Cyclic Disulfide Liposomes for Membrane Functionalization and Cellular Delivery

Liposomes are effective therapeutic delivery nanocarriers due to their ability to encapsulate and enhance the pharmacokinetic properties of a wide range of therapeutics. Two primary areas in which improvement is needed for liposomal drug delivery is to enhance the ability to infiltrate cells and to facilitate derivatization of the liposome surface. Herein, we report a liposome platform incorporating a cyclic disulfide lipid (CDL) for the dual purpose of enhancing cell entry and functionalizing the liposome membrane through thiol-disulfide exchange. In order to accomplish this, CDL-1 and CDL-2 , composed of lipoic acid (LA) or asparagusic acid (AA) appended to a lipid scaffold, were designed and synthesized. A fluorescence-based microplate immobilization assay was implemented to show that these compounds enable convenient membrane decoration through reaction with thiol-functionalized small molecules. Additionally, fluorescence microscopy experiments indicated dramatic enhancements in cellular delivery when CDLs were incorporated within liposomes. These results demonstrate that multifunctional CDLs serve as an exciting liposome system for surface decoration and enhanced cellular delivery.     

Dramatic influence of the orientation of linker between hydrophilic and hydrophobic lipid moiety in liposomal gene delivery

A number of prior studies have demonstrated that the DNA-binding and gene transfection efficacies of cationic amphiphiles crucially depend on their various structural parameters including hydrophobic chain lengths, headgroup functionalities, and the nature of the linker-functionality used in tethering the polar headgroup and hydrophobic tails. However, to date addressing the issue of linker orientation remains unexplored in liposomal gene delivery. Toward probing the influence of linker orientation in cationic lipid mediated gene delivery, we have designed and synthesized two structurally isomeric remarkably similar cationic amphiphiles 1 and 2 bearing the same hydrophobic tails and the same polar headgroups connected by the same ester linker group. The only structural difference between the cationic amphiphiles 1 and 2 is the orientation of their linker ester functionality. While lipid 1 showed high gene transfer efficacies in multiple cultured animal cells, lipid 2 was essentially transfection incompetent. Findings in both transmission electron microscopic and dynamic laser light scattering studies revealed no significant size difference between the lipoplexes of lipids 1 and 2. Findings in confocal microscopic and fluorescence resonance energy transfer (FRET) experiments, taken together, support the notion that the remarkably higher gene transfer efficacies of lipid 1 compared to those of lipid 2 presumably originate from higher biomembrane fusogenicity of lipid 1 liposomes. Differential scanning calorimetry (DSC) and fluorescence anisotropy studies revealed a significantly higher gel-to-liquid crystalline temperature for the lipid 2 liposomes than that for lipid 1 liposomes. Findings in the dye entrapment experiment were also consistent with the higher rigidity of lipid 2/cholesterol (1:1 mole ratio) liposomes. Thus, the higher biomembrane fusibility of lipid 1 liposomes than that of lipid 2 liposomes presumably originates from the more rigid nature of lipid 2 cationic liposomes. Taken together, the present findings demonstrate for the first time that even as minor a structural variation as linker orientation reversal in cationic amphiphiles can profoundly influence DNA-binding characteristics, membrane rigidity, membrane fusibility, cellular uptake, and consequently gene delivery efficacies of cationic liposomes.     

Flow cytometric characterization of interactions between U-937 human macrophages and positively charged catanionic vesicles

Catanionic vesicles are considered a potential alternative to liposomes for drug delivery systems because of their greater stability and lower cost. Before using catanionic vesicles in vivo, their interactions with macrophages must be fully understood because they are primarily removed from circulation by the macrophages of the mononuclear phagocyte system. Using flow cytometry, we examined the intracellular responses-reactive oxygen species (ROS) content, mitochondrial membrane potential, cell size and complexity, and cell cycle profiles-in U-937 human macrophages treated with positively charged catanionic vesicles. Kinetic hydrogen peroxide production initially increased at lower concentrations (4-10nM) but declined at higher concentrations (40 nM and 80 nM) over the entire incubation period. Superoxide content generation, however, increased over the entire concentration range and incubation period. Catanionic vesicles decreased mitochondrial membrane potential for every concentration after 4h of incubation but caused a significant fluctuation in mitochondrial membrane potential at 6h. After 6h of incubation, catanionic vesicles produced more changes in cell size and complexity than after 4h. The increase in the subG1 population of cells treated with catanionic vesicles at higher doses indicated that apoptosis progressed. Positively charged catanionic vesicles induced different activated patterns of ROS generation and changes in mitochondrial membrane potential than did cationic liposomes. The nature of the interactions between macrophages and catanionic vesicles is of great importance for the design of safer and more effective delivery systems for macrophages. Our findings contribute to a better understanding of the molecular action of catanionic vesicles in the cellular system.     

The role of Fe3+ on Fe2+-dependent lipid peroxidation in phospholipid liposomes

Fe2+-dependent lipid peroxidation in phosphatidylcholine (PC) liposomes, assessed by thiobarbituric acid-reactive substances (TBARS) production, was stimulated in the presence of Fe3+ in a concentration-dependent manner. The rates of nitroblue tetrazolium (NBT) reduction and Fe2+ oxidation (Fe2+ disappearance and Fe3+ formation) were also enhanced by the addition of Fe3+ to the reaction mixture, and there is a good linear relationship between these parameters. These results suggest that the facilitation of reactive oxygen species (ROS) production via Fe2+ oxidation is closely related to the onset of the stimulatory effect of Fe3+ on Fe2+-dependent lipid peroxidation. On the other hand, results using the liposomes containing various concentrations of endogenous lipid hydroperoxides (LOOH) indicated that endogenous LOOH is not directly involved in the onset of the Fe3+ stimulatory effect on Fe2+-dependent TBARS production and ROS production. This hypothesis was further confirmed by the evidence that Fe2+-dependent ROS production and Fe2+ oxidation of dipalmitoylphosphatidylcholine liposomes were also stimulated by the addition of Fe3+. The results with several antioxidants and radical scavengers suggested that ROS related to Fe2+-dependent lipid peroxidation and its stimulation by Fe3+ are ferrous-oxygen complexes rather than superoxide anion, hydrogen peroxide and hydroxyl radicals. Based on these results, we proposed a possible mechanism for the onset of the Fe3+ stimulation in Fe2+-dependent lipid peroxidation.     

New caged coumarin fluorophores with extraordinary uncaging cross sections suitable for biological imaging applications

Photocaged fluorescent molecules are important research tools for tracking molecular dynamics with high spatiotemporal resolution in biological systems. We have designed and synthesized a new class of caged coumarin fluorophores. These coumarin cages displayed more than 200-fold fluorescence enhancement after UV photolysis. Remarkably, the uncaging cross section of a 1-(2-nitrophenyl)ethyl (NPE)-caged coumarin is 6600 at wavelength of 365 nm, about 2 orders of magnitude higher than previously described caged fluorophores. Product analysis of the photolytic reaction showed clean conversion of NPE-caged coumarin to 2-nitrosoacetophenone and the parent coumarin, suggesting that the mechanism of the photolysis follows the known photochemical reaction pathway of the 2-nitrobenzyl group. We have also measured the two-photon uncaging cross sections of NPE-caged coumarins 2a and 5 at 740 nm to be near 1 Goeppert-Mayer (GM). The mechanistic study, together with the two-photon uncaging data, suggested that the coumarin moiety serves as an antenna to enhance the light harvesting efficiency of the coumarin cage and that the photonic energy absorbed by coumarin was utilized efficiently to photolyze the NPE group. Future explorations of this type of "substrate-assisted photolysis" may yield other cages of high uncaging cross sections. For cellular imaging applications, we prepared a cell permeable and caged coumarin fluorophore, NPE-HCCC2/AM (10), which can be loaded into fully intact cells to high concentrations. Initial tests of this probe in a number of cultured mammalian cells showed desired properties for the in vivo imaging applications. The combined advantages of robust fluorescence contrast enhancement, remarkably high uncaging cross sections, noninvasive cellular delivery, and flexible chemistry for bioconjugations should generate broad applications of these caged coumarins in biochemical and biological research.     

Fluorescence studies on radiation oxidative damage to membranes with implications to cellular radiosensitivity

Radiation oxidative damage to plasma membrane and its consequences to cellular radiosensitivity have received increasing attention in the past few years. This review gives a brief account of radiation oxidative damage in model and cellular membranes with particular emphasis on results from our laboratory. Fluorescence and ESR spin probes have been employed to investigate the structural and functional alterations in membranes after y-irradiation. Changes in the lipid bilayer in irradiated unilamellar liposomes prepared from egg yolk lecithin (EYL) were measured by using diphenylhexatriene (DPH) as a probe. The observed increase in DPH polarization and decrease in fluorescence intensity after γ-irradiation of liposomes imply radiation-induced decrease in bilayer fluidity. Inclusion of cholesterol in liposome was found to protect lipids against radiation damage, possibly by modulation of bilayer organization e.g. lipid packing. Measurements on dipalmitoyl phosphatidylcholine (DPPC) liposomes loaded with 6-carboxyfluorescein (CF) showed radiation dose-dependent release of the probe indicating radiation-induced increased permeability. Changes in plasma membrane permeability of thymocytes were monitored by fluorescein diacetate (FDA) and induced intracellular reactive oxygen species (ROS) were determined by 2,7-dichlorodihydro fluorescein diacetate (DCH-FDA). Results suggest a correlation between ROS generation and membrane permeability changes induced by radiation within therapeutic doses (0-10 Gy). It is concluded that increase in membrane permeability was the result of ROS-mediated oxidative reactions, which might trigger processes leading to apoptotic cell death after radiation exposure.     

Efficient delivery of streptavidin to mammalian cells: clathrin-mediated endocytosis regulated by a synthetic ligand

The efficient delivery of macromolecules to living cells presents a formidable challenge to the development of effective macromolecular therapeutics and cellular probes. We describe herein a novel synthetic ligand termed "Streptaphage" that enables efficient cellular uptake of the bacterial protein streptavidin by promoting noncovalent interactions with cholesterol and sphingolipid-rich lipid raft subdomains of cellular plasma membranes. The Streptaphage ligand comprises an N-alkyl derivative of 3 beta-cholesterylamine linked to the carboxylate of biotin through an 11-atom tether. Molecular recognition between streptavidin and this membrane-bound ligand promotes clathrin-mediated endocytosis, which renders streptavidin partially intracellular within 10 min and completely internalized within 4 h of protein addition. Analysis of protein uptake in Jurkat lymphocytes by epifluorescence microscopy and flow cytometry revealed intracellular fluorescence enhancements of over 300-fold (10 microM ligand) with >99% efficiency and low toxicity. Other mammalian cell lines including THP-1 macrophages, MCF-7 breast cancer cells, and CHO cells were similarly affected. Structurally related ligands bearing a shorter linker or substituting the protonated steroidal amine with an isosteric amide were ineffective molecular transporters. Confocal fluorescence microscopy revealed that Streptaphage-induced uptake of streptavidin functionally mimics the initial cellular penetration steps of Cholera toxin, which undergoes clathrin-mediated endocytosis upon binding to the lipid raft-associated natural product ganglioside GM1. The synthetic ligand described herein represents a designed cell surface receptor capable of targeting streptavidin conjugates into diverse mammalian cells by hijacking the molecular machinery used to organize cellular membranes. This technology has potential applications in DNA delivery, tumor therapy, and stimulation of immune responses.     

Targeted drug delivery utilizing protein-like molecular architecture

Nanotechnology-based drug delivery systems (nanoDDSs) have seen recent popularity due to their favorable physical, chemical, and biological properties, and great efforts have been made to target nanoDDSs to specific cellular receptors. CD44/chondroitin sulfate proteoglycan (CSPG) is among the receptors overexpressed in metastatic melanoma, and the sequence to which it binds within the type IV collagen triple-helix has been identified. A triple-helical "peptide-amphiphile" (alpha1(IV)1263-1277 PA), which binds CD44/CSPG, has been constructed and incorporated into liposomes of differing lipid compositions. Liposomes containing distearoyl phosphatidylcholine (DSPC) as the major bilayer component, in combination with distearoyl phosphatidylglycerol (DSPG) and cholesterol, were more stable than analogous liposomes containing dipalmitoyl phosphatidylcholine (DPPC) instead of DSPC. When dilauroyl phosphatidylcholine (DLPC):DSPG:cholesterol liposomes were prepared, monotectic behavior was observed. The presence of the alpha1(IV)1263-1277 PA conferred greater stability to the DPPC liposomal systems and did not affect the stability of the DSPC liposomes. A positive correlation was observed for cellular fluorophore delivery by the alpha1(IV)1263-1277 PA liposomes and CD44/CSPG receptor content in metastatic melanoma and fibroblast cell lines. Conversely, nontargeted liposomes delivered minimal fluorophore to these cells regardless of the CD44/CSPG receptor content. When metastatic melanoma cells and fibroblasts were treated with exogeneous alpha1(IV)1263-1277, prior to incubation with alpha1(IV)1263-1277 PA liposomes, to potentially disrupt receptor/liposome interactions, a dose-dependent decrease in the amount of fluorophore delivered was observed. Overall, our results suggest that PA-targeted liposomes can be constructed and rationally fine-tuned for drug delivery applications based on lipid composition. The selectivity of alpha1(IV)1263-1277 PA liposomes for CD44/CSPG-containing cells represents a targeted-nanoDDS with potential for further development and application.     

Antioxidant Effect of Human Selenium-containing Single-chain Fv in Rat Cardiac Myocytes

Reactive oxygen species(ROS) plays a key role in human heart diseases.Glutathione peroxidase(GPX) functions as an antioxidant as it catalyzes the reduction of hydroperoxide.In order to investigate the antioxidant effect of human selenium-containing single-chain Fv(Se-scFv-B3),a new mimic of GPX,a model system of hydrogen peroxide(H2O2)-induced rat cardiac myocyte damage was established.The cardiac myocyte damage was characterized in terms of cell viability,lipid peroxidation,cell membrane integrity,and intracellular H2O2 level.The Se-scFv-B3 significantly reduced H2O2-induced cell damage as shown by the increase of cell viability,the decline of malondialdehyde(MDA) production,lactate dehydrogenase(LDH) release,and intracellular H2O2 level.So Se-scFvB3 may have a great potential in the treatment of human heart diseases induced by ROS.     

Synthesis and preliminary investigations of the siRNA delivery potential of novel, single-chain rigid cationic carotenoid lipids

The success of nucleic acid delivery requires the development of safe and efficient delivery vectors that overcome cellular barriers for effective transport. Herein we describe the synthesis of a series of novel, single-chain rigid cationic carotenoid lipids and a study of their preliminary in vitro siRNA delivery effectiveness and cellular toxicity. The efficiency of siRNA delivery by the single-chain lipid series was compared with that of known cationic lipid vectors, 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]-cholesterol (DC-Chol) and 1,2-dimyristoyl-sn-glyceryl-3-phosphoethanolamine (EPC) as positive controls. All cationic lipids (controls and single-chain lipids) were co-formulated into liposomes with the neutral co-lipid, 1,2-dioleolyl-sn-glycerol-3-phosphoethanolamine (DOPE). Cationic lipid-siRNA complexes of varying (+/-) molar charge ratios were formulated for delivery into HR5-CL11 cells. Of the five single-chain carotenoid lipids investigated, lipids 1, 2, 3 and 5 displayed significant knockdown efficiency with HR5-CL11 cells. In addition, lipid 1 exhibited the lowest levels of cytotoxicity with cell viability greater than 80% at all (+/-) molar charge ratios studied. This novel, single-chain rigid carotenoid-based cationic lipid represents a new class of transfection vector with excellent cell tolerance, accompanied with encouraging siRNA delivery efficiency.     

Triggered Liposomal Release through a Synthetic Phosphatidylcholine Analogue Bearing a Photocleavable Moiety Embedded within the sn‐2 Acyl Chain

Liposomes represent promising carriers for drug delivery applications. To maximize this potential, there has been significant interest in developing liposomal systems encapsulating molecular cargo that are highly stable until their contents are released remotely in a controlled manner. Herein, we describe the design, synthesis, and analysis of a photocleavable analogue of the ubiquitous lipid phosphoatidylcholine (PC) for the development of highly stable and controllable photodisruptable membranes. Our strategy was to develop a lipid that closely mimics the structure of PC to optimize favorable properties including biocompatibility and stability of subsequent liposomes when mixed with lipids possessing a broad range of physicochemical properties. Thus, NB‐PC was designed, which contains a photocleavable 2‐nitrobenzyl group embedded within the acyl chain at the sn‐2 position. Following the synthesis of NB‐PC , liposome disruption efficacy was evaluated through photolysis studies involving the detection of nile red release. Studies performed using a range of liposomes with different percentages of NB‐PC , PC, phosphatidylethanolamine (PE), cholesterol, and polyethylene glycol‐PE (PEG‐PE) demonstrated minimal background release in controls, release efficacies that correlate directly with the amount of NB‐PC incorporation, and that release is only minimally impacted by the inclusion of the lipids PE and cholesterol that possess disparate properties. These results demonstrate that the NB‐PC system is a highly stable, flexible, and tunable system for photoinitiated release from liposomal systems.     

Development of a highly sensitive fluorescence probe for hydrogen peroxide

Hydrogen peroxide is believed to play a role in cellular signal transduction by reversible oxidation of proteins. Here, we report the design and synthesis of a novel fluorescence probe for hydrogen peroxide, utilizing a photoinduced electron transfer strategy based on benzil chemistry to control the fluorescence. The practical value of this highly sensitive and selective fluorescence probe, NBzF, was confirmed by its application to imaging of hydrogen peroxide generation in live RAW 264.7 macrophages. NBzF was also employed for live cell imaging of hydrogen peroxide generated as a signaling molecule in A431 human epidermoid carcinoma cells.     

Fluorescent lipids: functional parts of fusogenic liposomes and tools for cell membrane labeling and visualization

In this paper a rapid and highly efficient method for controlled incorporation of fluorescent lipids into living mammalian cells is introduced. Here, the fluorescent molecules have two consecutive functions: First, they trigger rapid membrane fusion between cellular plasma membranes and the lipid bilayers of their carrier particles, so called fusogenic liposomes, and second, after insertion into cellular membranes these molecules enable fluorescence imaging of cell membranes and membrane traffic processes. We tested the fluorescent derivatives of the following essential membrane lipids for membrane fusion: Ceramide, sphingomyelin, phosphocholine, phosphatidylinositol-bisphosphate, ganglioside, cholesterol, and cholesteryl ester. Our results show that all probed lipids could more efficiently be incorporated into the plasma membrane of living cells than by using other methods. Moreover, labeling occurred in a gentle manner under classical cell culture conditions reducing cellular stress responses. Staining procedures were monitored by fluorescence microscopy and it was observed that sphingolipids and cholesterol containing free hydroxyl groups exhibit a decreased distribution velocity as well as a longer persistence in the plasma membrane compared to lipids without hydroxyl groups like phospholipids or other artificial lipid analogs. After membrane staining, the fluorescent molecules were sorted into membranes of cell organelles according to their chemical properties and biological functions without any influence of the delivery system.     

芯片毛细管电泳评估脂质体介导超氧化物歧化酶减低细胞内氧化应激

超氧化物歧化酶(SOD)可用作抗氧化的药物。它能催化并清除细胞内的活性氧组分(ROS),保护细胞免受自由基的氧化破坏。但是由于SOD分子量较大,难以透过细胞膜进入细胞内,显著降低了SOD的药效。本研究用激光共聚焦荧光显微镜拍摄的荧光图像说明,纳米脂质体可介导SOD进入细胞。用芯片毛细管电泳激光诱导荧光分析法(MCE-LIF)测定单细胞中ROS和谷胱甘肽(GSH)的荧光信号强度,评估了用脂质体包裹的SOD与细胞作用的抗氧化效果。用脂质体包裹的SOD与肝癌细胞共培养2h,与直接用SOD作用于肝癌细胞相比较,细胞内ROS明显降低,GSH明显提高。实验结果说明,用脂质体包裹SOD是一种减低细胞内氧化应激的有效给药途径。     

Swallow-tailed perylene derivative: a new tool for fluorescent imaging of lipid hydroperoxides

A swallow-tailed perylene derivative including a triphenylphosphine moiety was synthesized and applied to the detection and the live-cell imaging of lipid hydroperoxides. The novel probe, named Spy-LHP, reacted rapidly and quantitatively with lipid hydroperoxides to form the corresponding oxide, Spy-LHPOx, which emits extremely strong fluorescence (Phi approximately 1) in the visible range (lambda(em) = 535 nm, 574 nm). Spy-LHP was highly selective for lipid hydroperoxides, and the addition of other reactive oxygen species (ROS) including hydrogen peroxides, hydroxyl radical, superoxide anion, nitric oxide, peroxynitrite, and alkylperoxyl radical, caused no significant increase in the fluorescence intensity. The probe exhibited good localization to cellular membranes and was successfully applied to the confocal laser scanning microscopy (CLSM) imaging of lipid hydroperoxides in live J774A.1 cells, in which lipid peroxidation was proceeded by the stimulation of 2,2-azobis(2-amidinopropane)dihydrochloride (AAPH). These findings establish Spy-LHP as a promising new tool for investigating the physiology of lipid hydroperoxides.     

Acid- and Oxidatively-Labile Vinyl Ether Surfactants: Synthesis and Drug Delivery Applications

Liposomes have now evolved into a commercially-important drug delivery vehicle by overcoming a host of problems that were initially encountered with first generation liposomes. In spite of these impressive advances, the great potential of liposomes as drug delivery vehicles will not be fully realized until more effective targeting and membrane fusion mechanisms have been incorporated into their formulations. Our laboratory has developed several plasmenyl-type lipids for use in acid- or photooxidatively-triggerable liposomes. This review summarizes our progress toward the design, synthesis, and triggered release of encapsulated agents upon acid-catalyzed hydrolysis or photosensitized oxidation of plasmenyl-type lipid systems. Application of these materials in cascade triggering and intracellular drug delivery schemes is also described.     

Boronate-based fluorescent probes for imaging cellular hydrogen peroxide

The syntheses, properties, and biological applications of the Peroxysensor family, a new class of fluorescent probes for hydrogen peroxide, are presented. These reagents utilize a boronate deprotection mechanism to provide high selectivity and optical dynamic range for detecting H2O2 in aqueous solution over similar reactive oxygen species (ROS) including superoxide, nitric oxide, tert-butyl hydroperoxide, hypochlorite, singlet oxygen, ozone, and hydroxyl radical. Peroxyresorufin-1 (PR1), Peroxyfluor-1 (PF1), and Peroxyxanthone-1 (PX1) are first-generation probes that respond to H2O2 by an increase in red, green, and blue fluorescence, respectively. The boronate dyes are cell-permeable and can detect micromolar changes in H2O2 concentrations in living cells, including hippocampal neurons, using confocal microscopy and two-photon microscopy. The unique combination of ROS selectivity, membrane permeability, and a range of available excitation/emission colors establishes the potential value of PR1, PF1, PX1, and related probes for interrogating the physiology and pathology of cellular H2O2.     

Carbohydrate-Derived Metal-Chelator-Triggered Lipids for Liposomal Drug Delivery

Liposomes are versatile three-dimensional, biomaterial-based frameworks that can spatially enclose a variety of organic and inorganic biomaterials for advanced targeted-delivery applications. Implementation of external-stimuli-controlled release of their cargo will significantly augment their wide application for liposomal drug delivery. This paper presents the synthesis of a carbohydrate-derived lipid, capable of changing its conformation depending on the presence of Zn²⁺: an active state in the presence of Zn²⁺ ions and back to an inactive state in the absence of Zn²⁺ or when exposed to Na₂EDTA, a metal chelator with high affinity for Zn²⁺ ions. This is the first report of a lipid triggered by the presence of a metal chelator. Total internal reflection fluorescence microscopy and a single-liposome study showed that it indeed was possible for the lipid to be incorporated into the bilayer of stable liposomes that remained leakage-free for the fluorescent cargo of the liposomes. On addition of EDTA to the liposomes, their fluorescent cargo could be released as a result of the membrane-incorporated lipids undergoing a conformational change.     

Electrochemistry and chemiluminescence techniques compared in the detection of NADPH oxidase activity in phagocyte cells

Several methodologies have been used in clinical chemistry for real-time assessment of NADPH oxidase primary product superoxide anion which dismutases to hydrogen peroxide. Among these methodologies, isoluminol chemiluminescence (CL) is considered to be one of the more sensitive and reliable techniques for the assessment of NADPH oxidase activity in neutrophils. The electrochemical technique was recently designed and also applied for real-time detection of NADPH oxidase activity in neutrophils but its reliability and sensitivity has not been investigated so far. In this study, isoluminol CL and electrochemical techniques were investigated and compared by monitoring the generation of superoxide and hydrogen peroxide in both PLB 985 cell line differentiated into neutrophil-like cells and human neutrophils. The electrochemical technique was shown to be as sensitive as that of CL and able to detect the reactive oxygen species (ROS) release of as low as 500 cells. Thus, the electrochemical technique could be used as an alternative to optical techniques for the evaluation of extracellular ROS in phagocyte cells.     

Alleviating Cellular Oxidative Stress through Treatment with Superoxide-Triggered Persulfide Prodrugs

Overproduction of superoxide anion (O₂^.−), the primary cellular reactive oxygen species (ROS), is implicated in various human diseases. To reduce cellular oxidative stress caused by overproduction of superoxide, we developed a compound that reacts with O₂^.− to release a persulfide (RSSH), a type of reactive sulfur species related to the gasotransmitter hydrogen sulfide (H₂S). Termed SOPD-NAC , this persulfide donor reacts specifically with O₂^.−, decomposing to generate N-acetyl cysteine (NAC) persulfide. To enhance persulfide delivery to cells, we conjugated the SOPD motif to a short, self-assembling peptide (Bz-CFFE-NH₂) to make a superoxide-responsive, persulfide-donating peptide ( SOPD-Pep ). Both SOPD-NAC and SOPD-Pep delivered persulfides/H₂S to H9C2 cardiomyocytes and lowered ROS levels as confirmed by quantitative in vitro fluorescence imaging studies. Additional in vitro studies on RAW 264.7 macrophages showed that SOPD-Pep mitigated toxicity induced by phorbol 12-myristate 13-acetate (PMA) more effectively than SOPD-NAC and several control compounds, including common H₂S donors.