In situ photo-crosslinkable hyaluronic acid-based hydrogel embedded with GHK peptide nanofibers for bioactive wound healing.

A versatile hydrogel was developed for enhancing bioactive wound healing by introducing the amphiphilic GHK peptide (GHK-C16) into a photo-crosslinkable tyramine-modified hyaluronic acid (HA-Ty). GHK-C16 self-assembled into GHK nanofibers (GHK NF) in HA-Ty solution, which underwent in situ gelation after the wound area was filled with precursor solution. Blue light irradiation (460-490 nm), with riboflavin phosphate as a photoinitiator, was used to trigger crosslinking, which enhanced the stability of the highly degradable hyaluronic acid and enabled sustained release of the nanostructured GHK derivatives. The hydrogels provided a microenvironment that promoted the proliferation of dermal fibroblasts and the activation of cytokines, leading to reduced inflammation and increased collagen expression during wound healing. The complexation of Cu2+ into GHK nanofibers resulted in superior wound healing capabilities compared with non-lipidated GHK peptide with a comparable level of growth factor (EGF). Additionally, nanostructured Cu-GHK improved angiogenesis through vascular endothelial growth factor (VEGF) activation, which exerted a synergistic therapeutic effect. Furthermore, in vivo wound healing experiments revealed that the Cu-GHK NF/HA-Ty hydrogel accelerated wound healing through densely packed remodeled collagen in the dermis and promoting the growth of denser fibroblasts. HA-Ty hydrogels incorporating GHK NF also possessed improved mechanical properties and a faster wound healing rate, making them suitable for advanced bioactive wound healing applications. STATEMENT OF SIGNIFICANCE: By combining photo-crosslinkable tyramine-modified hyaluronic acid with self-assembled Cu-GHK-C16 peptide nanofibers (Cu-GHK NF), the Cu-GHK NF/HA-Ty hydrogel offers remarkable advantages over conventional non-structured Cu-GHK for wound healing. It enhances cell proliferation, migration, and collagen remodeling-critical factors in tissue regeneration. The incorporation of GHK nanofibers complexed with copper ions imparts potent anti-inflammatory effects, promoting cytokine activation and angiogenesis during wound healing. The Cu-GHK NF/hydrogel's unique properties, including in situ photo-crosslinking, ensure high customization and potency in tissue regeneration, providing a cost-effective alternative to growth factors. In vivo experiments further validate its efficacy, demonstrating significant wound closure, collagen remodeling, and increased fibroblast density. Overall, the Cu-GHK NF/HA-Ty hydrogel represents an advanced therapeutic option for wound healing applications.

Bioapplications of Nanomaterials.

Nanobiotechnology is known as the application of nanoscaled techniques in biology which bridges natural science to living organism for improving the quality of life of humans. Nanotechnology was first issued in 1959 and has been rapidly developed, supplying numerous benefits to basic scientific academy and to clinical application including human healthcare, specifically in cancer therapy. This chapter discusses recent advances and potentials of nanotechnology in pharmaceutics, therapeutics, biosensing, bioimaging, and gene delivery that demonstrate the multifunctionality of nanotechnology.

Template-Assisted Plasmonic Nanogap Shells for Highly Enhanced Detection of Cancer Biomarkers.

We present a template-assisted method for synthesizing nanogap shell structures for biomolecular detections based on surface-enhanced Raman scattering. The interior nanogap-containing a silver shell structure, referred to as a silver nanogap shell (Ag NGS), was fabricated on silver nanoparticles (Ag NPs)-coated silica, by adsorbing small aromatic thiol molecules on the Ag NPs. The Ag NGSs showed a high enhancement factor and good signal uniformity, using 785-nm excitation. We performed in vitro immunoassays using a prostate-specific antigen as a model cancer biomarker with a detection limit of 2 pg/mL. To demonstrate the versatility of Ag NGS nanoprobes, extracellular duplex surface-enhanced Raman scattering (SERS) imaging was also performed to evaluate the co-expression of cancer biomarkers, human epidermal growth factor-2 (HER2) and epidermal growth factor receptor (EGFR), in a non-small cell lung cancer cell line (H522). Developing highly sensitive Ag NGS nanoprobes that enable multiplex biomolecular detection and imaging can open up new possibilities for point-of-care diagnostics and provide appropriate treatment options and prognosis.

Proton-enabled activation of peptide materials for biological bimodal memory.

The process of memory and learning in biological systems is multimodal, as several kinds of input signals cooperatively determine the weight of information transfer and storage. This study describes a peptide-based platform of materials and devices that can control the coupled conduction of protons and electrons and thus create distinct regions of synapse-like performance depending on the proton activity. We utilized tyrosine-rich peptide-based films and generalized our principles by demonstrating both memristor and synaptic devices. Interestingly, even memristive behavior can be controlled by both voltage and humidity inputs, learning and forgetting process in the device can be initiated and terminated by protons alone in peptide films. We believe that this work can help to understand the mechanism of biological memory and lay a foundation to realize a brain-like device based on ions and electrons.

Injectable Single-Component Peptide Depot: Autonomously Rechargeable Tumor Photosensitization for Repeated Photodynamic Therapy.

The general practice of photodynamic therapy (PDT) comprises repeated multiple sessions, where photosensitizers are repeatedly administered prior to each operation of light irradiation. To address potential problems arising from the total overdose of photosensitizer by such repeated injections, we here introduce an internalizing RGD peptide (iRGD) derivative (Ppa-iRGDC-BK01) that self-aggregates into an injectable single-component supramolecular depot. Ppa-iRGDC-BK01 is designed as an in situ self-implantable photosensitizer so that it forms a depot by itself upon injection, and its molecular functions (cancer cell internalization and photosensitization) are activated by sustained release, tumor targeting, and tumor-selective proteolytic/reductive cleavage of the iRGD segment. The experimental and theoretical studies revealed that when exposed to body temperature, Ppa-iRGDC-BK01 undergoes thermally accelerated self-assembly to form a supramolecular depot through the hydrophobic interaction of the Ppa pendants and the reorganization of the interpeptide hydrogen bonding. It turned out that the self-aggregation of Ppa-iRGDC-BK01 into a depot exerts a multiple-quenching effect on the photosensitivity to effectively prevent nonspecific phototoxicity and protect it from photobleaching outside the tumor, while enabling autonomous tumor rephotosensitization by long sustained release, tumor accumulation, and intratumoral activation over time. We demonstrate that depot formation through a single peritumoral injection and subsequent quintuple laser irradiations at intervals resulted in complete eradication of the tumor. During the repeated PDT, depot-implanted normal tissues around the tumor exhibited no phototoxic damage under laser exposure. Our approach of single-component photosensitizing supramolecular depot, combined with a strategy of tumor-targeted therapeutic activation, would be a safer and more precise operation of PDT through a nonconventional protocol composed of one-time photosensitizer injection and multiple laser irradiations.

Caffeoyl-Prolyl-Histidine Amide Inhibits Fyn and Alleviates Atopic Dermatitis-Like Phenotypes via Suppression of NF-κB Activation.

Caffeic acid (CA) is produced from a variety of plants and has diverse biological functions, including anti-inflammation activity. It has been recently demonstrated that caffeoyl-prolyl-histidine amide (CA-PH), which is CA conjugated with proline-histidine dipeptide, relieves atopic dermatitis (AD)-like phenotypes in mouse. In this study, we investigated the molecular mechanism underlying CA-PH-mediated alleviation of AD-like phenotypes using cell line and AD mouse models. We confirmed that CA-PH suppresses AD-like phenotypes, such as increased epidermal thickening, infiltration of mast cells, and dysregulated gene expression of cytokines. CA-PH suppressed up-regulation of cytokine expression through inhibition of nuclear translocation of NF-κB. Using a CA-PH affinity pull-down assay, we found that CA-PH binds to Fyn. In silico molecular docking and enzyme kinetic studies revealed that CA-PH binds to the ATP binding site and inhibits Fyn competitively with ATP. CA-PH further suppressed spleen tyrosine kinase (SYK)/inhibitor of nuclear factor kappa B kinase (IKK)/inhibitor of nuclear factor kappa B (IκB) signaling, which is required for nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation. In addition, chronic application of CA-PH, in contrast with that of glucocorticoids, did not induce up-regulation of regulated in development and DNA damage response 1 (REDD1), reduction of mammalian target of rapamycin (mTOR) signaling, or skin atrophy. Thus, our study suggests that CA-PH treatment may help to reduce skin inflammation via down-regulation of NF-κB activation, and Fyn may be a new therapeutic target of inflammatory skin diseases, such as AD.

Tumor microenvironment-responsive fluorogenic nanoprobe for ratiometric dual-channel imaging of lymph node metastasis.

Fluorogenic nanoprobes capable of providing microenvironmental information have extensively been developed to improve the diagnostic accuracy for early or metastatic cancer detection. In cancer-associated microenvironment, matrix metalloproteinase-2,9 (MMP-2,9) has drawn attention as a representative enzymatic marker for diagnosis, prognosis, and prediction of various cancers, which is overexpressed in the primary site as well as metastatic regions. Here, we devised dual-emissive fluorogenic nanoprobe (DFNP) emitting both MMP-2,9-sensitive and insensitive fluorescence signals, for accurate monitoring of the MMP-2,9 activity in metastatic regions. DFNP was nanoscopically constructed by amphiphilic self-assembly between a constantly fluorescent polymer surfactant labeled with Cy7 (F127-Cy7) and an initially nonfluorescent hydrophobic peptide (Cy5.5-MMP-Q) that is fluorogenic in response to MMP-2,9. Ratiometric readout (Cy5.5/Cy7) by dual-channel imaging could normalize the enzyme-responsive sensing signal relative to the constantly emissive internal reference that reflects the probe amount, allowing for semi-quantitative analysis on the MMP-2,9-related tissue microenvironment. In addition to the dual-channel emission, the nanoconstructed colloidal structure of DFNP enabled efficient accumulation to lymph node in vivo. Because of these two colloidal characteristics, when injected intradermally to a mouse model of lymph node metastasis, DFNP could produce reliable ratiometric signals to provide information on the MMP-2,9 activity in the lymph nodes depending on metastatic progression, which corresponded well to the temporal histologic analysis. Furthermore, ratiometric lymph node imaging with DFNP after photodynamic therapy allowed for monitoring a therapeutic response to the given cancer treatment, demonstrating diagnostic and prognostic potential of the nanoconstructed colloidal sensor of tumor microenvironment in cancer treatment.

Theranostic iRGD peptide containing cisplatin prodrug: Dual-cargo tumor penetration for improved imaging and therapy.

In theranostics, peptide-based platforms have widely been exploited owing to their unique biological functions and chemical versatilities. As a tumor-homing ligand, internalizing RGD peptide (iRGD), composed of a tumor-targeting sequence (RGD) and a cell-penetrating C-end Rule (CendR) motif, is known to facilitate the tumor-specific delivery of payloads that are covalently conjugated on its N-terminal fragment or co-administered without any covalent linkages. However, theranostic uses of the iRGD-based platform remain in its infancy with its full potential unexplored; for instance, detailed mechanism of iRGD fragmentation during internalization, strategies for the tumor-specific release of payloads from iRGD and the role of the C-terminal iRGD fragment in delivery have yet to be revealed. In this study, we designed a dual-channel fluorescent cyclic iRGD (TAMRA-iRGDC-Cy5.5) to track each of the N- and C-terminal fragments separately during the tumor internalization process. It turned out that both fragments undergo translocation into cancer cells together and are localized within endosomal-lysosomal compartments. The resulting co-internalization of both iRGD fragments allowed us to develop a new theranostic peptide platform (Cy5.5-iRGDC-Pt(IV)) by conjugating a fluorescent dye and a cisplatin prodrug on each terminus of cyclic iRGD for simultaneous cancer-targeted imaging and therapy. Compared to a control peptide having a non-iRGD sequence, the Cy5.5-iRGDC-Pt(IV) did not only provide a better tumor imaging contrast but also induced tumor-specific apoptosis leading to efficacious tumor suppression. Besides the outstanding cancer imaging and therapeutic performance, the Cy5.5-iRGDC-Pt(IV) revealed negligible systemic toxicity, holding potential to be applied for theranostic uses.

Effect of Alkylamines on Morphology Control of Silver Nanoshells for Highly Enhanced Raman Scattering.

Morphology control of the surface of a nanostructure is a key issue in modulating its surface plasmon resonance and scattering properties. Here, we studied the effect of alkylamines on morphology control during the one-step fabrication of silver nanoshells (NSs) for highly enhanced Raman scattering. Various types of alkylamines were used to study the effects of chain length, existence of hydroxyl groups, and degree of alkyl chains on the surface morphology of silver NSs. The alkylamines influenced the silver ion reduction and the growth of silver domains, resulting in distinctive morphology changes. The optical properties of the silver NSs of different surface morphologies were characterized by surface-enhanced Raman spectra. Especially, when long alkylamines were used, intense and uniform surface-enhanced Raman scattering signals were obtained at the visible and near-infrared (NIR) region, and their enhancement factor was ∼107. To detect cancer biomarkers in vivo, as a feasibility test, silver NSs were modified to highly NIR-active nanoprobes and successfully applied to detect colon cancer without causing nonspecific interactions. Our one-step fabrication method of silver NSs is simple and can overcome various hurdles of morphology control and can be extended to other metal nanostructures of controlled surface morphologies or shape.

Two-dimensional SERS encoding method for on-bead peptide sequencing in high-throughput bioanalysis.

We developed a ready-to-read on-bead peptide encoding method for high-throughput screening bioassays. With two-dimensional surface-enhanced Raman scattering nano-identifiers (2D-SERS IDs) which are concurrently labelled with two SERS codes (coupling steps and kinds of amino acid), we could possibly generate more than 10 trillion codes with only 30 Raman label compounds.

Assembly of Plasmonic and Magnetic Nanoparticles with Fluorescent Silica Shell Layer for Tri-functional SERS-Magnetic-Fluorescence Probes and Its Bioapplications.

In this study, we report on the fabrication of multilayered tri-functional magnetic-SERS-fluorescence nanoprobes (MF-SERS particles) containing clustered superparamagnetic Fe3O4 nanoparticles (NPs), silver NPs, and a fluorescent silica layer. The MF-SERS particles exhibited strong SERS signals from the silver NPs as well as both superparamagnetism and fluorescence. MF-SERS particles were uptaken by cells, allowing successful separation using an external magnetic field. SERS and fluorescence signals could be detected from the NP-containing cells, and CD44 antibody-conjugated MF-SERS particles selectively targeted MDA-MB-231 cells. Based on these properties, MF-SERS particles proved to be a useful nanoprobe for multiplex detection and separation of cancer cells.

ß-Lactoglobulin Peptide Fragments Conjugated with Caffeic Acid Displaying Dual Activities for Tyrosinase Inhibition and Antioxidant Effect.

The regulation of tyrosinase activity and reactive oxygen species is of great importance for the prevention of dermatological disorders in the fields of medicine and cosmetics. Herein, we report a strategy based on solid-phase peptide chemistry for the synthesis of ß-lactoglobulin peptide fragment/caffeic acid (CA) conjugates (CA-Peps) with dual activities of tyrosinase inhibition and antioxidation. The purity of the prepared conjugates, CA-MHIR, CA-HIRL, and CA-HIR, significantly increased to 99%, as acetonide-protected CA was employed in solid-phase coupling reactions on Rink amide resins. The tyrosinase inhibitory activities of all CA-Pep derivatives were higher than the activity of kojic acid, and CA-MHIR exhibited the highest tyrosinase inhibition activity (IC50 = 47.9 µM). Moreover, CA-Pep derivatives displayed significantly enhanced antioxidant activities in the peroxidation of linoleic acid as compared to the pristine peptide fragments. All CA-Pep derivatives showed no cytotoxicity against B16-F1 melanoma cells.

Preparation of tri(ethylene glycol) grafted core-shell type polymer support for solid-phase peptide synthesis.

A core-shell type polymer support for solid-phase peptide synthesis has been developed for high coupling efficiency of peptides and versatile applications such as on-bead bioassays. Although various kinds of polymer supports have been developed, they have their own drawbacks including poor accessibility of reagents and incompatibility in aqueous solution. In this paper, we prepared hydrophilic tri(ethylene glycol) (TEG) grafted core-shell type polymer supports (TEG SURE) for efficient solid-phase peptide synthesis and on-bead bioassays. TEG SURE was prepared by grafting TEG derivative on the surface of AM PS resin via biphasic diffusion control method and subsequent acetylation of amine groups which are located at the core region of AM PS resin. The performance of TEG SURE was evaluated by synthesizing several peptides. Three points can be highlighted: (1) easy control of loading level of TEG, (2) improved efficiency of peptide synthesis compared with the conventional resins, and (3) applicability of on-bead bioassays.

Ultrasensitive NIR-SERRS Probes with Multiplexed Ratiometric Quantification for In Vivo Antibody Leads Validation.

Immunotargeting ability of antibodies may show significant difference between in vitro and in vivo. To select antibody leads with high affinity and specificity, it is necessary to perform in vivo validation of antibody candidates following in vitro antibody screening. Herein, a robust in vivo validation of anti-tetraspanin-8 antibody candidates against human colon cancer using ratiometric quantification method is reported. The validation is performed on a single mouse and analyzed by multiplexed surface-enhanced Raman scattering using ultrasensitive and near infrared (NIR)-active surface-enhanced resonance Raman scattering nanoprobes (NIR-SERRS dots). The NIR-SERRS dots are composed of NIR-active labels and Au/Ag hollow-shell assembled silica nanospheres. A 93% of NIR-SERRS dots is detectable at a single-particle level and signal intensity is 100-fold stronger than that from nonresonant molecule-labeled spherical Au NPs (80 nm). The result of SERRS-based antibody validation is comparable to that of the conventional method using single-photon-emission computed tomography. The NIR-SERRS-based strategy is an alternate validation method which provides cost-effective and accurate multiplexing measurements for antibody-based drug development.

Starbon/High-Amylose Corn Starch-Supported N-Heterocyclic Carbene-Iron(III) Catalyst for Conversion of Fructose into 5-Hydroxymethylfurfural.

Iron-N-heterocyclic carbene complexes (Fe-NHCs) have come to prominence because of their applicability in diverse catalytic reactions, ranging from C-C cross-coupling and C-X bond formation to substitution, reduction, polymerization, and dehydration reactions. The detailed synthesis, characterization, and application of novel heterogeneous Fe-NHC catalysts immobilized on mesoporous expanded high-amylose corn starch (HACS) and Starbon 350 (S350) for facile fructose conversion into 5-hydroxymethylfurfural (HMF) is reported. Both catalyst types showed good performance for the dehydration of fructose to HMF when the reaction was tested at 100 °C with varying time (10 min, 20 min, 0.5 h, 1 h, 3 h and 6 h). For Fe-NHC/S350, the highest HMF yield was 81.7 % (t=0.5 h), with a TOF of 169 h-1 , fructose conversion of 95 %, and HMF selectivity of 85.7 %, whereas for Fe-NHC/expanded HACS, the highest yield was 86 % (t=0.5 h), with a TOF of 206 h-1 , fructose conversion of 87 %, and HMF selectivity of 99 %. Iron loadings of 0.26 and 0.30 mmol g-1 were achieved for Fe-NHC/expanded starch and Fe-NHC/S350, respectively.

A dual modal silver bumpy nanoprobe for photoacoustic imaging and SERS multiplexed identification of in vivo lymph nodes.

Multimodal imaging can provide complementary biomedical information which has huge potential in pre-clinical and clinical imaging and sensing. In this study, we introduce dual modal NIR silver bumpy nanoprobes for in vivo imaging and multiplexed detection of biomolecules by both photoacoustic imaging (PAI) and surface-enhanced Raman scattering (SERS) techniques. For this study, we used silica-coated silver bumpy nanoshell probes (AgNS@SiO2). AgNS@SiO2 have strong NIR-absorption and scattering properties compared with other nanostructures, and therefore, can be a good candidate for photoacoustic (PA) and SERS multimodal imaging. We obtained PA images of the skin and SLNs of rats by injecting various kinds of Raman-labeled AgNS@SiO2. Multiplexed identification of the injected AgNS@SiO2 was achieved by measuring SERS signals. AgNS@SiO2 have the potential to be applied in detecting cancer biomarkers by locating biomarkers quickly using PA imaging, and identification by multiplexed target measurement using SERS signals in vivo.

Adenosine Triphosphate-Encapsulated Liposomes with Plasmonic Nanoparticles for Surface Enhanced Raman Scattering-Based Immunoassays.

In this study, we prepared adenosine triphosphate (ATP) encapsulated liposomes, and assessed their applicability for the surface enhanced Raman scattering (SERS)-based assays with gold-silver alloy (Au@Ag)-assembled silica nanoparticles (NPs; SiO2@Au@Ag). The liposomes were prepared by the thin film hydration method from a mixture of l-α-phosphatidylcholine, cholesterol, and PE-PEG2000 in chloroform; evaporating the solvent, followed by hydration of the resulting thin film with ATP in phosphate-buffered saline (PBS). Upon lysis of the liposome, the SERS intensity of the SiO2@Au@Ag NPs increased with the logarithm of number of ATP-encapsulated liposomes after lysis in the range of 8 × 106 to 8 × 1010. The detection limit of liposome was calculated to be 1.3 × 10-17 mol. The successful application of ATP-encapsulated liposomes to SiO2@Au@Ag NPs based SERS analysis has opened a new avenue for Raman label chemical (RCL)-encapsulated liposome-enhanced SERS-based immunoassays.

Simultaneous Detection of EGFR and VEGF in Colorectal Cancer using Fluorescence-Raman Endoscopy.

Fluorescence endomicroscopy provides quick access to molecular targets, while Raman spectroscopy allows the detection of multiple molecular targets. Using a simultaneous fluorescence-Raman endoscopic system (FRES), we herein demonstrate its potential in cancer diagnosis in an orthotopically induced colorectal cancer (CRC) xenograft model. In the model, epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) were targeted with antibody-conjugated fluorescence and surface-enhanced Raman scattering (F-SERS) dots. FRES demonstrated fast signal detection and multiplex targeting ability using fluorescence and Raman signals to detect the F-SERS dots. In addition, FRES showed a multiplex targeting ability even on a subcentimeter-sized CRC after spraying with a dose of 50 µg F-SERS dots. In conclusion, molecular characteristics of tumor cells (EGFR in cancer cell membranes) and tumor microenvironments (VEGF in the extracellular matrix) could be simultaneously investigated when performing a colonoscopy.

Water-Floating Giant Nanosheets from Helical Peptide Pentamers.

One of the important challenges in the development of protein-mimetic materials is understanding the sequence-specific assembly behavior and dynamic folding change. Conventional strategies for constructing two-dimensional (2D) nanostructures from peptides have been limited to using ß-sheet forming sequences as building blocks due to their natural tendency to form sheet-like aggregations. We have identified a peptide sequence (YFCFY) that can form dimers via a disulfide bridge, fold into a helix, and assemble into macroscopic flat sheets at the air/water interface. Due to the large driving force for 2D assembly and high elastic modulus of the resulting sheet, the peptide assembly induces flattening of the initially round water droplet. Additionally, we found that stabilization of the helix by dimerization is a key determinant for maintaining macroscopic flatness over a few tens of centimeters even with a uniform thickness of <10 nm. Furthermore, the ability to transfer the sheets from a water droplet to another substrate allows for multiple stacking of 2D peptide nanostructures, suggesting possible applications in biomimetic catalysis, biosensors, and 2D related electronic devices.

Activatable iRGD-based peptide monolith: Targeting, internalization, and fluorescence activation for precise tumor imaging.

A disulfide-bridged cyclic RGD peptide, named iRGD (internalizing RGD, c(CRGDK/RGPD/EC)), is known to facilitate tumor targeting as well as tissue penetration. After the RGD motif-induced targeting on αv integrins expressed near tumor tissue, iRGD encounters proteolytic cleavage to expose the CendR motif that promotes penetration into cancer cells via the interaction with neuropilin-1. Based on these proteolytic cleavage and internalization mechanism, we designed an iRGD-based monolithic imaging probe that integrates multiple functions (cancer-specific targeting, internalization and fluorescence activation) within a small peptide framework. To provide the capability of activatable fluorescence signaling, we conjugated a fluorescent dye to the N-terminal of iRGD, which was linked to the internalizing sequence (CendR motif), and a quencher to the opposite C-terminal. It turned out that fluorescence activation of the dye/quencher-conjugated monolithic peptide probe requires dual (reductive and proteolytic) cleavages on both disulfide and amide bond of iRGD peptide. Furthermore, the cleavage of the iRGD peptide leading to fluorescence recovery was indeed operative depending on the tumor-related angiogenic receptors (αvß3 integrin and neuropilin-1) in vitro as well as in vivo. Compared to an 'always fluorescent' iRGD control probe without quencher conjugation, the dye/quencher-conjugated activatable monolithic peptide probe visualized tumor regions more precisely with lower background noise after intravenous injection, owing to the multifunctional responses specific to tumor microenvironment. All these results, along with minimal in vitro and in vivo toxicity profiles, suggest potential of the iRGD-based activatable monolithic peptide probe as a promising imaging agent for precise tumor diagnosis.