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The achievement of smart pharmaceuticals whose bioactivity can be spatiotemporally controlled by light stimuli is known as photopharmacology, an emerging area aimed at improving the therapeutic outcome and minimizing side effects. This is especially attractive for antibiotics, for which the inevitable development of multidrug resistance and the dwindling of new clinically approved drugs represent the main drawbacks. Here, we show that nitrosation of the fluoroquinolone norfloxacin (NF), a broad-spectrum antibiotic, leads to the nitrosated bioconjugate NF-NO, which is inactive at the typical minimum inhibitory concentration of NF. Irradiation of NF-NO with visible blue light triggers the simultaneous release of NF and nitric oxide (NO). The photouncaging process is accompanied by the revival of the typical fluorescence emission of NF, quenched in NF-NO, which acts as an optical reporter. This permits the real-time monitoring of the photouncaging process, even within bacteria cells where antibacterial activity is switched on exclusively upon light irradiation. The mechanism of photorelease seems to occur through a two-step hopping electron transfer mediated by the lowest triplet state of NF-NO and the phosphate buffer ions or aminoacids such as tyrosine. Considering the well-known role of NO as an "unconventional" antibacterial, the NF-NO conjugate may represent a potential bimodal antibacterial weapon activatable on demand with high spatio-temporal control.
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Antibacterianos , Óxido Nítrico , Norfloxacino , Antibacterianos/farmacologia , Antibacterianos/química , Óxido Nítrico/metabolismo , Norfloxacino/farmacologia , Norfloxacino/química , Fluorescência , Processos Fotoquímicos , Fluoroquinolonas/química , Fluoroquinolonas/farmacologia , Testes de Sensibilidade Microbiana , Luz , Estrutura Molecular , Escherichia coli/efeitos dos fármacosRESUMO
Several studies have highlighted the presence of nitration damage following neuroinflammation in Alzheimer's disease (AD). Accordingly, post-transcriptional modifications of ß-amyloid (Aß), including peptide nitration, have been explored as a marker of the disease. However, the implications of Aß nitration in terms of aggregation propensity and neurotoxicity are still debated. Here, we show new data obtained using a photoactivatable peroxynitrite generator (BPT-NO) to overcome the limitations associated with chemical nitration methods. We found that the photoactivation of BPT-NO with the highly biocompatible red light selectively induces the nitration of tyrosine 10 of freshly solubilized full-length Aß1-42. Photonitrated Aß1-42 was, therefore, investigated for aggregation states and functions. It resulted that photonitrated Aß1-42 did not aggregate into small oligomers but rather self-assembled into large amorphous aggregates. When tested on neuronal-like SH-SY5Y cells and microglial C57BL/6 BV2 cells, photonitrated Aß1-42 showed to be free of neurotoxicity and able to induce phagocytic microglia cells. We propose that light-controlled nitration of the multiple forms in which Aß occurs (i.e., monomers, oligomers, fibrils) could be a tool to assess in real-time the impact of tyrosine nitration on the amyloidogenic and toxic properties of Aß1-42.
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Peptídeos beta-Amiloides , Luz , Fragmentos de Peptídeos , Tirosina , Peptídeos beta-Amiloides/metabolismo , Tirosina/metabolismo , Tirosina/análogos & derivados , Tirosina/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Humanos , Animais , Microglia/metabolismo , Microglia/efeitos dos fármacos , Ácido Peroxinitroso/metabolismo , Camundongos , Agregados Proteicos/fisiologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Neurônios/metabolismo , Neurônios/efeitos dos fármacosRESUMO
The multifaceted role nitric oxide (NO) plays in human physiology and pathophysiology has opened new scenarios in biomedicine by exploiting this free radical as an unconventional therapeutic against important diseases. The difficulties in handling gaseous NO and the strict dependence of the biological effects on its doses and location have made the light-activated NO precursors, namely NO photodonors (NOPDs), very appealing by virtue of their precise spatiotemporal control of NO delivery. The covalent integration of NOPDs and additional functional components within the same molecular skeleton through suitable linkers can lead to an intriguing class of multifunctional photoactivatable molecular hybrids. In this Perspective, we provide an overview of the recent advances in these molecular constructs, emphasizing those merging NO photorelease with targeting, fluorescent reporting, and phototherapeutic functionalities. We will highlight the rational design behind synthesizing these molecular hybrids and critically describe the advantages, drawbacks, and opportunities they offer in biomedical research.
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A hydrophobic nitric oxide (NO) photodonor integrating both nitroso and nitro functionalities within its chromophoric skeleton has been synthesized. Excitation of this compound with blue light triggers the release of two NO molecules from the nitroso and the nitro functionalities via a stepwise mechanism. Encapsulation of the NO photodonor within biocompatible neutral, cationic, and anionic ß-cyclodextrin branched polymers as suitable carriers leads to supramolecular nanoassemblies, which exhibit the same nature of the photochemical processes but NO photorelease performances enhanced by about 1 order of magnitude when compared with the free guest. Antibacterial tests carried out with methicillin-resistant Staphylococcus aureus and Acinetobacter baumannii demonstrate an effective antibacterial activity exclusively under light activation and point out a differentiated role of the polymeric nanocarriers in determining the outcome of the antibacterial photodynamic action.
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The strict dependence of the biological effects of nitric oxide (NO) on its concentration and generation site requires this inorganic free radical to be delivered with precise spatiotemporal control. Light-activation by suitable NO photoprecursors represents an ideal approach. Developing strategies to activate NO release using long-wavelength excitation light in the therapeutic window (650-1300 nm) is challenging. In this contribution, we demonstrate that NO release by a blue-light activatable NO photodonor (NOPD) with self-fluorescence reporting can be triggered catalytically by the much more biocompatible red light exploiting a supramolecular photosensitization process. Different red-light absorbing photosensitizers (PSs) are co-entrapped with the NOPD within different biocompatible nanocarriers such as Pluronic® micelles, microemulsions and branched cyclodextrin polymers. The intra-carrier photosensitized NO release, involving the lowest, long-lived triplet state of the PS as the key intermediate and its quenching by the NOPD, is competitive with that by molecular oxygen. This allows NO to be released with good efficacy, even under aerobic conditions. Therefore, the adopted general strategy provides a valuable tool for generating NO from an already available NOPD, otherwise activatable with the poorly biocompatible blue light, without requiring any chemical modification and using sophisticated and expensive irradiation sources.
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Materiais Biocompatíveis , Luz , Óxido Nítrico , Fármacos Fotossensibilizantes , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Portadores de Fármacos/química , Fluorescência , Nanopartículas/química , Humanos , Tamanho da PartículaRESUMO
Age-related reduction in muscle stem cell (MuSC) regenerative capacity is associated with cell-autonomous and non-cell-autonomous changes caused by alterations in systemic and skeletal muscle environments, ultimately leading to a decline in MuSC number and function. Previous studies demonstrated that STAT3 plays a key role in driving MuSC expansion and differentiation after injury-activated regeneration, by regulating autophagy in activated MuSCs. However, autophagy gradually declines in MuSCs during lifespan and contributes to the impairment of MuSC-mediated regeneration of aged muscles. Here, we show that STAT3 inhibition restores the autophagic process in aged MuSCs, thereby recovering MuSC ability to promote muscle regeneration in geriatric mice. We show that STAT3 inhibition could activate autophagy at the nuclear level, by promoting transcription of autophagy-related genes, and at the cytoplasmic level, by targeting STAT3/PKR phosphorylation of eIF2α. These results point to STAT3 inhibition as a potential intervention to reverse the age-related autophagic block that impairs MuSC ability to regenerate aged muscles. They also reveal that STAT3 regulates MuSC function by both transcription-dependent and transcription-independent regulation of autophagy.
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Envelhecimento , Autofagia , Músculo Esquelético , Regeneração , Fator de Transcrição STAT3 , Fator de Transcrição STAT3/metabolismo , Animais , Camundongos , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Músculo Esquelético/citologia , Envelhecimento/fisiologia , Envelhecimento/metabolismo , Camundongos Endogâmicos C57BL , Células-Tronco/metabolismo , Células-Tronco/citologia , Fosforilação , Masculino , Diferenciação Celular , Transdução de SinaisRESUMO
Nitric oxide (NO) is a diatomic inorganic free radical ubiquitous in mammalian tissues and cells that plays a multifaceted role in a variety of physiological and pathophysiological processes. The strict dependence of the biological effects of NO on its concentration makes its real-time monitoring crucial. In view of the reactivity of NO with multiple bio-targets, the development of NO sensors that associate a fast response rate with selectivity and sensitivity is very challenging. Herein we report a fluorescent NO probe based on a BODIPY fluorogenic unit covalently linked to a trimethoxy aniline derivative through a flexible spacer. NO leads to effective nitrosation of the highly electron-rich amino active site of the probe through the secondary oxide N2O3, resulting in an increase of BODIPY fluorescence quantum yield from Φf = 0.06 to Φf = 0.55, accompanied by significant changes in the relative amplitude of the fluorescence lifetimes. In situ generation of NO, achieved by a tailored light-activatable NO releaser, allows the real-time detection of NO as a function of its concentration and permits demonstrating that the probe exhibits a very fast response time, being ≤0.1 s. This remarkable data combines with the high sensitivity of the probe to NO (LOD = 35 nM), responsiveness also to ONOO-, the other important secondary oxide of NO, independence from the fluorescence response within a wide pH range, good selectivity towards different analytes and small interference by typical physiological concentrations of glutathione. Validation of this probe in melanoma cell lines is also reported.
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Compostos de Boro , Corantes Fluorescentes , Óxido Nítrico , Óxido Nítrico/análise , Óxido Nítrico/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Humanos , Compostos de Boro/química , Compostos de Boro/farmacologia , Estrutura Molecular , Linhagem Celular TumoralRESUMO
The achievement of biocompatible platforms for multimodal therapies is one of the major challenges in the burgeoning field of nanomedicine. Here, we report on a mixed ß- and γ-cyclodextrin-based branched polymeric material (ßγCD-NOPD) covalently integrating a nitric oxide (NO) photodonor (NOPD) within its macromolecular scaffold, and its supramolecular ensemble with a singlet oxygen (1O2) photosensitizer (PS) Zn(II) phthalocyanine (ZnPc) and the chemodrug Lenvatinib (LVB). This polymer is highly water-soluble and generates NO under visible blue light stimuli with an efficiency of more than 1 order of magnitude higher than that of the single NOPD. The PS, which in an aqueous solution is aggregated and non-photoresponsive, can be entangled in the polymeric network as a photoresponsive monomeric species. In addition, the poorly water-soluble LVB can be co-encapsulated within the polymeric host, which increases the drug solubility by more than 30-fold compared to the free drug and more than 2-fold compared with a similar branched polymer containing only ßCD units. The supramolecular nanoensemble, ca. 15 nm in diameter, retains well the photochemical properties of both the NOPD and PS, which can operate in parallel under light stimuli of different energies. Irradiation with blue and red light results in the photogeneration of NO and 1O2 associated with red fluorescence emission, without inducing any photodegradation of LVB. This result is not trivial and is due to the absence of significant, mutual interactions between the NOPD, the PS and LVB both in the ground and excited states, despite these components are confined in the same host. The proposed polymeric nanoplatform may represent a potential trimodal nanomedicine for biomedical research studies, since it combines the double photodynamic action of NO and 1O2, two species that do not suffer multidrug resistance, with the therapeutic activity of a conventional chemodrug.
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Developing biocompatible nitric oxide (NO) photoreleasing nanoconstucts is of great interest in view of the large variety of biological roles that NO plays and the unique advantage light offers in controlling NO release in space and time. In this contribution, we report the supramolecular assemblies of two NO photodonors (NOPDs), NBF-NO and RHD-NO, as water-dispersible nanogels, ca. 10 nm in diameter, based on γ-cyclodextrins (γ-CDng). These NOPDs, containing amino-nitro-benzofurazan and rhodamine chromophores as light harvesting antennae, can be activated by visible light, are highly hydrophobic and can be effectively entrapped within the γ-CDng. Despite being confined in a very restricted environment, neither NOPD suffer self-aggregation and preserve their photochemical and photophysical properties well. The blue light excitation of the weakly fluorescent γ-CDng/NBF-NO complex results in effective NO release and the concomitant generation of the highly green, fluorescent co-product, which acts as an optical NO reporter. Moreover, the green light excitation of the persistent red fluorescent γ-CDng/RHD-NO triggers NO photorelease without significantly modifying the emission properties. The activatable and persistent fluorescence emissions of the NOPDs are useful for monitoring their interactions with the Gram-positive methicillin-resistant Staphylococcus aureus, whose growth is significantly inhibited by γ-CDng/RHD-NO upon green light irradiation.
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Ciclodextrinas , Staphylococcus aureus Resistente à Meticilina , Óxido Nítrico/química , Nanogéis , Doadores de Óxido Nítrico/farmacologia , CorantesRESUMO
Curcumin (CUR) is a naturally occurring pigment extensively studied due to its therapeutic activity and delivered by suitable nanocarriers to overcome poor solubility in aqueous media. The significant absorption of CUR in the visible blue region has prompted its use as a potential phototherapeutic agent in treating infectious and cancer diseases, although the mechanism underlying the phototoxic effects is still not fully understood. This contribution investigates the photobehaviour of CUR within polymeric micelles, microemulsions, and zein nanoparticles, chosen as biocompatible nanocarriers, and human serum albumin as a representative biomolecule. Spectroscopic studies indicate that in all host systems, the enolic tautomeric form of CUR is converted in a significant amount of the diketo form because of the perturbation of the intramolecular hydrogen bond. This leads to intermolecular H-abstraction from the host components by the lowest excited triplet state of CUR with the formation of the corresponding ketyl radical, detected by nanosecond laser flash photolysis. This radical is oxidized by molecular oxygen, likely generating peroxyl and hydroperoxyl radical species, unless in Zein, reasonably due to the poor availability of oxygen in the closely packed structure of this nanocarrier. In contrast, no detectable formation of singlet oxygen was revealed in all the systems. Overall these results highlight the key role of the H-abstraction process over singlet oxygen sensitization as a primary photochemical pathway strictly dictated by the specific features of the microenvironment, providing new insights into the photoreactivity of CUR in biocompatible hosts that can also be useful for a better understanding of its phototoxicity mechanism.
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Curcumina , Zeína , Humanos , Curcumina/química , Fotólise , Oxigênio Singlete , Oxigênio/químicaRESUMO
The role of nitric oxide (NO) as an "unconventional" therapeutic and the strict dependence of biological effects on its concentration require the generation of NO with precise spatiotemporal control. The development of precursors and strategies to activate NO release by excitation in the so-called "therapeutic window" with highly biocompatible and tissue-penetrating red light is desirable and challenging. Herein, we demonstrate that one-photon red-light excitation of Verteporfin, a clinically approved photosensitizer (PS) for photodynamic therapy, activates NO release, in a catalytic fashion, from an otherwise blue-light activatable NO photodonor (NOPD) with an improvement of about 300 nm toward longer and more biocompatible wavelengths. Steady-state and time-resolved spectroscopic and photochemical studies combined with theoretical calculations account for an NO photorelease photosensitized by the lowest triplet state of the PS. In view of biological applications, the water-insoluble PS and NOPD have been co-entrapped within water-dispersible, biodegradable polymeric nanoparticles (NPs) of mPEG-b-PCL (about 84 nm in diameter), where the red-light activation of NO release takes place even more effectively than in an organic solvent solution and almost independently by the presence of oxygen. Moreover, the ideal spectroscopic prerequisites and the restricted environment of the NPs permit the green-fluorescent co-product formed concomitantly to NO photorelease to communicate with the PS via Förster resonance energy transfer. This leads to an enhancement of the typical red emission of the PS offering the possibility of a double color optical reporter useful for the real-time monitoring of the NO release through fluorescence techniques. The suitability of this strategy applied to the polymeric NPs as potential nanotherapeutics was evaluated through biological tests performed by using HepG2 hepatocarcinoma and A375 melanoma cancer cell lines. Fluorescence investigation in cells and cell viability experiments demonstrates the occurrence of the NO release under one-photon red-light illumination also in the biological environment. This confirms that the adopted strategy provides a valuable tool for generating NO from an already available NOPD, otherwise activatable with the poorly biocompatible blue light, without requiring any chemical modification and the use of sophisticated irradiation sources.
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Several strategies have been explored with the attempt of improving the safety and feasibility of umbilical cord blood transplantation (UCBT) in adults. The aim of this retrospective analysis was to examine the safety and efficacy of intrabone transplantation of a single unwashed cord blood unit in an antithymocyte globulin-free, sirolimus-based graft-versus-host disease prophylaxis platform. We collected data for all consecutive UCBTs infused intrabone (IB) and unwashed at San Raffaele Hospital in Milan between 2012 and 2021. Thirty-one consecutive UCBTs were identified. All but 3 UCB units had a high-resolution HLA typing on 8 loci at the time of selection. At the time of cryopreservation, the median CD34+ cell count was 1 × 105/kg (range, .6 to 12.0 × 105/kg) and the median total nucleated cell (TNC) count was 2.8 × 107/kg (range, 1.48 to 5.6 × 107/kg). Eighty-seven percent of patients received myeloablative conditioning, and 77% underwent transplantation for acute myeloid leukemia. The median duration of follow-up among survivors was 38.2 months (range, 10.4 to 123.6 months). No adverse events were related to the IB infusion at bedside under short-conscious periprocedural sedation or to the no wash technique. After thawing, median CD34+ cell and TNC counts were .8 × 105/kg (range, .1 to 2.3 × 105/kg) and 1.42 × 107/kg (range, .69 to 3.2 × 107/kg). The median time to engraftment was 27 days for neutrophils and 53 days for platelets. One patient experienced graft rejection and was subsequently rescued with a salvage transplantation. The median time to a CD3+ cell count >100/µL was 30 days. The 100-day cumulative incidence of grade III-IV acute graft-versus-host disease (GVHD) was 12.9% (95% confidence interval [CI], 4% to 27.3%), and the 2-year cumulative incidence of moderate-to-severe chronic GVHD (cGVHD) was 11.8% (95% CI, 2.7% to 28.3%). At 2 years, overall survival (OS) was 52.7% (95% CI, 33% to 69%), relapse incidence was 30.7% (95% CI, 13.7% to 49.6%), and transplantation-related mortality was 29% (95% CI, 14.3% to 45.6%). In univariate analysis, infused CD34+ cell count did not impact transplantation outcomes. In patients who underwent transplantation in first complete remission, relapse rate was 13%, with a 2-year OS >90%. In our cohort, IB infusion of a single cord blood unit was feasible, with no adverse reactions related to the no wash/IB infusion, low rates of cGVHD and disease relapse, and rapid immune reconstitution.
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Doença Enxerto-Hospedeiro , Reconstituição Imune , Adulto , Humanos , Sangue Fetal , Soro Antilinfocitário/uso terapêutico , Sirolimo/uso terapêutico , Estudos Retrospectivos , Doença Enxerto-Hospedeiro/prevenção & controle , RecidivaRESUMO
Striated muscle is a highly organized structure composed of well-defined anatomical domains with integrated but distinct assignments. So far, the lack of a direct correlation between tissue architecture and gene expression has limited our understanding of how each unit responds to physio-pathologic contexts. Here, we show how the combined use of spatially resolved transcriptomics and immunofluorescence can bridge this gap by enabling the unbiased identification of such domains and the characterization of their response to external perturbations. Using a spatiotemporal analysis, we follow changes in the transcriptome of specific domains in muscle in a model of denervation. Furthermore, our approach enables us to identify the spatial distribution and nerve dependence of atrophic signaling pathway and polyamine metabolism to glycolytic fibers. Indeed, we demonstrate that perturbations of polyamine pathway can affect muscle function. Our dataset serves as a resource for future studies of the mechanisms underlying skeletal muscle homeostasis and innervation.
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Atrofia Muscular , Transcriptoma , Humanos , Atrofia Muscular/metabolismo , Transcriptoma/genética , Músculo Esquelético/metabolismo , Perfilação da Expressão Gênica , Poliaminas/metabolismoRESUMO
The design, synthesis, photochemical properties, and biological evaluation of a novel molecular dyad with double photodynamic action and its formulation within biodegradable polymeric nanoparticles (NPs) are reported. A BODIPY-based singlet oxygen (1O2) photosensitizer (PS) and a nitric oxide (NO) photodonor (NOPD) based on an amino-nitro-benzofurazan moiety have been covalently joined in a new molecular dyad, through a flexible alkyl spacer. Excitation of the dyad with visible light in the range 400-570 nm leads to the concomitant generation of the cytotoxic 1O2 and NO with effective quantum yields, being ΦΔ = 0.49 ± 0.05 and ΦNO = 0.18 ± 0.01, respectively. Besides, the non-fluorescent NOPD unit becomes highly fluorescent after the NO release, acting as an optical reporter for the NO photogenerated. The dyad is not soluble in water medium but can be effectively entrapped in water-dispersible, biodegradable polymeric NPs made of mPEG-PCL, ca. 66 nm in diameter. The polymeric nano-environment affects in an opposite way the photochemical performances of the dyad, reducing ΦΔ to 0.16 ± 0.02 and increasing ΦNO to 0.92 ± 0.03, respectively. The NPs effectively deliver the photoactive cargo into the cytoplasm of HepG2 hepatocellular carcinoma cells. A remarkable level of cell mortality is observed for the loaded NPs at very low concentrations of the dyad (1-5 µM) and very low light doses (≤0.8 J cm-2) more likely as the result of the combined photodynamic action of 1O2 and NO.
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Nanopartículas , Neoplasias , Fotoquimioterapia , Linhagem Celular Tumoral , Nanopartículas/química , Óxido Nítrico , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Polímeros/química , ÁguaRESUMO
The biological activity of a molecular hybrid (DXNO-GR) joining doxorubicin (DOX) and an N-nitroso moiety releasing nitric oxide (NO) under irradiation with the biocompatible green light has been investigated against DOX-sensitive (MCF7) and -resistant (MDA-MB-231) breast cancer cells in vitro. DXNO-GR shows significantly higher cellular internalization than DOX in both cell lines and, in contrast to DOX, does not experience cell efflux in MDR overexpressing MDA-MB-231 cells. The higher cellular internalization of the DXNO-GR hybrid seems to be mediated by bovine serum albumin (BSA) as a suitable carrier among serum proteins, according to the high binding constant measured for DXNO-GR, which is more than one order of magnitude larger than that reported for DOX. Despite the higher cellular accumulation, DXNO-GR is not toxic in the dark but induces remarkable cell death following photoactivation with green light. This lack of dark toxicity is strictly related to the different cellular compartmentalization of the molecular hybrid that, different from DOX, does not localize in the nucleus but is mainly confined in the Golgi apparatus and endoplasmic reticulum and therefore does not act as a DNA intercalator. The photochemical properties of the hybrid are not affected by binding to BSA as demonstrated by the direct detection of NO photorelease, suggesting that the reduction of cell viability observed under light irradiation is a combined effect of DOX phototoxicity and NO release which, ultimately, inhibits MDR1 efflux pump in DOX-resistant cells.
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Hematopoietic stem and progenitor cell (HSPC)-based gene therapy (GT) requires the collection of a large number of cells. While bone marrow (BM) is the most common source of HSPCs in pediatric donors, the collection of autologous peripheral blood stem cells (PBSCs) is an attractive alternative for GT. We present safety and efficacy data of a 10-year cohort of 45 pediatric patients who underwent PBSC collection for backup and/or purification of CD34+ cells for ex vivo gene transfer. Median age was 3.7 years and median weight 15.8 kg. After mobilization with lenograstim/plerixafor (n = 41) or lenograstim alone (n = 4) and 1-3 cycles of leukapheresis, median collection was 37 × 106 CD34+ cells/kg. The procedures were well tolerated. Patients who collected ≥7 and ≥13 × 106 CD34+ cells/kg in the first cycle had pre-apheresis circulating counts of at ≥42 and ≥86 CD34+ cells/µL, respectively. Weight-adjusted CD34+ cell yield was positively correlated with peripheral CD34+ cell counts and influenced by female gender, disease, and drug dosage. All patients received a GT product above the minimum target, ranging from 4 to 30.9 × 106 CD34+ cells/kg. Pediatric PBSC collection compares well to BM harvest in terms of CD34+ cell yields for the purpose of GT, with a favorable safety profile.
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Nitric oxide (NO) plays a multifaceted role in human physiology and pathophysiology, and its controlled delivery has great prospects in therapeutic applications. The light-activated uncaging of NO from NO caging compounds allows this free radical to be released with accurate control of site and dosage, which strictly determine its biological effects. Molecular constructs able to activate fluorescence concomitantly to NO release offer the important advantage of easy and real-time tracking of the amount of NO uncaged in a non-invasive fashion even in the cell environment. This contribution provides an overview of the advances in photoactivatable NO releasers bearing fluorescent reporting functionalities achieved in our and other laboratories, highlighting the rationale design and their potential therapeutic applications.
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Corantes , Óxido Nítrico , Fluorescência , Corantes Fluorescentes , Radicais Livres , HumanosRESUMO
The generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) as "unconventional" therapeutics with precise spatiotemporal control by using light stimuli may open entirely new horizons for innovative therapeutic modalities. Among ROS and RNS, peroxynitrite (ONOO-) plays a dominant role in chemistry and biology in view of its potent oxidizing power and cytotoxic action. We have designed and synthesized a molecular hybrid based on benzophenothiazine as a red light-harvesting antenna joined to an N-nitroso appendage through a flexible spacer. Single photon red light excitation of this molecular construct triggers the release of nitric oxide (ËNO) and simultaneously produces superoxide anions (O2Ë-). The diffusion-controlled reaction between these two radical species generates ONOO-, as confirmed by the use of fluorescein-boronate as a highly selective chemical probe. Besides, the red fluorescence of the hybrid allows its tracking in different types of cancer cells where it is well-tolerated in the dark but induces remarkable cell mortality under irradiation with red light in a very low concentration range, with very low light doses (ca. 1 J cm-2). This ONOO- generator activatable by highly biocompatible and tissue penetrating single photon red light can open up intriguing prospects in biomedical research, where precise and spatiotemporally controlled concentrations of ONOO- are required.
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Emapalumab, a fully human anti-IFNγ monoclonal antibody, has been approved in the US as second-line treatment of primary hemophagocytic lymphohistiocytosis (HLH) patients and has shown promise in patients with graft failure (GF) requiring a second allogeneic hematopoietic stem cell transplantation (HSCT). The blockade of IFNγ activity may increase the risk of severe infections, including fatal mycobacteriosis. We report a case of secondary HLH-related GF in the context of HLA-haploidentical HSCT successfully treated with emapalumab in the presence of concomitant life-threatening infections, including disseminated tuberculosis (TB). A 4 years old girl with Adenosine Deaminase-Severe Combined Immunodeficiency complicated by disseminated TB came to our attention for ex-vivo hematopoietic stem cell-gene therapy. After engraftment failure of gene corrected cells, she received two HLA-haploidentical T-cell depleted HSCT from the father, both failed due to GF related to concomitant multiple infections and secondary HLH. Emapalumab administration allowed to control HLH, as well as to prevent GF after a third haplo-HSCT from the mother. Remarkably, all infections improved with antimicrobial medications and disseminated TB did not show any reactivation. This seminal case supports emapalumab use for treatment of secondary HLH and prevention of GF in patients undergoing haplo-HSCT even in the presence of multiple infections, including TB.
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Transplante de Células-Tronco Hematopoéticas , Linfo-Histiocitose Hemofagocítica , Imunodeficiência Combinada Severa , Tuberculose , Adenosina Desaminase , Agamaglobulinemia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes , Vacina BCG , Pré-Escolar , Feminino , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Linfo-Histiocitose Hemofagocítica/tratamento farmacológico , Linfo-Histiocitose Hemofagocítica/etiologia , Imunodeficiência Combinada Severa/complicações , Imunodeficiência Combinada Severa/tratamento farmacológico , Tuberculose/complicações , Tuberculose/tratamento farmacológicoRESUMO
A novel molecular hybrid has been designed and synthesized in which acridine orange (AO) is covalently linked to an N-nitrosoaniline derivative through an alkyl spacer. Photoexcitation of the AO antenna with the highly biocompatible green light results in intense fluorescence emission and triggers NO detachment from the N-nitroso appendage via an intramolecular electron transfer. The presence of the AO moiety encourages the binding with DNA through both external and partially intercalative fashions, depending on the DNA:molecular hybrid molar ratio. Importantly, this dual-mode binding interaction with the biopolymer does not preclude the NO photoreleasing performances of the molecular hybrid, permitting NO to be photogenerated nearby DNA with an efficiency similar to that of the free molecule. These properties make the presented compound an intriguing candidate for fundamental and potential applicative research studies where NO delivery in the DNA proximity precisely regulated by harmless green light is required.