Multifunction-Harnessed Afterglow Nanosensor for Molecular Imaging of Acute Kidney Injury In Vivo.

Afterglow is superior to other optical modalities for biomedical applications in that it can exclude the autofluorescence background. Nevertheless, afterglow has rarely been applied to the high-contrast "off-to-on" activatable sensing scheme because the complicated afterglow systems hamper the additional inclusion of sensory functions while preserving the afterglow luminescence. Herein, a simple formulation of a multifunctional components-incorporated afterglow nanosensor (MANS) is developed for the superoxide-responsive activatable afterglow imaging of cisplatin-induced kidney injury. A multifunctional iridium complex (Ir-OTf) is designed to recover its photoactivities (phosphorescence and the ability of singlet oxygen-generating afterglow initiator) upon exposure to superoxide. To construct the nanoscopic afterglow detection system (MANS), Ir-OTf is incorporated with another multifunctional molecule (rubrene) in the polymeric micellar nanoparticle, where rubrene also plays dual roles as an afterglow substrate and a luminophore. The multiple functions covered by Ir-OTf and rubrene renders the composition of MANS quite simple, which exhibits superoxide-responsive "off-to-on" activatable afterglow luminescence for periods longer than 11 min after the termination of pre-excitation. Finally, MANS is successfully applied to the molecular imaging of cisplatin-induced kidney injury with activatable afterglow signals responsive to pathologically overproduced superoxide in a mouse model without autofluorescence background.

Photoechogenic Inflatable Nanohybrids for Upconversion-Mediated Sonotheranostics.

Hybrid nanostructures are promising for ultrasound-triggered drug delivery and treatment, called sonotheranostics. Structures based on plasmonic nanoparticles for photothermal-induced microbubble inflation for ultrasound imaging exist. However, they have limited therapeutic applications because of short microbubble lifetimes and limited contrast. Photochemistry-based sonotheranostics is an attractive alternative, but building near-infrared (NIR)-responsive echogenic nanostructures for deep tissue applications is challenging because photolysis requires high-energy (UV-visible) photons. Here, we report a photochemistry-based echogenic nanoparticle for in situ NIR-controlled ultrasound imaging and ultrasound-mediated drug delivery. Our nanoparticle has an upconversion nanoparticle core and an organic shell carrying gas generator molecules and drugs. The core converts low-energy NIR photons into ultraviolet emission for photolysis of the gas generator. Carbon dioxide gases generated in the tumor-penetrated nanoparticle inflate into microbubbles for sonotheranostics. Using different NIR laser power allows dual-modal upconversion luminescence planar imaging and cross-sectional ultrasonography. Low-frequency (10 MHz) ultrasound stimulated microbubble collapse, releasing drugs deep inside the tumor through cavitation-induced transport. We believe that the photoechogenic inflatable hierarchical nanostructure approach introduced here can have broad applications for image-guided multimodal theranostics.

Lung-targeted delivery of TGF-ß antisense oligonucleotides to treat pulmonary fibrosis.

Pulmonary fibrosis is a serious respiratory disease, with limited therapeutic options. Since TGF-ß is a critical factor in the fibrotic process, downregulation of this cytokine has been considered a potential approach for disease treatment. Herein, we designed a new lung-targeted delivery technology based on the complexation of polymeric antisense oligonucleotides (pASO) and dimeric human ß-defensin 23 (DhBD23). Antisense oligonucleotides targeting TGF-ß mRNA were polymerized by rolling circle amplification and complexed with DhBD23. After complexation with DhBD23, pASO showed improved serum stability and enhanced uptake by fibroblasts in vitro and lung-specific accumulation upon intravenous injection in vivo. The pASO/DhBD23 complex delivered into the lung downregulated target mRNA, and subsequently alleviated lung fibrosis in mice, as demonstrated by western blotting, quantitative reverse-transcriptase PCR (qRT-PCR), immunohistochemistry, and immunofluorescence imaging. Moreover, as the complex was prepared only with highly biocompatible materials such as DNA and human-derived peptides, no systemic toxicity was observed in major organs. Therefore, the pASO/DhBD23 complex is a promising gene therapy platform with lung-targeting ability to treat various pulmonary diseases, including pulmonary fibrosis, with low side effects.

Heterochiral Assembly of Amphiphilic Peptides Inside the Mitochondria for Supramolecular Cancer Therapeutics.

Self-assembly of peptides containing both l- and d-isomers often results in nanostructures with enhanced properties compared to their enantiomeric analogues, such as faster kinetics of formation, higher mechanical strength, and enzymatic stability. However, occurrence and consequences of the heterochiral assembly in the cellular microenvironment are unknown. In this study, we monitored heterochiral assembly of amphiphilic peptides inside the cell, specifically mitochondria of cancer cells, resulting in nanostructures with refined morphological and biological properties owing to the superior interaction between the backbones of opposite chirality. We have designed a mitochondria penetrating tripeptide containing a diphenyl alanine building unit, named as Mito-FF due to their mitochondria targeting ability. The short peptide amphiphile, Mito-FF co-assembled with its mirror pair, Mito-ff, induced superfibrils of around 100 nm in diameter and 0.5-1 µm in length, while enantiomers formed only narrow fibers of 10 nm in diameter. The co-administration of Mito-FF and Mito-ff in the cell induced drastic mitochondrial disruption both in vitro and in vivo. The experimental and theoretical analyses revealed that pyrene capping played a major role in inducing superfibril morphology upon the co-assembly of racemic peptides. This work shows the impact of chirality control over the peptide self-assembly inside the biological system, thus showing a potent strategy for fabricating promising peptide biomaterials by considering chirality as a design modality.

Dual-color fluorescent nanoparticles showing perfect color-specific photoswitching for bioimaging and super-resolution microscopy.

Dual-emissive systems showing color-specific photoswitching are promising in bioimaging and super-resolution microscopy. However, their switching efficiency has been limited because a delicate manipulation of all the energy transfer crosstalks in the systems is unfeasible. Here, we report a perfect color-specific photoswitching, which is rationally designed by combining the complete off-to-on fluorescence switching capability of a fluorescent photochromic diarylethene and the frustrated energy transfer to the other fluorescent dye based on the excited-state intramolecular proton transfer (ESIPT) process. Upon alternation of UV and visible light irradiations, the system achieves 100% switching on/off of blue emission from the diarylethene while orange emission from the ESIPT dye is unchanged in the polymer film. By fabricating this system into biocompatible polymer nanoparticles, we demonstrate microscopic imaging of RAW264.7 macrophage cells with reversible blue-color specific fluorescence switching that enables super-resolution imaging with a resolution of 70 nm.

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.

Potential Protective Effect of Nitric Oxide-Releasing Nanofibers in Hypoxia/Reoxygenation-Induced Cardiomyocyte Injury.

Nitric oxide (NO) is involved in several physiological processes including vasodilation, angiogenesis, immune response, and wound healing, as well as preventing ischemia/reperfusion injury in many organs such as the heart, liver, lungs, and kidneys. Recently, various NO delivery systems such as nanoparticles, nanorods, and nanofibers have been widely studied as potential therapeutic agents. In particular, NO-releasing nanofibers have been attracting much attention for various medicinal applications including regenerative medicine, wound dressings, and coatings for implantable medical devices, due to their flexible and open architectures. In this study, we prepared biocompatible NO-releasing nanofibers by electrospinning using mixed solutions of polymers and methylaminopropyltrimethoxysilane (MAP3), which was modified with N-diazeniumdiolate as an NO donor. In addition, we evaluated their protective effects on hypoxia/reoxygenation (HR) injury in H9c2 cells. The total NO amount released from the resulting MAP3 nanofibers was 1.26 µmol ·mg-1. From the cytotoxicity evaluation of various weights of NO-releasing nanofibers (0 to 2 mg), we selected 1 mg NO-releasing nanofibers for the subsequent experiments. Pre-treatment with NO-releasing nanofibers before hypoxia induction could provide a cytoprotective effect against HR-induced injury in H9c2 cells. The nanofibers could also effectively inhibit the generation of hydrogen peroxide, which was one major contributor to oxidative damage, as well as 8-hydroxyl-2-deoxyguanosine level as an indicator of oxidative DNA damage. In addition, pre-treatment with NO-releasing nano-fibers in a wound model showed wound healing effects similar to those of normal cells. As a result, N-diazeniumdiolate-modified MAP3 nanofibers might protect H9c2 cells from DNA damage by inhibiting the generation of oxidative stress in HR injury. Therefore, we expect that NO-releasing nanofibers could be utilized as a therapeutic strategy for protecting cardiomyocytes from HR injury.

Gold-stabilized carboxymethyl dextran nanoparticles for image-guided photodynamic therapy of cancer.

Photodynamic therapy (PDT) has been extensively investigated to treat cancer since it induces cell death through the activation of photosensitizers by light. However, its success has been hampered by the insufficient selectivity of photosensitizers to tumor tissues. In an attempt to increase the therapeutic efficacy of PDT by targeting the photosensitizer specifically to the tumor site, we prepared chlorin e6 (Ce6)-loaded gold-stabilized carboxymethyl dextran nanoparticles (Ce6-GS-CNPs). Ce6-GS-CNPs possessed highly stable nanostructures and no significant change was observed in their particle size in the presence of serum for 6 days. When Ce6-GS-CNPs were intravenously injected into tumor-bearing mice, they exhibited prolonged circulation in the body and gradually accumulated in the tumor tissue. Under laser irradiation of the tumor site which could be recognized by the near-infrared fluorescence imaging system, Ce6-GS-CNPs effectively suppressed tumor growth. Overall, Ce6-GS-CNPs might have potential as nanomedicine for image-guided photodynamic cancer therapy.