Photo-triggered Release Research Articles

Pillar[5]arene stabilized gold nanoparticles for the enhanced light-triggered nitric oxide release with antibacterial and antibiofilm activities

The reactivity of guests confined in well-defined host cavities act significantly different from that in bulk systems. In this work, pillar[5]arene not only works as a stabilizer for the fabrication of gold nanoparticles, but also act as an accelerator for the enhanced photo-triggered release of gaseous nitric oxide (NO) from encapsulated NO donor. The host–guest complex can dramatically improve the photoactivity of loaded guest molecule to generate antibacterial NO molecules through the confinement effect of pillar[5]arene cavity. The synergistic photo-triggered localized NO release and photo-thermal effect of host–guest complex demonstrated impressive antibacterial behaviors against Gram-negative bacterial strain Escherichia coli, Gram-positive bacterial strain Staphylococcus aureus and methicillin-resistant Staphylococcus aureus. Moreover, this biocompatible photo-responsive complex can also inhibit bacterial biofilm formation and disrupt the pre-existing biofilms.

Light-Activated Nanodiamond-Based Drug Delivery Systems for Spatiotemporal Release of Antisense Oligonucleotides.

Nanodiamonds (NDs) are considered promising delivery platforms, but inaccurate and uncontrolled release of drugs at target sites is the biggest challenge of NDs in precision medicine. This study presents the development of phototriggerable ND-based drug delivery systems, utilizing ortho-nitrobenzyl (o-NB) molecules as photocleavable linkers between drugs and nanocarriers. UV irradiation specifically cleaved o-NB molecules and then was followed by releasing antisense oligonucleotides from ND-based carriers in both buffer and cellular environments. This ND system carried cell nonpermeable therapeutic agents for bypassing lysosomal trapping and degradation. The presence of fluorescent nitrogen-vacancy centers also allowed NDs to serve as biological probes for tracing in cells. We successfully demonstrated phototriggered release of antisense oligonucleotides from ND-based nanocarriers, reactivating their antisense functions. This highlights the potential of NDs, photocleavable linkers, and light stimuli to create advanced drug delivery systems for controlled drug release in disease therapy, opening possibilities for targeted and personalized treatments.

Radiometallation and photo-triggered release of ready-to-inject radiopharmaceuticals from the solid phase.

The efficient, large-scale synthesis of radiometallated radiopharmaceuticals represents an emerging clinical need which, to date, is inherently limited by time consuming, sequential procedures to conduct isotope separation, radiochemical labeling and purification prior to formulation for injection into the patient. In this work, we demonstrate that a solid-phase based, concerted separation and radiosynthesis strategy followed by photochemical release of radiotracer in biocompatible solvents can be employed to prepare ready-to-inject, clinical grade radiopharmaceuticals. Optimization of resin base, resin loading, and radiochemical labeling capacity are demonstrated with 67Ga and 64Cu radioisotopes using a short model peptide sequence and further validated using two peptide-based radiopharmaceuticals with clinical relevance, targeting the gastrin-releasing peptide and the prostate specific membrane antigen. We also demonstrate that the solid-phase approach enables separation of non-radioactive carrier ions Zn2+ and Ni2+ present at 105-fold excess over 67Ga and 64Cu by taking advantage of the superior Ga3+ and Cu2+ binding affinity of the solid-phase appended, chelator-functionalized peptide. Finally, a proof of concept radiolabeling and subsequent preclinical PET-CT study with the clinically employed positron emitter 68Ga successfully exemplifies that Solid Phase Radiometallation Photorelease (SPRP) allows the streamlined preparation of radiometallated radiopharmaceuticals by concerted, selective radiometal ion capture, radiolabeling and photorelease.

Acyl-caged rhodamines: photo-controlled and self-calibrated generation of acetyl radicals for neural function recovery in early AD mice.

The biological function of radicals is a broad continuum from signaling to killing. Yet, biomedical exploitation of radicals is largely restricted to the theme of healing-by-killing. To explore their potential in healing-by-signaling, robust radical generation methods are warranted. Acyl radicals are endogenous, exhibit facile chemistry and elicit matrix-dependent biological outcomes. Their implications in health and disease remain untapped, primarily due to the lack of a robust generation method with spatiotemporal specificity. Fusing the Norrish chemistry into the xanthene scaffold, we developed a novel general and modular molecular design strategy for photo-triggered generation of acyl radicals, i.e., acyl-caged rhodamine (ACR). A notable feature of ACR is the simultaneous release of a fluorescent probe for cell redox homeostasis allowing real-time monitoring of the biological outcome of acyl radicals. With a donor of the endogenous acetyl radical (ACR575a), we showcased its capability in precise and continuous modulation of the cell redox homeostasis from signaling to stress, and induction of a local oxidative burst to promote differentiation of neural stem cells (NSCs). Upon intracerebral-injection of ACR575a and subsequent fiber-optical activation, early AD mice exhibited enhanced differentiation of NSCs toward neurons, reduced formation of Aβ plaques, and significantly improved cognitive abilities, including learning and memory.

Single-Component Photo-Responsive Template for the Controlled Release of NO and H2S2.

Redox signaling molecules include a number of reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive sulfur species (RSS). These molecules work collectively in the regulation of many physiological processes. Understanding the crosstalk mechanisms in these signaling molecules is important but challenging. The development of donor compounds of ROS/RNS/RSS will aid the advances in this field. While many donors that can release one ROS/RNS/RSS have been developed, dual donors that can release two signaling species and facilitate their crosstalk studies are still very rare. Those limited examples lack the ability to precisely control the timing of two releases. In this work, a 2-methoxy-6-naphthacyl-derived tertiary SNO compound, Naph-SNO, was designed and evaluated as the dual donor for NO and H2S2. The 2-methoxy-6-naphthacyl structure was demonstrated to be a novel photoremovable protecting group that could directly uncage C-S bonds. Under the irradiation of lights with different wavelengths (visible or UV), Naph-SNO could release NO and H2S2 in a stepwise manner, or simultaneously (i.e., likely producing the crosstalk product HSNO/HSSNO). In addition, the release of payloads from the donor also produced an end product with blue fluorescence. Therefore, the release process could be easily monitored in "real time." This controllable photo-triggered release strategy has the potential to be used in the design of other RNS/RSS dual donors.

Erythrocyte-Derived Nanoparticles with Folate Functionalization for Near Infrared Pulsed Laser-Mediated Photo-Chemotherapy of Tumors.

Despite its common side effects and varying degrees of therapeutic success, chemotherapy remains the gold standard method for treatment of cancer. Towards developing a new therapeutic approach, we have engineered nanoparticles derived from erythrocytes that contain indocyanine green as a photo-activated agent that enables near infrared photothermal heating, and doxorubicin hydrochloride (DOX) as a chemotherapeutic drug. We hypothesize that milliseconds pulsed laser irradiation results in rapid heating and photo-triggered release of DOX, providing a dual photo-chemo therapeutic mechanism for tumor destruction. Additionally, the surface of the nanoparticles is functionalized with folate to target the folate receptor-α on tumor cells to further enhance the therapeutic efficacy. Using non-contract infrared radiometry and absorption spectroscopy, we have characterized the photothermal response and photostability of the nanoparticles to pulsed laser irradiation. Our in vitro studies show that these nanoparticles can mediate photo-chemo killing of SKOV3 ovarian cancer cells when activated by pulsed laser irradiation. We further demonstrate that this dual photo-chemo therapeutic approach is effective in reducing the volume of tumor implants in mice and elicits an apoptotic response. This treatment modality presents a promising approach in destruction of small tumor nodules.

Thermoresponsive Liposomes for Photo-Triggered Release of Hypericin Cyclodextrin Inclusion Complex for Efficient Antimicrobial Photodynamic Therapy.

Antimicrobial strategies with high efficacy against bacterial infections are urgently needed. The development of effective therapies to control bacterial infections is still a challenge. Herein, near-infrared (NIR)-activated thermosensitive liposomes (TSL) were loaded with the NIR-dye 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR) and the water-soluble hypericin (Hyp) β-cyclodextrin inclusion complex (Hyp-βCD). DiR and Hyp-βCD loaded thermosensitive liposomes (DHβCD-TSL) are functionalized for photothermal triggered release and synergistic photodynamic therapy to eliminate the gram-positive Staphylococcus saprophyticus. The dually active liposomes allow the production of heat and singlet oxygen species with the help of DiR and Hyp, respectively. The elevated temperature, generated by the NIR irradiation, irreversibly damages the bacterial membrane, increases the permeation, and melts the liposomes via a phase-transition mechanism, which allows the release of the Hyp-βCD complex. The photodynamic effect of Hyp-βCD eradicates the bacterial cells owing to its toxic oxygen species production. DHβCD-TSL measured the size of 130 nm with an adequate encapsulation efficiency of 81.3% of Hyp-βCD. They exhibited a phase transition temperature of 42.3 °C, while they remained stable at 37 °C, and 44% of Hyp-βCD was released after NIR irradiation (T > 47 °C). The bacterial viability dropped significantly after the synergistic treatment (>4 log10), indicating that the NIR-activated TSL have immense therapeutic potential to enhance the antibacterial efficacy. The liposomes showed good biocompatibility, which was confirmed by the cellular viability of mouse fibroblasts (L929).

A universally dispersible graphene-based ink modifier facilitates 3D printing of multi-functional tissue-engineered scaffolds

3D-printed bioscaffolds for the realization of individualized tissue regeneration remains challenging due to the limitation in terms of current biomaterial inks. Exploring a universal ink modifier to enhance the properties of the arbitrary inks for 3D printing of multi-functional scaffolds is therefore an alternative. Herein, a universally dispersible graphene oxide-based polyurethane (GO-PU) ink modifier with network structure constituting amphiphilic polyethylene glycol (PEG) and nanoscale GO (nGO) is presented for the first time. GO-PU can be stably dispersed in various organic and aqueous solutions for 24 h without obvious aggregation, far superior to pure nGO. The excellent printability of GO-PU is demonstrated to fabricate pure GO-PU and GO-PU modified composite scaffolds in which GO-PU is used as ink modifiers. The addition of GO-PU with 5% (ww%/wt%) contents into PLGA or PEGDA can not only improve their mechanical properties without decrease printability, but also endow the additional performances with the resulting scaffolds from the incorporated functional nGO segments, like photo-triggered release ability. In addition, the results of in vitro and in vivo toxicity tests confirmed that GO-PU is biocompatible, indicating that this facile and universal approach for introducing graphene materials into 3D-printed scaffolds is with a great potential for tissue regeneration.

Nitroimidazole-Based Ruthenium(II) Complexes: Playing with Structural Parameters to Design Photostable and Light-Responsive Antibacterial Agents.

5-Nitroimidazole (5NIMH), chosen as a molecular model of nitroimidazole derivatives, which represent a broad-spectrum class of antimicrobials, was incorporated into the ruthenium complexes [Ru(tpy)(phen)(5NIM)]PF6 (1) and [Ru(tpy)(dmp)(5NIM)]PF6 (2) (tpy = terpyridine, phen = phenanthroline, dmp = 2,9-dimethyl-1,10-phenanthroline). Besides the uncommon metal coordination of 5-nitroimidazole in its imidazolate form (5NIM), the different architectures of the spectator ligands (phen and dmp) were exploited to tune the "mode of action" of the resulting complexes, passing from a photostable compound where the redox properties of 5NIMH are preserved (1) to one suitable for the nitroimidazole phototriggered release (2) and whose antibacterial activity against B. subtilis, chosen as cellular model, is effectively improved upon light exposure. This study may provide a fundamental knowledge on the use of Ru(II)-polypyridyl complexes to incorporate and/or photorelease biologically relevant nitroimidazole derivatives in the design of a novel class of antimicrobials.

Multifunctional photo-responsive liposomes for tumor imaging and phototherapy to enhance the antitumor efficacy and reduce the hepatotoxicity of methotrexate

Methotrexate (MTX) is a chemotherapy drug widely applied in clinical treatment for various tumors. However, side effects caused by MTX greatly affect the therapeutic efficacy. Hence, photo-triggered release liposomes were constructed by amphiphilic photosensitizers ZnPc(PEG)4 and phospholipid materials DPPC to load MTX for improved antitumor effect. It was demonstrated that ZnPc(PEG)4 integrated into lipid layers endowed final product ZnPc(PEG)4:MTX@LiPOs improved stability. ZnPc(PEG)4:MTX@LiPOs was verified to have excellent characteristics in terms of photo-triggered release, ROS generation and heat production. In particular, cellular experiments indicated that ZnPc(PEG)4:MTX@LiPOs had synergistic cytotoxicity of phototherapy and chemotherapy. The cell uptake assay demonstrated that the internalization of MTX into tumor cells was significantly increased by the encapsulation of ZnPc(PEG)4@LiPOs. ZnPc(PEG)4:MTX@LiPOs was confirmed to possess excellent tumor targeting reflected in faster accumulation, higher drug level and longer retention at tumor sites than free ZnPc(PEG)4. In addition, ZnPc(PEG)4:MTX@LiPOs had significantly enhanced tumor inhibition rate. What's more, liver histopathology and serum enzyme tests indicated that ZnPc(PEG)4:MTX@LiPOs had distinctly reduced hepatotoxicity than MTX. The constructed liposomes with photo-triggered release can be applied to deliver other chemotherapy drugs for synergistic antitumor ability and reduced side effect.

Membrane composition is a functional determinant of NIR-activable liposomes in orthotopic head and neck cancer.

Near-infrared (NIR)-activable liposomes containing photosensitizer (PS)-lipid conjugates are emerging as tunable, high-payload, and tumor-selective platforms for photodynamic therapy (PDT)-based theranostics. To date, the impact that the membrane composition of a NIR-activable liposome (the chemical nature and subsequent conformation of PS-lipid conjugates) has on their in vitro and in vivo functionality has not been fully investigated. While their chemical nature is critical, the resultant physical conformation dictates their interactions with the immediate biological environments. Here, we evaluate NIR-activable liposomes containing lipid conjugates of the clinically-used PSs benzoporphyrin derivative (BPD; hydrophobic, membrane-inserting conformation) or IRDye 700DX (hydrophilic, membrane-protruding conformation) and demonstrate that membrane composition is critical for their function as tumor-selective PDT-based platforms. The PS-lipid conformations were primarily dictated by the varying solubilities of the two PSs and assisted by their lipid conjugation sites. Conformation was further validated by photophysical analysis and computational predictions of PS membrane partitioning (topological polar surface area [tPSA], calculated octanol/water partition [cLogP], and apparent biomembrane permeability coefficient [Papp]). Results show that the membrane-protruding lipo-IRDye700DX exhibits 5-fold more efficient photodynamic generation of reactive molecular species (RMS), 12-fold expedited phototriggered burst release of entrap-ped agents, and 15-fold brighter fluorescence intensity as compared to the membrane-inserting lipo-BPD-PC (phosphatidylcholine conjugate). Although the membrane-inserting lipo-BPD-PC exhibits less efficient photo-dynamic generation of RMS, it allows for more sustained phototriggered release, 10-fold greater FaDu cancer cell phototoxicity, and 7.16-fold higher tumor-selective delivery in orthotopic mouse FaDu head and neck tumors. These critical insights pave the path for the rational design of emerging NIR-activable liposomes, whereby functional consequences of membrane composition can be tailored toward a specific therapeutic purpose.

A photo-triggered antifungal nanoplatform with efflux pump and heat shock protein reversal activity for enhanced chemo-photothermal synergistic therapy.

Drug-resistant pathogens are less sensitive to traditional antibiotics in many stubborn infections. It is imminently desirable to have an effective alternative therapeutic agent for combating drug-resistant pathogen infections. Herein, a photo-triggered multifunctional nanoplatform (TMOB/FLU@PCM NPs) with efflux pump and heat shock protein expression reversal activity is developed for the highly effective eradication of drug-resistant fungi. Upon 808 nm laser excitation, the hyperthermia originating from a BODIPY derivative (TMOB) can not only melt the phase-change material (PCM) vehicle consisting of hexadecanol and cis-2-dodecenoic acid (BDSF) to on-demand release the quorum sensing molecule BDSF and the antifungal drug fluconazole (FLU), but can also destroy the integrity of the C. albicans cell membrane. Thanks to the release of BDSF from TMOB/FLU@PCM NPs, the expression of drug efflux pumps (MDR1, CDR2, CDR4) and thermotolerant proteins (HSP12, HSP21, HSP60, HSP90) is inhibited, which further boosts the therapeutic effect of chemo/photothermal therapy. Moreover, the hyphal and biofilm formation of C. albicans can be blocked by TMOB/FLU@PCM NPs under 808 nm laser irradiation. In vitro and in vivo results indicate that TMOB/FLU@PCM NPs with good biosafety can efficiently eliminate clinical azole-resistant C. albicans. Thus, TMOB/FLU@PCM NPs exhibits a promising future in the treatment of azole-resistant C. albicans infection.

Spatiotemporally Controlling the Release of Biological Effectors Enhances Their Effects on Cell Migration and Neurite Outgrowth.

It is a major challenge to coordinate topographic cues from scaffolds with the on-demand, sustained release of biological effectors to maximize their performance in tissue regeneration. Here, a system involving masked, photo-triggered release of biological effectors from a temperature-sensitive scaffold for augmented cell migration and neurite outgrowth is reported. The scaffold contains microparticles of a phase-change material (PCM) sandwiched between two layers of electrospun fibers. The biological effectors are co-loaded with a photothermal dye in the PCM microparticles. Under irradiation with a near-infrared laser, the PCM will be melted to swiftly release the biological effectors. By imposing a photomask between the scaffold and the laser, only those microparticles in the irradiated region are melted, enabling a spatial control over the release. By adjusting the photomask, different regions of the scaffold can be sequentially irradiated at designated times, realizing on-demand and sustained release of the biological effectors with spatiotemporal controls. In one demonstration, this method is used to accelerate the directional migration of NIH-3T3 fibroblasts along the uniaxial or radial direction of fiber alignment by controlling the release of epidermal growth factor. In another demonstration, the release of nerve growth factor is managed to significantly promote neurite outgrowth from PC12 cells.

Photosensitive Poly-l-lysine/Heparin Interpolyelectrolyte Complexes for Delivery of Genetic Drugs.

Photo-triggered release of biopharmaceutical drugs inside the cells is a challenging direction of modern science, which requires obtaining new polymeric systems. The interpolyelectrolyte complexes (IPECs) of poly-l-lysine with heparin capable of encapsulation of genetic constructions—such as model oligonucleotide, siRNA, and pDNA—were obtained. Poly-l-lysine to heparin ratios were optimized to provide the appropriate release kinetics of genetic material from the polyplex. In order to impart the obtained IPEC with photosensitive properties, the linker was synthesized as based on 4-brommethyl-3-nitrobenzoic acid. The conditions and kinetics of photosensitive linker destruction were carefully studied. The colloid particles of IPEC were modified with Cy3 probe and their cellular internalization was investigated by flow cytometry method. The efficacy of photosensitive IPECs as siRNA and pDNA delivery system was evaluated.

Abstract B78: NIR light-triggered photoimmunotherapy selectively destroy tumor cells in a clinically relevant desmoplastic heterocellular model

Abstract Near-infrared light-triggered photoimmunotherapy, utilizing the simultaneous presence of light and targeted conjugates, has emerged as a promising approach to impart selective cytotoxicity, especially for more complex nanoconstructs. Targeted immunoconjugates promise unique capabilities of combining multiple therapeutic agents within a single nanoplatform and promise great advances in revolutionizing cancer treatments, promoting selective drug localization and photo-triggered release of secondary agents to tumors cells that overexpress specific receptors. This study has utilized smart, liposome-based photoactivatable multiagent nanoconjugates, doped with a photoactivatable chromophore benzoporphyrin derivative (BPD) labeled with an active targeting ligand cetuximab to target the EGFR receptor (overexpressed on various cancer cells) for quantitative molecular imaging to measure the true in vivo receptor specificity and localized photodynamic therapy following NIR activation, that further can trigger the release of encapsulated payload to pancreatic cancer spheroids. In an in vivo model, these targeted nanoconstructs demonstrate substantial pancreatic tumor necrosis in a receptor-specific manner and modulate the collagen content in desmoplastic model of pancreatic ductal adenocarcinoma (MIA PaCa-2) in the presence of patient-derived pancreatic cancer-associated fibroblasts (PCAFs). This study establishes a tunable path towards optimally effective multiagent nanoconjugates for tumor-specific accumulation and targeted destruction of cancer cells in complex cancer model to enhance the therapeutic index of the administrated drugs. Citation Format: Shazia Bano. NIR light-triggered photoimmunotherapy selectively destroy tumor cells in a clinically relevant desmoplastic heterocellular model [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B78.

Photo-triggered release of doxorubicin from liposomes formulated by amphiphilic phthalocyanines for combination therapy to enhance antitumor efficacy.

Multidrug combination therapy based on drug delivery systems (DDSs) has great potential for cancer treatment. Stimuli-sensitive DDSs further enhance therapeutic efficacy by providing controllable drug delivery. Herein, the phospholipid compound DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) was used to construct thermosensitive liposomes to load the photosensitizer ZnPc(PEG)4 (zinc phthalocyanine substituted by tetraethylene glycol) for molecular imaging, and photodynamic and photothermal therapy, together with doxorubicin (DOX) for chemotherapy. Interestingly, ZnPc(PEG)4 as an amphipathic molecule was found to be important in the construction of the liposomes, and it provided liposomes with improved stability. The thus-obtained liposomes ZnPc(PEG)4:DOX@LiPOs were demonstrated to have enhanced ROS production capacity, heat generation properties and a photo-triggered doxorubicin release effect, and, in cellular experiments, increased cytotoxicity and apoptotic cell proportions, compared to ZnPc(PEG)4@LiPOs and DOX@LiPOs. ZnPc(PEG)4 loaded in lipid bilayers showed stronger intracellular ROS production ability compared to free ZnPc(PEG)4. In vivo studies indicated that ZnPc(PEG)4:DOX@LiPOs exhibited enhanced tumor accumulation, increased anti-cancer effects and reduced liver retention. These photo-triggered liposomes constructed by the photosensitizer ZnPc(PEG)4 can also be used to package other cargo for combined target tumor therapy and molecular imaging.

A dual signal amplification nanosensor based on SERS technology for detection of tumor-related DNA.

A dual signal amplification method based on surface-enhanced Raman scattering (SERS) is developed by photo-triggered release of SERS probes from mesoporous silica-coated Au nanorods (SiO2@Au) and the use of a specially-designed SERS substrate with an internal reference. Two metal carbonyl (metal-CO) labels (Os-SCO and Re-SCO) are proposed here as novel interference-free labels. Results demonstrate that tumor-related DNA can be quantitatively detected by this reliable and ultra-sensitive SERS platform.

Visible to NIR Light Photoactivation of Hydrogen Sulfide for Biological Targeting.

The synthesis and photochemical properties of H2S-releasing BODIPY thiocarbamate photocage scaffolds activatable by visible-to-NIR (up to 700 nm) light to release carbonyl sulfide (COS), which is transformed to H2S using either isolated or natural carbonic anhydrase, is reported. The excellent uncaging cross section and high H2S release yields in in vitro experiments, including live-cell imaging, suggest that these photocages can serve as a platform for the bio-orthogonal phototriggered release within the tissue-transparent window.

Light-Controlled Simultaneous "On Demand" Release of Cytotoxic Combinations for Bimodal Killing of Cancer Cells.

In this contribution, we report a novel entirely photocontrolled nanoplatform comprising a binary mixture of pluronic copolymers capable of self-assembling into core-shell micelles and co-entrapping two photoactivatable components: a benzoporphyrin photosensitizer for photodynamic therapy (PDT) and coumarin-photocaged chemotherapeutic agent Chlorambucil (CAB). The resulting supramolecular micellar assembly is about 30 nm in diameter with a polydispersity index <0.1, stable for more than 72 h, and exhibits excellent preservation of the photochemical properties of the two photoresponsive components, even though they are confined within the same host nanocarrier. Appropriate regulation of the relative concentrations of these components makes them capable of absorbing visible light in comparable amounts, leading to effective simultaneous photogeneration of singlet oxygen and photo-triggered release of CAB. This "on demand" release of cytotoxic combinations results in amplified anticancer activity against MCF-7 human breast adenocarcinoma cells.

Super-Resolution Monitoring of Mitochondrial Dynamics upon Time-Gated Photo-Triggered Release of Nitric Oxide.

Nitric oxide (NO) potentially plays a regulatory role in mitochondrial fusion and fission, which are vital to cell survival and implicated in health, disease, and aging. Molecular tools facilitating the study of the relationship between NO and mitochondrial dynamics are in need. We have recently developed a novel NO donor (NOD550). Upon photoactivation, NOD550 decomposes to release two NO molecules and a fluorophore. The NO release could be spatially mapped with subdiffraction resolution and with a temporal resolution of 10 s. Due to the preferential localization of NOD550 at mitochondria, morphology and dynamics of mitochondria could be monitored upon NO release from NOD550.