Design and Synthesis of a Calcium‐Sensitive Photocage

Photolabile protecting groups (or “photocages”) enable precise spatiotemporal control of chemical functionality and facilitate advanced biological experiments. Extant photocages exhibit a simple input–output relationship, however, where application of light elicits a photochemical reaction irrespective of the environment. Herein, we refine and extend the concept of photolabile groups, synthesizing the first Ca²⁺‐sensitive photocage. This system functions as a chemical coincidence detector, releasing small molecules only in the presence of both light and elevated [Ca²⁺]. Caging a fluorophore with this ion‐sensitive moiety yields an “ion integrator” that permanently marks cells undergoing high Ca²⁺ flux during an illumination‐defined time period. Our general design concept demonstrates a new class of light‐sensitive material for cellular imaging, sensing, and targeted molecular delivery.     

A Zinc(II) Photocage Based on a Decarboxylation Metal Ion Release Mechanism for Investigating Homeostasis and Biological Signaling

Metal ion signaling in biology has been studied extensively with ortho‐nitrobenzyl photocages; however, the low quantum yields and other optical properties are not ideal for these applications. We describe the synthesis and characterization of NTAdeCage, the first member in a new class of Zn²⁺ photocages that utilizes a light‐driven decarboxylation reaction in the metal ion release mechanism. NTAdeCage binds Zn²⁺ with sub‐pM affinity using a modified nitrilotriacetate chelator and exhibits an almost 6 order of magnitude decrease in metal binding affinity upon uncaging. In contrast to other metal ion photocages, NTAdeCage and the corresponding Zn²⁺ complex undergo efficient photolysis with quantum yields approaching 30 %. The ability of NTAdeCage to mediate the uptake of ⁶⁵Zn²⁺ by Xenopus laevis oocytes expressing hZIP4 demonstrates the viability of this photocaging strategy to execute biological assays.     

Photo‐ and pH‐Responsive Polycarbonate Block Copolymer Prodrug Nanomicelles for Controlled Release of Doxorubicin

Photo/pH dual‐responsive amphiphilic diblock copolymers with alkyne functionalized pendant o‐nitrobenzyl ester group are synthesized using poly(ethylene glycol) as a macroinitiator. The pendant alkynes are functionalized as aldehyde groups by the azide‐alkyne Huisgen cycloaddition. The anticancer drug doxorubicin (DOX) molecules are then covalently conjugated through acid‐sensitive Schiff‐base linkage. The resultant prodrug copolymers self‐assemble into nanomicelles in aqueous solution. The prodrug nanomicelles have a well‐defined morphology with an average size of 20–40 nm. The dual‐stimuli are applied individually or simultaneously to study the release behavior of DOX. Under UV light irradiation, nanomicelles are disassembled due to the ONB ester photocleavage. The light‐controlled DOX release behavior is demonstrated using fluorescence spectroscopy. Due to the pH‐sensitive imine linkage the DOX molecules are released rapidly from the nanomicelles at the acidic pH of 5.0, whereas only minimal amount of DOX molecules is released at the pH of 7.4. The DOX release rate is tunable by applying the dual‐stimuli simultaneously. In vitro studies against colon cancer cells demonstrate that the nanomicelles show the efficient cellular uptake and the intracellular DOX release, indicating that the newly designed copolymers with dual‐stimuli‐response have significant potential applications as a smart nanomedicine against cancer.     

Coumarin‐based Fluorescent Probes for Selectively Targeting and Imaging the Endoplasmic Reticulum in Mammalian Cells

Developing improved fluorescent probes for imaging the endoplasmic reticulum (ER) is necessary for structure‐activity studies of this dynamic organelle. Two coumarin‐based compounds with sulfonamide side groups were synthesized and characterized as ER‐targeting probes. Their selectivity to target the ER in HeLa and GM07373 mammalian cells was shown with co‐localization experiments using commercially available probes that localize in the ER, mitochondria, or lysozymes. The hydrophobicity of the coumarin‐based probes was comparable to known probes that partition into the ER membrane. Their cytotoxicity in mammalian cells was low with IC50 values that range from 205 to 252 μm . The fluorescent quantum yields of the coumarin‐based probes when excited with 400 nm light were 0.60, and they have a much narrower emission spectrum (from 435 to 525 nm in methanol) than that of the only commercially available ER probe that is exited with 400 nm light (ER‐Tracker? Blue‐White DPX). Thus, the coumarin‐based probes are more useful for multicolor imaging with yellow and red emitting fluorophores. In addition to the above benefits, ER labeling was achieved with the coumarin‐based probes in both live cells and fixed cells, revealing their versatility for a wide range of cellular imaging applications.     

Prometabolites of 5‐Diphospho‐myo‐inositol Pentakisphosphate

Diphospho‐myo‐inositol phosphates (PP‐InsP_y) are an important class of cellular messengers. Thus far, no method for the transport of PP‐InsP_y into living cells is available. Owing to their high negative charge density, PP‐InsP_y will not cross the cell membrane. A strategy to circumvent this issue involves the generation of precursors in which the negative charges are masked with biolabile groups. A PP‐InsP_y prometabolite would require twelve to thirteen biolabile groups, which need to be cleaved by cellular enzymes to release the parent molecules. Such densely modified prometabolites of phosphate esters and anhydrides have never been reported to date. This study discloses the synthesis of such agents and an analysis of their metabolism in tissue homogenates by gel electrophoresis. The acetoxybenzyl‐protected system is capable of releasing 5‐PP‐InsP₅ in mammalian cell/tissue homogenates within a few minutes and can be used to release 5‐PP‐InsP₅ inside cells. These molecules will serve as a platform for the development of fundamental tools required to study PP‐InsP_y physiology.     

Near‐Infrared Light Triggered‐Release in Deep Brain Regions Using Ultra‐photosensitive Nanovesicles

Remote and minimally‐invasive modulation of biological systems with light has transformed modern biology and neuroscience. However, light absorption and scattering significantly prevents penetration to deep brain regions. Herein, we describe the use of gold‐coated mechanoresponsive nanovesicles, which consist of liposomes made from the artificial phospholipid Rad‐PC‐Rad as a tool for the delivery of bioactive molecules into brain tissue. Near‐infrared picosecond laser pulses activated the gold‐coating on the surface of nanovesicles, creating nanomechanical stress and leading to near‐complete vesicle cargo release in sub‐seconds. Compared to natural phospholipid liposomes, the photo‐release was possible at 40 times lower laser energy. This high photosensitivity enables photorelease of molecules down to a depth of 4 mm in mouse brain. This promising tool provides a versatile platform to optically release functional molecules to modulate brain circuits.     

Resonance Raman Spectral Imaging of Intracellular Uptake of β‐Carotene Loaded Poly(D,L‐lactide‐co‐glycolide) Nanoparticles

The use of nanoparticles for drug delivery has been drawing considerable attention in pharmaceutical research. With increasing diversity and potential of various carrier systems, it is important to study the impact of nanocarriers on sub‐cellular metabolic processes and organelles, since the delivery of a drug usually involves intra‐cellular internalization. Herein, we employ Raman microscopy as a non‐invasive method for cellular and sub‐cellular imaging, to monitor the uptake and translocation patterns of particles based on poly(D,L‐lactide‐co‐glycolide) over time. As the technique detects inherent signals from the molecules of interest, it does not rely on external labels or dyes, which is an advantage over fluorescence labeling. For this purpose, the particles were loaded with β‐carotene. The conjugated π‐system of the molecule has a large Raman scattering cross‐section and gives rise to resonance Raman effects, which can enhance the sensitivity by orders of magnitude. β‐Carotene as a provitamin is not soluble in water and is thus usually of low bioavailability, which is enhanced by encapsulation into the nanoparticles.     

A Cyanine Photooxidation/β‐Elimination Sequence Enables Near‐infrared Uncaging of Aryl Amine Payloads

Uncaging strategies that use near‐infrared wavelengths can enable the highly targeted delivery of biomolecules in complex settings. Many methods, including an approach we developed using cyanine photooxidation, are limited to phenol‐containing payloads. Given the critical role of amines in diverse biological processes, we sought to use cyanine photooxidation to initiate the release of aryl amines. Heptamethine cyanines substituted with an aryl amine at the C4′ position undergo only inefficient release, likely due electronic factors. We then pursued the hypothesis that the carbonyl products derived from cyanine photooxidation could undergo efficient β‐elimination. After examining both symmetrical and unsymmetrical scaffolds, we identify a merocyanine substituted with indolenine and coumarin heterocycles that undergoes efficient photooxidation and aniline uncaging. In total, these studies provide a new scheme—cyanine photooxidation followed by β‐elimination—through which to design photocages with efficient uncaging properties.     

Chemo‐Enzymatic Synthesis of Position‐Specifically Modified RNA for Biophysical Studies including Light Control and NMR Spectroscopy

The investigation of non‐coding RNAs requires RNAs containing modifications at every possible position within the oligonucleotide. Here, we present the chemo‐enzymatic RNA synthesis containing photoactivatable or ¹³C,¹⁵N‐labelled nucleosides. All four ribonucleotides containing ortho‐nitrophenylethyl (NPE) photocages, photoswitchable azobenzene C‐nucleotides and ¹³C,¹⁵N‐labelled nucleotides were incorporated position‐specifically in high yields. We applied this approach for the synthesis of light‐inducible 2′dG‐sensing riboswitch variants and detected ligand‐induced structural reorganization upon irradiation by NMR spectroscopy. This chemo‐enzymatic method opens the possibility to incorporate a wide range of modifications at any desired position of RNAs of any lengths beyond the limits of solid‐phase synthesis.     

Water‐Mediated Recognition of Simple Alkyl Chains by Heart‐Type Fatty‐Acid‐Binding Protein

Long‐chain fatty acids (FAs) with low water solubility require fatty‐acid‐binding proteins (FABPs) to transport them from cytoplasm to the mitochondria for energy production. However, the precise mechanism by which these proteins recognize the various lengths of simple alkyl chains of FAs with similar high affinity remains unknown. To address this question, we employed a newly developed calorimetric method for comprehensively evaluating the affinity of FAs, sub‐Angstrom X‐ray crystallography to accurately determine their 3D structure, and energy calculations of the coexisting water molecules using the computer program WaterMap. Our results clearly showed that the heart‐type FABP (FABP3) preferentially incorporates a U‐shaped FA of C10–C18 using a lipid‐compatible water cluster, and excludes longer FAs using a chain‐length‐limiting water cluster. These mechanisms could help us gain a general understanding of how proteins recognize diverse lipids with different chain lengths.     

Synthesis and structures of photoactive manganese–carbonyl complexes derived from 2‐(pyridin‐2‐yl)‐1,3‐benzothiazole and 2‐(quinolin‐2‐yl)‐1,3‐benzothiazole

PhotoCORMs (photo‐active CO‐releasing molecules) have emerged as a class of CO donors where the CO release process can be triggered upon illumination with light of appropriate wavelength. We have recently reported an Mn‐based photoCORM, namely [MnBr(pbt)(CO)₃] [pbt is 2‐(pyridin‐2‐yl)‐1,3‐benzothiazole], where the CO release event can be tracked within cellular milieu by virtue of the emergence of strong blue fluorescence. In pursuit of developing more such trackable photoCORMs, we report herein the syntheses and structural characterization of two Mn^I–carbonyl complexes, namely fac‐tricarbonylchlorido[2‐(pyridin‐2‐yl)‐1,3‐benzothiazole‐κ²N ,N ′]manganese(I), [MnCl(C₁₂H₈N₂S)(CO)₃], (1), and fac‐tricarbonylchlorido[2‐(quinolin‐2‐yl)‐1,3‐benzothiazole‐κ²N ,N ′]manganese(I), [MnCl(C₁₆H₁₀N₂S)(CO)₃], (2). In both complexes, the Mn^I center resides in a distorted octahedral coordination environment. Weak intermolecular C—H…Cl contacts in complex (1) and Cl…S contacts in complex (2) consolidate their extended structures. These complexes also exhibit CO release upon exposure to low‐power broadband visible light. The apparent CO release rates for the two complexes have been measured to compare their CO donating capacity. The fluorogenic 2‐(pyridin‐2‐yl)‐1,3‐benzothiazole and 2‐(quinolin‐2‐yl)‐1,3‐benzothiazole ligands provide a convenient way to track the CO release event through the `turn‐ON' fluorescence which results upon de‐ligation of the ligands from their respective metal centers following CO photorelease.     

Novel Cyclometalated Fe(II) Complex with NCN Pincer and BODIPY‐Appended 4'‐Ethynyl‐2,2':6',2”‐terpyridine as Mitochondria‐Targeted Photodynamic Anticancer Agents

BODIPY (boron dipyrromethene) derivatives and iron complexes are two types of functional compounds that have found wide applications in the fields of biology and medicine. The new class of cyclometalated Fe(II) complex with NCN pincer and meso‐phenyl‐4'‐ethynyl‐2,2':6',2”‐terpyridine BODIPY ligands of formula [Fe(L)(tpy‐BODIPY)] , 1, in which HL:5‐methoxy‐1,3‐bis (1‐methyl‐1H‐benzo[d]imidazol‐2‐yl)benzene, tpy‐BODIPY: 8‐(4‐phenyl‐4'‐ethynyl‐2,2':6',2”‐terpyridine) BODIPY, has been synthesized and studied as mitochondria‐targeted photodynamic therapy (PDT). Complex 1 showed photocytotoxicity in HeLa cells at 500 nm with low dark toxicity. The phototoxicity of complex 1 on the nontumorigenic MRC‐5 cell line showed the same trend observed for HeLa cells, that is moderately photocytotoxic against the nontumorigenic MRC‐5 cell line (IC₅₀ = 36.21 μM). Moreover, complex 1 selectively localizes into mitochondria of the HeLa cells. The photophysical properties, cellular uptake, reactive oxygen species (ROS) generation, and cellular apoptosis of complex 1 have also been studied.Overall, the new Fe(II) complex with BODIPY moiety is significantly photocytotoxic in HeLa cells when irradiated with visible light of 500 nm giving as mitochondria targeting. Therefore, we present cyclometalated Fe(II) pincer complex induced mitochondria‐targeted PDT involving the BODIPY moiety that develops persuasively designed photoactivatable Fe(II) complexes.     

Fluorescent Triazole Urea Activity‐Based Probes for the Single‐Cell Phenotypic Characterization of Staphylococcus aureus

Phenotypically distinct cellular (sub)populations are clinically relevant for the virulence and antibiotic resistance of a bacterial pathogen, but functionally different cells are usually indistinguishable from each other. Herein, we introduce fluorescent activity‐based probes as chemical tools for the single‐cell phenotypic characterization of enzyme activity levels in Staphylococcus aureus. We screened a 1,2,3‐triazole urea library to identify selective inhibitors of fluorophosphonate‐binding serine hydrolases and lipases in S. aureus and synthesized target‐selective activity‐based probes. Molecular imaging and activity‐based protein profiling studies with these probes revealed a dynamic network within this enzyme family involving compensatory regulation of specific family members and exposed single‐cell phenotypic heterogeneity. We propose the labeling of enzymatic activities by chemical probes as a generalizable method for the phenotyping of bacterial cells at the population and single‐cell level.     

Light Might Directly Affect Retinal Ganglion Cell Mitochondria to Potentially Influence Function†

Visible light (360–760 nm) entering the eye impinges on the many ganglion cell mitochondria in the non‐myelinated part of their axons. The same light also disrupts isolated mitochondrial function in vitro and kills cells in culture with the blue light component being particularly lethal whereas red light has little effect. Significantly, a defined light insult only affects the survival of fibroblasts in vitro that contain functional mitochondria supporting the view that mitochondrial photosensitizers are influenced by light. Moreover, a blue light insult to cells in culture causes a change in mitochondrial structure and membrane potential and results in a release of cytochrome c. Blue light also causes an alteration in mitochondria located components of the OXPHOS (oxidative phosphorylation system). Complexes III and IV as well as complex V are significantly upregulated whereas complexes I and II are slightly but significantly up‐ and downregulated, respectively. Also, blue light causes Dexras1 and reactive oxygen species to be upregulated and for mitochondrial located apoptosis‐inducing factor to be activated. A blue light detrimental insult to cells in culture does not involve the activation of caspases but is known to be attenuated by necrostatin‐1, typical of a death mechanism named necroptosis.     

Cellular pharmacokinetics and pharmacodynamics mechanisms of ginkgo diterpene lactone and its modulation of P‐glycoprotein expression in human SH‐SY5Y cells

Ginkgo diterpene lactone (GDL) is the raw material for ginkgo diterpene lactone meglumine injection, which is used for treating cerebral ischemia. The aims of this study were to explore the cellular pharmacokinetics of GDL in whole cells and subcellular fractions, and detect cellular pharmacodynamics on the human SH‐SY5Y cells induced by oxygen–glucose deprivation and reoxygenation (OGD/R). Firstly, a simple, sensitive and reliable liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for assessing the amount of ginkgolide A (GA), B (GB) and K (GK) in cellular/subcellular samples. Then, phosphatidylserine and mitochondria membrane potential were assayed to evaluate the extent of apoptosis effect. The study showed that the cellular/subcellular accumulation of GA and GB were increased in a concentration‐dependent manner; the levels of GA and GB in cytosol were the highest among these subcellular organelles. Meanwhile, GDL also attenuated the OGD/R‐induced increases in the percentage of apoptotic and mitochondria membrane potential. In addition, verapamil increased the rate and amount of GA and GB entering cellular/subcellular compartments through inhibition of P‐glycoprotein activity, and promoted the protective effect of GDL. The present study reports the cellular pharmacokinetics profiles of GA and GB in normal and OGD/R‐induced SH‐SY5Y cells in vitro for the first time, which provided valuable information for clinical safety application.     

Mitochondria‐Targeted Cancer Therapy Using a Light‐Up Probe with Aggregation‐Induced‐Emission Characteristics

Subcellular organelle‐specific reagents for simultaneous tumor targeting, imaging, and treatment are of enormous interest in cancer therapy. Herein, we present a mitochondria‐targeting probe (AIE‐mito‐TPP) by conjugating a triphenylphosphine (TPP) with a fluorogen which can undergo aggregation‐induced emission (AIE). Owing to the more negative mitochondrial membrane potential of cancer cells than normal cells, the AIE‐mito‐TPP probe can selectively accumulate in cancer‐cell mitochondria and light up its fluorescence. More importantly, the probe exhibits selective cytotoxicity for studied cancer cells over normal cells. The high potency of AIE‐mito‐TPP correlates with its strong ability to aggregate in mitochondria, which can efficiently decrease the mitochondria membrane potential and increase the level of intracellular reactive oxygen species (ROS) in cancer cells. The mitochondrial light‐up probe provides a unique strategy for potential image‐guided therapy of cancer cells.     

Optogenetic Apoptosis: Light‐Triggered Cell Death

An optogenetic Bax has been designed that facilitates light‐induced apoptosis. We demonstrate that mitochondrial recruitment of a genetically encoded light‐responsive Bax results in the release of mitochondrial proteins, downstream caspase‐3 cleavage, changes in cellular morphology, and ultimately cell death. Mutagenesis of a key phosphorylatable residue or modification of the C‐terminus mitigates background (dark) levels of apoptosis that result from Bax overexpression. The mechanism of optogenetic Bax‐mediated apoptosis was explored using a series of small molecules known to interfere with various steps in programmed cell death. Optogenetic Bax appears to form a mitochondrial apoptosis‐induced channel analogous to that of endogenous Bax.     

Molecule‐Membrane Interactions in Biological Cells Studied with Second Harmonic Light Scattering

The nonlinear optical phenomenon second harmonic light scattering (SHS) can be used for detecting molecules at the membrane surfaces of living biological cells. Over the last decade, SHS has been developed for quantitatively monitoring the adsorption and transport of small and medium size molecules (both neutral and ionic) across membranes in living cells. SHS can be operated with both time and spatial resolution and is even capable of isolating molecule‐membrane interactions at specific membrane surfaces in multi‐membrane cells, such as bacteria. In this review, we discuss select examples from our lab employing time‐resolved SHS to study real‐time molecular interactions at the plasma membranes of biological cells. We first demonstrate the utility of this method for determining the transport rates at each membrane/interface in a Gram‐negative bacterial cell. Next, we show how SHS can be used to characterize the molecular mechanism of the century old Gram stain protocol for classifying bacteria. Additionally, we examine how membrane structures and molecular charge and polarity affect adsorption and transport, as well as how antimicrobial compounds alter bacteria membrane permeability. Finally, we discuss adaptation of SHS as an imaging modality to quantify molecular adsorption and transport in sub‐cellular regions of individual living cells.     

Characterization of the Cytochrome c Membrane‐Binding Site Using Cardiolipin‐Containing Bicelles with NMR

Cytochrome (cyt) c transports electrons from Complex III to Complex IV in mitochondria. Cyt c is ordinarily anchored to the mitochondrial membrane through interaction with cardiolipin (CL), however its release into the cytosol initiates apoptosis. The cyt c interaction site with CL‐containing bicelles was characterized by NMR spectroscopy. Chemical shift perturbations in cyt c signals upon interaction with bicelles revealed that a relatively wide region, which includes the A‐site, the CXXCH motif, and the N‐ and C‐terminal helices, and contains multiple Lys residues, interacts cooperatively with CL. The specific cyt c–CL interaction increased with increasing CL molecules in the bicelles. The location of the cyt c interaction site for CL was similar to those for Complex III and Complex IV, thus indicating that cyt c recognizes lipids and partner proteins in a similar way. In addition to elucidating the cyt c membrane‐binding site, these results provide insight into the dynamic aspect of cyt c interactions in mitochondria.     

Gold Nanorods with Phase‐Changing Polymer Corona for Remotely Near‐Infrared‐Triggered Drug Release

Herein, we report a new drug‐delivery system (DDS) that is comprised of a near‐infrared (NIR)‐light‐sensitive gold‐nanorod (GNR) core and a phase‐changing poly(ε‐caprolactone)‐b‐poly(ethylene glycol) polymer corona (GNR@PCL‐b‐PEG). The underlying mechanism of the drug‐loading and triggered‐release behaviors involves the entrapment of drug payloads among the PCL crystallites and a heat‐induced phase change, respectively. A low premature release of the pre‐loaded doxorubicin was observed in PBS buffer (pH 7.4) at 37 °C (<10 % of the entire payload after 48 h). However, release could be activated within 30 min by conventional heating at 50 °C, above the T_m of the crystalline PCL domain (43.5 °C), with about 60 % release over the subsequent 42 h at 37 °C. The NIR‐induced heating of an aqueous suspension of GNR@PCL‐b‐PEG under NIR irradiation (802 nm) was investigated in terms of the irradiation period, power, and concentration‐dependent heating behavior, as well as the NIR‐induced shape‐transformation of the GNR cores. Remotely NIR‐triggered release was also explored upon NIR irradiation for 30 min and about 70 % release was achieved in the following 42 h at 37 °C, with a mild warming (<4 °C) of the surroundings. The cytotoxicity of GNR@PCL‐b‐PEG against the mouse fibroblastic‐like L929 cell‐line was assessed by MTS assay and good compatibility was confirmed with a cell viability of over 90 % after incubation for 72 h. The cellular uptake of GNR@PCL‐b‐PEG by melanoma MEL‐5 cells was also confirmed, with an averaged uptake of 1250(±110) particles cell^?1 after incubation for 12 h (50 μg mL^?1). This GNR@PCL‐b‐PEG DDS is aimed at addressing the different requirements for therapeutic treatments and is envisaged to provide new insights into DDS targeting for remotely triggered release by NIR activation.