peptide. For SDS mixtures below molar unity and concentrations below CMC for C12TAB, V6K was able to dominate adsorption at the interface. Above molar unity, no adsorption was detected for SDS/V6K mixtures. In contrast, C12TAB gradually replaced the peptide and became dominant at the interface. These results thus elucidate the adsorption behaviour of V6K, which was found to dominate interfacial adsorption but its exact adsorbed amount and distribution were affected by interfacial hydrophobicity and interactions with conventional surfactants.

Interfacial structure of immobilized antibodies and perdeuterated HSA in model pregnancy tests measured with neutron reflectivity.

Experimental studies of antibody adsorption and antigen binding that mimicked pregnancy test immunoassays have been performed using neutron reflectivity studies of a model antibody/antigen system immobilized on the silica/water interface. The study revealed the nature of the antibody/antigen interaction and also the importance of a blocking protein, in this case human serum albumin (HSA), that enhances the immunoassay's specificity and efficiency. Of central importance to this study has been the use of a perdeuterated human serum albumin (d-HSA), providing contrast that highlights the orientation and position of the blocking agent within the adsorbed layer. It was found that the adsorbed HSA filled the gaps between the preadsorbed antibodies on the substrate, with decreased adsorption occurring as a function of increased antibody surface coverage. In addition, the antigen binding capacity of the adsorbed antibodies was investigated as a function of antibody surface coverage. The amount of specifically bound antigen was found to saturate at approximately 0.17 mg/m(2) and became independent of the antibody surface coverage. The ratio of bound antigen to immobilized antibody decreased with increased antibody surface coverage. These results are of importance for a full understanding of immunoassay systems that are widely used in clinical tests and in the detection of environmental contaminants.

Antimicrobial peptide A9K as a gene delivery vector in cancer cells.

Designed peptides are promising biomaterials for biomedical applications. The amphiphilic cationic antimicrobial peptide (AMP), A9K, can self-assemble into nano-rod structures and has shown cancer cell selectivity and could therefore be a promising candidate for therapeutic delivery into cancer cells. In this paper, we investigate the selectivity of A9K for cancer cell models, examining its effect on two human cancer cell lines, A431 and HCT-116. Little or no activity was observed on the control, human dermal fibroblasts (HDFs). In the cancer cell lines the peptide inhibited cellular growth through changes in mitochondrial morphology and membrane potential while remaining harmless towards HDFs. In addition, the peptide can bind to and protect nucleic acids while transporting them into both 2D cultures and 3D spheroids of cancer cells. A9K showed high efficiency in delivering siRNA molecules into the centre of the spheroids. A9K was also explored in vivo, using a zebrafish (Danio rerio) development toxicity assay, showing that the peptide is safe at low doses. Finally, a high-content imaging screen, using RNA interference (RNAi) targeted towards cellular uptake, in HCT-116 cells was carried out. Our findings suggest that active cellular uptake is involved in peptide internalisation, mediated through clathrin-mediated endocytosis. These new discoveries make A9K attractive for future developments in clinical and biotechnological applications.

Sensitive colorimetric detection of Escherichia coli in milk using Au@Ag core-shell nanoparticles.

Escherichia coli (E. coli) is a prevalent pathogen that is frequently associated with the foodborne illness. It causes various infections and poses a significant threat to human health. A rapid and sensitive assay for detecting E. coli is essential for timely diagnosis. Herein, a simple and sensitive colorimetric analysis method for detecting E. coli was developed based on the formation of Au@Ag core-shell nanoparticles facilitated by p-benzoquinone (BQ). E. coli reduced p-benzoquinone to generate hydroquinone (HQ), which could reduce the added Tollens' reagent to silver elemental and grow on the surface of gold nanoparticles (AuNPs). As the E. coli concentration increased, the silver layer thickess on the AuNPs surface growed, resulting in a stronger silver absorption peak observed at 390 nm. The color of the solution changed from red to orange, which could be used to detect E. coli by the naked eye. As a result, E. coli was detected with a linear range from 1.0 × 101 to 1.0 × 107 CFU/mL based on the absorbance intensity. In addition, this method accurately detected E. coli in real milk sample, demonstrating promising applications in foodborne pathogen detection. With satisfactory accuracy, the proposed colorimetric method holds excellent prospects in detecting pathogens in actual food samples.

Synergistic bactericidal effect of antimicrobial peptides and copper sulfide-loaded zeolitic imidazolate framework-8 nanoparticles with photothermal therapy.

Antimicrobial resistance (AMR) has emerged as a significant threat to human health. Antimicrobial peptides (AMPs) have proven to be an effective strategy against antibiotic-resistant bacteria, given their capacity to swiftly disrupt microorganism membranes and alter cell morphology. A common limitation, however, lies in the inherent toxicity of many AMPs and their vulnerability to protease degradation within the body. Photothermal therapy (PTT) stands out as a widely utilized approach in combating antibiotic-resistant bacterial infections, boasting high efficiency and non-invasive benefits. To enhance the stability and antibacterial efficacy of AMPs, a novel approach involving the combination of AMPs and PTT has been proposed. This study focuses on the encapsulation of At10 (an AMP designed by our group), and copper sulfide nanoparticles (CuS NPs) within zeolitic imidazolate framework-8 (ZIF-8) to form nanocomposites (At10/CuS@ZIF-8). The encapsulated CuS NPs exhibit notable photothermal properties upon exposure to near-infrared radiation. This induces the cleavage of ZIF-8, facilitating the release of At10, which effectively targets bacterial membranes to exert its antibacterial effects. Bacteria treated with At10/CuS@ZIF-8 under light radiation exhibited not only membrane folding and intracellular matrix outflow but also bacterial fracture. This synergistic antibacterial strategy, integrating the unique properties of AMPs, CuS NPs, and pH responsiveness of ZIF-8, holds promising potential for widespread application in the treatment of bacterial infections.

Curcumin-shellac nanoparticle-loaded GelMA/SilMA hydrogel for colorectal cancer therapy.

In this study, a novel approach was employed to develop a therapeutic system for colorectal cancer treatment. Specifically, a GelMA/SilMA hydrogel loaded with curcumin-shellac nanoparticles (Cur@Lac NPs) was created. A microfluidic swirl mixer was utilized to formulate stable Cur@Lac NPs, ensuring their consistent and effective encapsulation. The pH-specific release of curcumin from the NPs demonstrated their potential for colon cancer treatment. By carefully regulating the ratio of GelMA (gelatin methacrylate) and SilMA (silk fibroin methacrylate), a GelMA/SilMA dual network hydrogel was generated, offering controlled release and degradation capabilities. The incorporation of SilMA notably enhanced the mechanical properties of the dual network matrix, improving compression resistance and mitigating deformation. This mechanical improvement is crucial for maintaining the structural integrity of the hydrogel during in vivo applications. In comparison to the direct incubation of curcumin, the strategy of encapsulating curcumin into NPs and embedding them within the GelMA/SilMA hydrogel resulted in more controlled release mechanisms. This controlled release was achieved through the disintegration of the NPs and the swelling and degradation of the hydrogel matrix. The encapsulating strategy also demonstrated enhanced cellular uptake of curcumin, leveraging the advantages of both NPs and in-situ hydrogel injection. This combination ensures a more efficient and sustained delivery of the therapeutic agent directly to the tumor site. Overall, this approach holds significant promise as a smart drug delivery system, potentially improving the efficacy of colorectal cancer treatments by providing targeted, controlled, and sustained drug release with enhanced mechanical stability and biocompatibility.

Co-assembling bioactive short peptide nanofibers coated silk scaffolds induce neurite outgrowth of PC12 cells.

Controlling biomolecular-cell interactions is crucial for the design of scaffolds for tissue engineering (TE). Regenerated silk fibroin (RSF) has been extensively used as TE scaffolds, however, RSF showed poor attachment of neuronal cells, such as rat pheochromocytoma (PC12) cells. In this work, amphiphilic peptides containing a hydrophobic isoleucine tail (I3) and laminin or fibronectin derived peptides (IKVAV, PDSGR, YIGSR, RGDS and PHSRN) were designed for promoting scaffold-cell interaction. Three of them (I3KVAV, I3RGDS and I3YIGSR) can self-assemble into nanofibers, therefore, were used to enhance the application of RSF in neuron TE. Live / dead assays revealed that the peptides exhibited negligible cytotoxicity against PC12 cells. The specific interaction between PC12 cells and the peptides were investigate using atomic force microscopy (AFM). The results indicated a synergistic effect in the designed peptides, promoting cellular attachment, proliferation and morphology changes. In addition, AFM results showed that co-assembling peptides I3KVAV and I3YIGSR possesses the best regulation of proliferation and attachment of PC12 cells, consistent with immunofluorescence staining results. Moreover, cell culture with hydrogels revealed that a mixture of peptides I3KVAV and I3YIGSR can also promote 3D neurites outgrowth. The approach of combining two different self-assembling peptides shows great potential for nerve regeneration applications.

Microfluidic formulation of food additives-loaded nanoparticles for antioxidation.

Browning has many important implications with nutrition and the shelf life of foods. Mitigating browning is of particular interest in food chemistry. The addition of antioxidants has been a common strategy to extend shelf life of drug and food products. In this work, we report a microfluidic technology for encapsulation of three common food additives (potassium metathionite (PMS), curcumin (CCM), and ß-carotene (ß-Car)) into nano-formulations using low-cost and readily available materials such as shellac. The food additives encapsulated nanoparticles provide a microenvironment that can prevent oxidation during daily storage. The results showed that the produced nanoparticles had a narrow size distribution with an average size of around 100 nm, were stable at conventional storage conditions (4 ºC) for 18 weeks, and had sustained release ability at 37 ºC, pH= 7.8, 160 rpm. In addition, further experiments showed that the formulation of hydrophobic additives, such as CCM and ß-Car did not only improve their bioavailability but also allowed for the encapsulation of a combination of ingredients. In addition, the antioxidants loaded nanoparticles demonstrated good biocompatibility, low toxicity to human cells. The longer release time of encapsulated food additives increases shelf life of foods and enhances consumer purchase preferences, which not only saves costs but also reduces waste. In summary, this study shows that such antioxidant-loaded nanoparticles provide a promising strategy in extending the shelf life of food products.

An enzyme-responsive double-locked amonafide prodrug for the treatment of glioblastoma with minimal side effects.

Amonafide (ANF), a topoisomerase II inhibitor and DNA intercalator, has exhibited promise in phase II trials but faces significant limitations due to adverse side effects. Here, we have developed a novel enzyme-triggered fluorogenic prodrug, AcKLP, that incorporates dual-locked enzyme activation, ensuring that the prodrug remains inactive until it confronts the unique enzymatic environment of glioblastoma cells. This approach minimizes premature activation and reduces toxicity to normal cells, with an IC50 > 100 µM for human umbilical vein endothelial cells (HUVEC) and ∼2.3 µM for human glioblastoma cells (U87). Upon activation of AcKLP by two distinct enzymes prevalent in glioblastoma cells, amonafide is released and emits a fluorescence signal response, facilitating treatment and the monitoring of real-time drug distribution. Mechanistic studies indicate that AcKLP mainly induces autophagic cell death in U87 cells. Moreover, three-dimensional multicellular U87 tumor spheroid assays and in vivo experiments confirm the potent antiproliferative activity of AcKLP against glioblastoma cells. This work demonstrates a novel de-caging strategy to improve the selectivity and efficacy of amonafide for cancer therapy.