In Situ Nanofiber Formation Blocks AXL and GAS6 Binding to Suppress Ovarian Cancer Development.

Anexelekto (AXL) is an attractive molecular target for ovarian cancer therapy because of its important role in ovarian cancer initiation and progression. To date, several AXL inhibitors have entered clinical trials for the treatment of ovarian cancer. However, the disadvantages of low AXL affinity and severe off-target toxicity of these inhibitors limit their further clinical applications. Herein, by rational design of a nonapeptide derivative Nap-Phe-Phe-Glu-Ile-Arg-Leu-Arg-Phe-Lys (Nap-IR), a strategy of in situ nanofiber formation is proposed to suppress ovarian cancer growth. After administration, Nap-IR specifically targets overexpressed AXL on ovarian cancer cell membranes and undergoes a receptor-instructed nanoparticle-to-nanofiber transition. In vivo and in vitro experiments demonstrate that in situ formed Nap-IR nanofibers efficiently induce apoptosis of ovarian cancer cells by blocking AXL activation and disrupting subsequent downstream signaling events. Remarkably, Nap-IR can synergistically enhance the anticancer effect of cisplatin against HO8910 ovarian tumors. It is anticipated that the Nap-IR can be applied in clinical ovarian cancer therapy in the near future.

ß-Galactosidase-activated near-infrared AIEgen for ovarian cancer imaging in vivo.

Near-infrared (NIR) aggregation induced-emission luminogens (AIEgens) circumvent the noisome aggregation-caused quenching (ACQ) effect in physiological milieu, thus holding high promise for real-time and sensitive imaging of biomarkers in vivo. ß-Galactosidase (ß-Gal) is a biomarker for primary ovarian carcinoma, but current AIEgens for ß-Gal sensing display emissions in the visible region and have not been applied in vivo. We herein propose an NIR AIEgen QM-TPA-Gal and applied it for imaging ß-Gal activity in vitro and in ovarian tumor model. After being internalized by ovarian cancer cells (e.g., SKOV3), the hydrophilic nonfluorescent QM-TPA-Gal undergoes hydrolyzation by ß-Gal to yield hydrophobic QM-TPA-OH, which subsequently aggregates into nanoparticles to turn NIR fluorescence "on" through the AIE mechanism. In vitro experimental results indicate that QM-TPA-Gal has a sensitive and selective response to ß-Gal with a limit of detection (LOD) of 0.21 U/mL. Molecular docking simulation confirms that QM-TPA-Gal has a good binding ability with ß-Gal to allow efficient hydrolysis. Furthermore, QM-TPA-Gal is successfully applied for ß-Gal imaging in SKOV3 cell and SKOV3-bearing living mouse models. It is anticipated that QM-TPA-Gal could be applied for early diagnosis of ovarian cancers or other ß-Gal-associated diseases in near future.

Three-in-One Peptide Prodrug with Targeting, Assembly and Release Properties for Overcoming Bacterium-Induced Drug Resistance and Potentiating Anti-Cancer Immune Response.

The presence of bacteria in tumor results in chemotherapeutic drug resistance and weakens the immune response in colorectal cancer. To overcome bacterium-induced chemotherapeutic drug resistance and potentiate antitumor immunity, herein a novel molecule Biotin-Lys(SA-Cip-OH)-Lys(SA-CPT)-Phe-Phe-Nap (Biotin-Cip-CPT-Nap) is rationally designed containing four functional motifs (i.e., a biotin motif for targeting, Phe-Phe(-Nap) motif for self-assembly, ciprofloxacin derivative (Cip-OH) motif for antibacterial effect, and camptothecin (CPT) motif for chemotherapy). Using the designed molecule, a novel strategy of intracellular enzymatic nanofiber formation and synergistic antibacterium-enhanced chemotherapy and immunotherapy is achieved. Under endocytosis mediated by highly expressed biotin receptor in colorectal cancer cell membrane and the catalysis of highly expressed carboxylesterase in the cytoplasm, this novel molecule can be transformed into Biotin-Nap, which self-assembled into nanofibers. Meanwhile, antibiotic Cip-OH and chemotherapeutic drug CPT are released, overcoming bacterium-induced drug resistance and enhancing the therapeutic efficacy of immunotherapy towards colorectal cancer. This work offers a feasible strategy for the design of novel multifunctional prodrugs to improve the efficiency of colorectal cancer treatment.

In situ peptide assemblies for bacterial infection imaging and treatment.

Bacterial infections, especially antibiotic-resistant ones, remain a major threat to human health. Advances in nanotechnology have led to the development of numerous antimicrobial nanomaterials. Among them, in situ peptide assemblies, formed by biomarker-triggered self-assembly of peptide-based building blocks, have received increasing attention due to their unique merits of good spatiotemporal controllability and excellent disease accumulation and retention. In recent years, a variety of "turn on" imaging probes and activatable antibacterial agents based on in situ peptide assemblies have been developed, providing promising alternatives for the treatment and diagnosis of bacterial infections. In this review, we introduce representative design strategies for in situ peptide assemblies and highlight the bacterial infection imaging and treatment applications of these supramolecular materials. Besides, current challenges in this field are proposed.

Intracellular Nitroreductase-Triggered "On" and "Enhanced" Photoacoustic Signals for Sensitive Imaging of Tumor Hypoxia.

Current molecular photoacoustic (PA) probes are designed with either stimulus-turned "on" or assembly-enhanced signals to trace biological analytes/events. PA probes based on the nature-derived click reaction between 2-cyano-6-aminobenzothiazole (CBT) and cysteine (Cys) (i.e., CBT-Cys click reaction) possess both "turn-on" and "enhanced" PA signals; and thus, should have higher sensitivity. Nevertheless, such PA probes, particularly those for sensitive imaging of tumor hypoxia, remain scarce. Herein, a PA probe NI-Cys(StBu)-Dap(IR780)-CBT (NI-C-CBT) is rationally designed, which after being internalized by hypoxic tumor cells, is cleaved by nitroreductase under the reduction condition to yield cyclic dimer C-CBT-Dimer to turn the PA signal "ON" and subsequently assembled into nanoparticles C-CBT-NPs with additionally enhanced PA signal ("Enhanced"). NI-C-CBT exhibits 1.7-fold "ON" and 3.2-fold overall "Enhanced" PA signals in vitro. Moreover, it provides 1.9-fold and 2.8-fold overall enhanced PA signals for tumor hypoxia imaging in HeLa cells and HeLa tumor-bearing mice, respectively. This strategy is expected to be widely applied to design more "smart" PA probes for sensitive imaging of important biological events in vivo in near future.

Enhanced Photoacoustic Imaging of Urokinase-Type Plasminogen Activator Activity in Tumors.

Photoacoustic (PA) imaging of urokinase-type plasminogen activator (uPA) activity in vivo holds high promise for early diagnosis of breast cancer. Molecular probes with resisted fluorescence (FL) emission for enhanced PA signals of uPA activity have not been reported. Herein, we proposed a molecular probe Cbz-Gly-Gly-Arg-Phe-Phe-IR775 (Z-GGRFF-IR775) which, upon uPA cleavage, assembled into nanoparticles FF-IR775-NP with quenched fluorescence but enhanced PA signals. Experimental results validated that, upon uPA activation, Z-GGRFF-IR775 exhibited 4.7-fold, 4.1-fold, and 2.9-fold higher PA signals over those in uPA inhibitor-treated control groups in vitro, in MDA-MB-231 cells, and in a tumor-bearing mouse model, respectively. We anticipate that this probe could be applied for highly sensitive PA imaging of uPA activity in early stage malignant tumors in the near future.

ß-Glucuronidase-Activated Bioluminescence Probe for In Vivo Tumor Imaging.

ß-Glucuronidase (GLU) is a hallmark enzyme for many malignant tumors, but bioluminescence (BL) probes that enable GLU imaging in vivo have not been reported. Herein, we rationally designed the BL probe Glc-Luc to address this issue. In vitro results demonstrated the specific responsiveness of Glc-Luc toward GLU with a calculated catalytic efficiency (kcat/Km) of 0.0109 µM-1 min-1 and a limit of detection (LOD) of 1.39 U/mL. Moreover, Glc-Luc rendered 3.1-fold and 15.9-fold higher BL intensities over the control groups in cell lysates and tumor-bearing mice, respectively. We anticipate that Glc-Luc could be further applied for the sensitive diagnosis of GLU-related diseases.

A Chemiluminescent Probe for Imaging Peroxynitrite in Inflammatory Cells and Tissues.

Peroxynitrite (ONOO-) is one type of important reactive oxygen/nitrogen species (ROS/RNS) and plays a vital role in many physiological activities. Excessive ONOO- is associated with many diseases including inflammation, arthritis, inflammatory bowel disease, cancer, and neurodegenerative diseases. However, a chemiluminescent probe capable of detecting endogenous ONOO- at the acidic condition that might be applied for sensitive diagnosis of inflammation-related disease has not been reported. Hence, we designed and synthesized a chemiluminescence (CL) probe, B-PD, to detect endogenous ONOO- both in vitro and in vivo. B-PD demonstrated a quick response toward ONOO- with a limit of detection of 201 nM. In vivo CL imaging results showed that, 30 min postinjection, B-PD could effectively locate early stage inflammation tissue with an imaging contrast up to 6.2. These results suggest that B-PD holds great promise for highly sensitive diagnosis of inflammation-related diseases in the future.

Intracellular Construction of Cathepsin B-Guided Gadolinium Nanoparticles for Enhanced T2 -Weighted MR Tumor Imaging.

Magnetic resonance imaging (MRI) is a superior and noninvasive imaging technique with unlimited tissue penetration depth and superb spatiotemporal resolution, however, using intracellular self-assembly of Gd-containing nanoparticles to enhance the T2 -weighted MR contrast of cancer cells in vivo for precise tumor MRI is rarely reported. The lysosomal cysteine protease cathepsin B (CTSB) is regarded as an attractive biomarker for the early diagnosis of cancers and metastasis. Herein, taking advantage of a biocompatible condensation reaction, a "smart" Gd-based CTSB-responsive small molecular contrast agent VC-Gd-CBT is developed, which can self-assemble into large intracellular Gd-containing nanoparticles by glutathione reduction and CTSB cleavage to enhance the T2 -weighted MR contrast of CTSB-overexpressing MDA-MB-231 cells at 9.4 T. In vivo T2 -weighted MRI studies using MDA-MB-231 murine xenografts show that the T2 -weighted MR contrast change of tumors in VC-Gd-CBT-injected mice is distinctly larger than the mice injected with the commercial agent gadopentetate dimeglumine, or co-injected with CTSB inhibitor and VC-Gd-CBT, indicating that the accumulation of self-assembled Gd-containing nanoparticles at tumor sites effectively enhances the T2 -weighted MR tumor imaging. Hence, this CTSB-targeted small molecule VC-Gd-CBT has the potential to be employed as a T2 contrast agent for the clinical diagnosis of cancers at an early stage.

Enzymatic Self-Assembly of Adamantane-Peptide Conjugate for Combating Staphylococcus aureus Infection.

Staphylococcus aureus (S. aureus) remains a leading cause of bacterial infections. However, eradication of S. aureus infections with common antibiotics is increasingly difficult due to outbreaks of drug resistance. Therefore, new antibiotic classes and antibacterial strategies are urgently in demand. Herein, it is shown that an adamantane-peptide conjugate, upon dephosphorylation by alkaline phosphatase (ALP) constitutively expressed on S. aureus, generates fibrous assemblies in situ to combat S. aureus infection. By attaching adamantane to a phosphorylated tetrapeptide Nap-Phe-Phe-Lys-Tyr(H2 PO3 )-OH, the rationally designed adamantane-peptide conjugate Nap-Phe-Phe-Lys(Ada)-Tyr(H2 PO3 )-OH (Nap-FYp-Ada) is obtained. Upon bacterial ALP activation, Nap-FYp-Ada is dephosphorylated and self-assembles into nanofibers on the surface of S. aureus. As revealed by cell assays, the assemblies of adamantane-peptide conjugates interact with cell lipid membrane and thereby disrupt membrane integrity to kill S. aureus. Animal experiments further demonstrate the excellent potential of Nap-FYp-Ada in the treatment of S. aureus infection in vivo. This work provides an alternative approach to design antimicrobial agents.

Apoptosis-Amplified Assembly of Porphyrin Nanofiber Enhances Photodynamic Therapy of Oral Tumor.

Oral squamous cell carcinoma (OSCC) is the most common oral cancer, having high recurrence and metastasis features. In addition to surgery, photodynamic therapy (PDT) is considered as another effective approach for OSCC treatment. The water solubility of currently available PDT photosensitizers (PSs) is poor, lowering their singlet oxygen (1O2) yield and consequent PDT efficiency. Strategies of PS assembly have been reported to increase 1O2 yield, but it is still possible to further enhance PDT efficiency. In this work, we utilized apoptosis to amplify the assembly of porphyrin nanofibers for enhanced PDT of OSCC. A water-soluble porphyrin derivative, Ac-Asp-Glu-Val-Asp-Asp-TPP (Ac-DEVDD-TPP), was designed for this purpose. Upon caspase-3 (Casp3, an activated enzyme during apoptosis) cleavage and laser irradiation, Ac-DEVDD-TPP was converted to D-TPP, which spontaneously self-assembled into porphyrin nanofibers, accompanied by 1.4-fold and 2.1-fold 1O2 generations in vitro and in cells, respectively. The as-formed porphyrin nanofiber induced efficient cell apoptosis and pyroptosis. In vivo experiments demonstrated that, compared with the scrambled control compound Ac-DEDVD-TPP, Ac-DEVDD-TPP led to 6.2-fold and 1.3-fold expressions of Casp3 in subcutaneous and orthotopic oral tumor models, respectively, and significantly suppressed the tumors. We envision that our strategy of apoptosis-amplified porphyrin assembly might be applied for OSCC treatment in the clinic in the near future.

Bioorthogonal surface-enhanced Raman scattering flower-like nanoprobe with embedded standards for accurate cancer cell imaging.

Developing precise and effective strategies for cancer identification and imaging is attractive due to their importance for early cancer detection, prognosis, and subsequent treatment. Herein, we reported a novel bioorthogonal surface-enhanced Raman scattering (SERS) nanoprobe for accurate cancer cell imaging. A novel core-molecule-shell nanoflower (Au@4-MBN@Au) with rich electromagnetic hot spots and enhanced Raman scattering was first synthesized by optimizing the embedded concentrations of 4-mercaptobenzonitrile (4-MBN). Then, Au@4-MBN@Au was further modified with FA-PEG-SH molecules to acquire the bioorthogonal SERS nanoprobe Au@4-MBN@Au-PEG-FA. The SERS nanoprobe illustrated a robust and stable nitrile stretching vibration Raman signal (2223 cm-1) in the cellular silent region, ensuring high sensitivity and ultra-accuracy SERS imaging of cancer cells. Furthermore, cell imaging results demonstrated Au@4-MBN@Au-PEG-FA could recognize FR-positive HeLa cells with high selectivity due to the high affinity between folate receptor and folic acid. More notably, Au@4-MBN@Au-PEG-FA has been applied to identify FR-positive Hela cells from co-cultured cancer cells with similar morphology by SERS imaging for the first time. With improved signal-to-background ratio, high selectivity, and excellent stability, we anticipate the SERS nanoprobe Au@4-MBN@Au-PEG-FA could be applied for FR-related cancer theranostics and clinical detection in the future.

Atg4B and Cathepsin B-Triggered in Situ Luciferin Formation for Precise Cancer Autophagy Bioluminescence Imaging.

Autophagy plays a crucial role in tumorigenesis and progression, but current approaches to visualize it in vivo show limited precision due to their single-analyte-responsive mode. Hence, by simultaneously employing dual autophagy enzymes Atg4B and cathepsin B to trigger the in situ formation of luciferin, we herein propose a strategy for precise autophagy bioluminescence imaging. An Atg4B-responsive peptide Ac-Thr-Phe-Gly-d-Cys (TFGC) and a cathepsin B-activatable compound Ac-Lys-Gly-Arg-Arg-CBT (KGRR-CBT) were rationally designed. During tumor autophagy, these two compounds were uptaken by cancer cells and cleaved by their corresponding enzymes to yield d-cysteine and 2-cyano-6-aminobenzothiazole, respectively, which underwent a CBT-Cys click reaction to yield d-aminoluciferin, turning the bioluminescence "on". The responsiveness of these two compounds toward the two enzymes was tested in vitro, and the ability to turn bioluminescence "on" was validated in living cancer cells and in vivo. We anticipate that our precise autophagy imaging strategy could be further applied for the diagnosis of autophagy-related diseases in the near future.

MMP 9-instructed assembly of bFGF nanofibers in ischemic myocardium to promote heart repair.

Background: The only effective treatment for myocardial infarction (MI) is the timely restoration of coronary blood flow in the infarcted area, but further reperfusion exacerbates myocardial injury and leads to distal coronary no-reflow, which affects patient prognosis. Angiogenesis could be an important therapeutic strategy for re-establishing the blood supply to save the ischemic myocardium after MI. Basic fibroblast growth factor (bFGF) has been shown to promote angiogenesis. However, direct intravenous administration of bFGF is not a viable option given its poor half-life in vivo. Methods: Herein, we developed a peptide Lys-Lys-Pro-Leu-Gly-Leu-Ala-Gly-Phe-Phe (K2) to encapsulate bFGF to form bFGF@K2 micelle and proposed an enzyme-instructed self-assembly (EISA) strategy to deliver and slowly release bFGF in the ischemic myocardium. Results: The bFGF@K2 micelle exerted a stronger cardioprotective effect than free bFGF in a rat model of myocardial ischemia-reperfusion (MI/R). In vitro results revealed that the bFGF@K2 micelle could be cleaved by matrix metallopeptidase 9 (MMP-9) to yield bFGF@Nanofiber through amphipathic changes. In vivo experiments indicated that intravenous administration of bFGF@K2 micelle could lead to their restructuring into bFGF@Nanofiber and long term retention of bFGF in the ischemic myocardium of rat due to high expression of MMP-9 and assembly-induced retention (AIR) effect, respectively. Twenty-eight days after MI/R model establishment, bFGF@K2 micelle treatment significantly reduced fibrosis and improved cardiac function of the rats. Conclusion: We predict that our strategy could be applied in clinic for MI treatment in the future.

Internal Standard Assisted Surface-Enhanced Raman Scattering Nanoprobe with 4-NTP as Recognition Unit for Ratiometric Imaging Hydrogen Sulfide in Living Cells.

Hydrogen sulfide (H2S), as the third endogenous gasotransmitter, is closely associated with various physiological and pathological processes, whereas many aspects of its functions remain unclear. Effective tools for the accurate detection of H2S in living organisms are urgently needed. We herein reported an internal standard assisted surface-enhanced Raman scattering (SERS) nanoprobe for ratiometric detection of H2S in vitro and in living cells based on the reduction of nitros with H2S. This nanoprobe consists of an internal standard (4-mercaptobenzonitrile, MPBN) embedded core-molecule-shell Au nanoflower (Au@MPBN@Au) as the high plasmonic active SERS substrate and the 4-nitrothiophenol (4-NTP) molecule immobilized on the surface as the H2S recognition unit. With the addition of H2S, the nitros peak (1329 cm-1) decreased. Meanwhile, three obvious new peaks appeared at 1139, 1387, and 1433 cm-1, which were related to the vibration of the dimerized product 4,4'-dimercaptoazobisbenzene (DMAB) of 4-aminothiophenol (4-ATP). However, the peak intensity at 2223 cm-1 derived from MPBN was not influenced by the outer environment. Thus, the H2S level was able to be determined based on the ratio of two peak intensities (I1139/I2223) with a detection limit as low as 0.24 µM. Notably, we have proved that SERS nanoprobe Au@MPBN@Au@4-NTP could ratiometrically image both the endogenous and exogenous H2S in living cells. We anticipate that Au@MPBN@Au@4-NTP could be applied for the study of H2S-related physiological function in the future.

Granzyme B Turns Nanoparticle Fluorescence "On" for Imaging Cytotoxic T Lymphocyte Activity in Vivo.

Cytotoxic T lymphocytes (CTLs) are important immune cells, and their activation is a key step for cancer immunotherapy. Precise evaluation of CTL activity in vivo provides a powerful tool for monitoring cancer-immunotherapeutic outcomes, yet it faces tremendous challenges. Herein, by rationally designing a near-infrared (NIR) fluorescence probe Cys(StBu)-Ile-Glu-Phe-Asp-Lys(Cy5.5)-CBT (Cy5.5-CBT) and employing a reduction-instructed CBT-Cys click condensation reaction, we developed the fluorescence "dual quenched" nanoparticles Cy5.5-CBT-NPs for imaging of granzyme B (GraB), a biomarker tightly associated with the tumoricidal activity of CTLs. Upon GraB cleavage, Cy5.5-CBT-NPs disassembled, subtly turning the fluorescence signal "on". With this fluorescence "turn-on" property, Cy5.5-CBT-NPs enabled sensitive and real-time monitoring of GraB-mediated CTL responses against cancer cells in vitro. Animal experiments demonstrated that, at 16 h post injection, the fluorescence imaging signal of Cy5.5-CBT-NPs showed a 3.1-fold increase on the tumor sites of mice treated by an immune-activating drug S-(2-boronoethyl)-L-cysteine hydrochloride. We envision that Cy5.5-CBT-NPs may provide a powerful tool for noninvasive and sensitive evaluation of immunotherapeutic efficacy of cancer in the near future.

Enzymatic Nanosphere-to-Nanofiber Transition and Autophagy Inducer Release Promote Tumor Chemotherapy.

Chemotherapy has remained an effective and predominant cancer treatment for the past decades, but is hampered by its low response rate and severe systemic toxicity. Combination chemotherapies are proposed to address these issues, yet their therapeutic outcomes are still far from satisfactory. Thus, it is urgent to develop novel strategies to promote tumor chemosensitivity while reducing toxic side effects of chemotherapeutics. Herein, employing a rationally designed peptide conjugate Nap-Phe-Phe-Lys(SA-AZD8055)-Tyr(H2 PO3 )-OH (Nap-AZD-Yp), a novel approach of simultaneous intracellular nanofiber formation and autophagy inducer release is proposed for selectively sensitizing tumor to chemotherapy. Upon sequential catalyses of alkaline phosphatase and carboxylesterase, Nap-AZD-Yp undergoes nanosphere-to-nanofiber transition accompanied by autophagy inducer AZD8055 release in cancer cells. Cell experiments show enhanced endocytosis of anticancer drug doxorubicin and inhibition of cell migration due to the intracellular nanofiber formation. The released AZD8055 further activates excessive autophagy of cancer cells, sensitizing them to chemotherapy. Animal experiment results suggest Nap-AZD-Yp can significantly enhance the therapeutic effects of doxorubicin on tumors while mitigate its toxic adverse effects on normal tissues. It is anticipated that the "smart" concept in this work c be widely employed to develop novel combinational therapies for the treatment of cancers and other diseases in near future.

FAP-α-Instructed Coumarin Excimer Formation for High Contrast Fluorescence Imaging of Tumor.

Emissive excimers, which are formed by planar polycyclic aromatic fluorophores (e.g., coumarin), enable high contrast tumor imaging. However, it is still challenging to "turn on" excimer fluorescence in physiological dilute solutions. The biocompatible CBT-Cys click condensation reaction enables both intra- and intermolecular aggregations of the as-loaded fluorophores on the probe molecules, which may promote the generation of emissive excimers in a synergistic manner. As a proof-of-concept, we herein design a fluorescence probe Cbz-Gly-Pro-Cys(StBu)-Lys(coumarin)-CBT (Cbz-GPC(StBu)K(Cou)-CBT), which can be activated by FAP-α under tumor-inherent reduction conditions, undergo a CBT-Cys click reaction, and self-assemble into coumarin nanoparticle Cou-CBT-NP to "turn on" the excimer fluorescence. In vitro and in vivo studies validate that this "smart" probe realizes efficient excimer fluorescence imaging of FAP-α-overexpressed tumor cells with high contrast and enhanced accumulation, respectively. We anticipate that this probe can be applied for diagnosis of FAP-α-related diseases in the clinic in near future.

Trident Molecule with Nanobrush-Nanoparticle-Nanofiber Transition Property Spatially Suppresses Tumor Metastasis.

Metastasis-induced high mortality of cancers urgently demands new approaches to simultaneously inhibit primary tumor metastasis and distant tumor growth. Herein, by rational design of a trident molecule Nap-Phe-Phe-Lys(SA-CPT)-Lys(SA-HCQ)-Tyr(H2PO3)-OH (Nap-CPT-HCQ-Yp) with three functional "spears" (i.e., a phosphotyrosine motif for enzymatic self-assembly, camptothecin (CPT) motif for chemotherapy, and hydroxychloroquine (HCQ) motif for autophagy inhibition) and nanobrush-nanoparticle-nanofiber transition property, we propose a novel strategy of intracellular enzymatic nanofiber formation and synergistic autophagy inhibition-enhanced chemotherapy and immunotherapy for spatial suppression of tumor metastasis. Under sequential alkaline phosphatase catalysis and carboxylesterase hydrolysis, Nap-CPT-HCQ-Yp undergoes nanobrush-nanoparticle-nanofiber transition, accompanied by the releases of CPT and HCQ. The formed intracellular nanofibers effectively inhibit the metastasis and invasion behaviors of cancer cells. Meanwhile, the released CPT and HCQ synergistically induce a prominent therapeutic effect through autophagy inhibition-enhanced chemotherapy. Furthermore, chemotherapy of Nap-CPT-HCQ-Yp enhances immunogenic cell death, resulting in the activation of toxic T-cells. Finally, a combination of checkpoint blockade therapy and Nap-CPT-HCQ-Yp-mediated chemotherapy elicits systemic antitumor immunity, thereby achieving efficient inhibitions of primary tumors as well as distant tumors in a breast tumor model. Our work offers a simple and feasible strategy for the design of "smart" multifunctional prodrugs to spatially suppress tumor metastasis.

Salt-Inducible Kinase 2-Triggered Release of Its Inhibitor from Hydrogel to Suppress Ovarian Cancer Metastasis.

Salt-inducible kinase 2 (SIK2) is a promising target for ovarian cancer therapy due to its critical role in tumorigenesis and progression. Currently available SIK2 inhibitors have shown remarkable therapeutic effects on ovarian cancers in preclinical studies. However, direct administration of the SIK2 inhibitors may bring significant off-target effect, limiting their clinical applications. In this work, by rational design of a hydrogelator Nap-Phe-Phe-Glu-Glu-Leu-Tyr-Arg-Thr-Gln-Ser-Ser-Ser-Asn-Leu-OH (Nap-S) to coassemble a SIK2 inhibitor HG-9-91-01 (HG), a SIK2-responsive supramolecular hydrogel (Gel Nap-S+HG) for local administration and SIK2-responsive release of HG is reported to efficiently suppress ovarian cancer metastasis. Under the activation of SIK2 overexpressed in ovarian cancers, Nap-S in the hydrogel is phosphorylated to yield hydrophilic Nap-Phe-Phe-Glu-Glu-Leu-Tyr-Arg-Thr-Gln-Ser(H2 PO3 )-Ser-Ser-Asn-Leu (Nap-Sp), triggering the disassembly of the hydrogel and a responsive release of the inhibitor. Cell experiments indicate that sustained release of HG from Gel Nap-S+HG induce a prominent therapeutic effect on cancer cells by inhibiting SIK2 and phosphorylation of their downstream signaling molecules. Animal experiments demonstrate that, compared with those tumor model mice treated with free HG, Gel Nap-S+HG-treatment mice show an enhanced inhibition on ovarian tumor growth and metastasis. It is anticipated that the Gel Nap-S+HG can be applied for ovarian cancer therapy in clinic in the near future.