A fluorescent probe with an ultra-rapid response to nitric oxide.

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.

Red-Light-Photosensitized NO Release and Its Monitoring in Cancer Cells with Biodegradable Polymeric Nanoparticles.

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.

On the photobehaviour of curcumin in biocompatible hosts: The role of H-abstraction in the photodegradation and photosensitization.

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.

Intrabone Transplantation of a Single Unwashed Umbilical Cord Blood Unit with Antithymocyte Globulin-Free and Sirolimus-Based Graft-versus-Host Disease Prophylaxis: Fast Immune Reconstitution and Long-Term Disease Control in Patients with High-Risk Diseases.

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.

A molecular dyad delivered by biodegradable polymeric nanoparticles for combined PDT and NO-PDT in cancer cells.

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.

Doxorubicin-NO Releaser Molecular Hybrid Activatable by Green Light to Overcome Resistance in Breast Cancer Cells.

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.

Peripheral blood stem and progenitor cell collection in pediatric candidates for ex vivo gene therapy: a 10-year series.

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.

A generator of peroxynitrite activatable with red light.

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 (˙NO) and simultaneously produces superoxide anions (O2˙-). 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.

Emapalumab treatment in an ADA-SCID patient with refractory hemophagocytic lymphohistiocytosis-related graft failure and disseminated bacillus Calmette-Guérin infection.

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.

NO release regulated by doxorubicin as the green light-harvesting antenna.

We report for the first time a NO photodonor (NOPD) operating with the widely used chemotherapeutic agent doxorubicin (DOX) as the light-harvesting antenna. This permits NO uncaging from an N-nitroso appendage upon selective excitation of DOX with highly biocompatible green light, without precluding its typical red emission. This NOPD effectively binds DNA and photodelivers NO nearby, representing an intriguing candidate for potential multimodal therapeutic applications based on the combination of DOX and NO.

Fluorescent Nitric Oxide Photodonors Based on BODIPY and Rhodamine Antennae.

Two novel NO photodonors (NOPDs) based on BODIPY and Rhodamine antennae activatable with the highly biocompatible green light are reported. Both NOPDs exhibit considerable fluorescence emission and release NO with remarkable quantum efficiencies. The combination of the photoreleasing and emissive performance for both compounds is superior to those exhibited by other NOPDs based on similar light-harvesting centres, making them very intriguing for image-guided phototherapeutic applications. Preliminary biological data prove their easy visualization in cell environment due to the intense green and orange-red fluorescence and their photodynamic action on cancer cells due to the NO photo-liberated.

A molecular hybrid producing simultaneously singlet oxygen and nitric oxide by single photon excitation with green light.

Combination of photosensitizers (PS) for photodynamic therapy with NO photodonors (NOPD) is opening intriguing horizons towards new and still underexplored multimodal anticancer and antibacterial treatments not based on "conventional" drugs and entirely controlled by light stimuli. In this contribution, we report an intriguing molecular hybrid based on a BODIPY light-harvesting antenna that acts simultaneously as PS and NOPD upon single photon excitation with the highly biocompatible green light. The presented hybrid offers a combination of superior advantages with respect to the other rare cases reported to date, meeting most of the key criteria for both PSs and NOPDs in the same molecular entity such as: (i) capability to generate 1O2 and NO with single photon excitation of biocompatible visible light, (ii) excellent 1O2 quantum yield and NO quantum efficiency, (iii) photogeneration of NO independent from the presence of oxygen, (iv) large light harvesting properties in the green region. Furthermore, this compound together with its stable photoproduct, is well tolerated by both normal and cancer cells in the dark and exhibits bimodal photomortality of cancer cells under green light excitation due to the combined action of the cytotoxic 1O2 and NO.

Clinical impact of a new automated system employed for peripheral blood stem cell collection.

At the moment, PBSC collections can be performed using semi-automated or automated cell separator devices. The automated methods offer the advantages of a decreased working load for dedicated personnel and high standardization of the collection procedure. Herein we report our single institutional experience in 80 PBSC collections employing the new automated COM.TEC Fresenius autoMNC program that provides the ability to predict the total number of CD34(+) cells collected, based on the pre-leukapheresis CD34(+) cell count in peripheral blood. Fourty-eight patients and 21 healthy donors were mobilized with chemotherapy + G-CSF or G-CSF alone, respectively. Eighty leukapheresis collections were performed starting with a CD34(+) cell count in peripheral blood at least of 20/microL. Collection parameters and related side effects were evaluated. The mean CD34(+) cell collection efficiency in patients and donors was 81.8% (sd 27.6) and 95.1% (sd 15.6), respectively. The mean difference between real and predicted CD34(+) cells was +30.2% (sd 92.9) for patients and +4.6% (sd 30.3) for donors. The mean leukapheresis bag volume was 240.7 ml (sd 67.3) and 310.3 ml (sd 86.8) with a mean HCT of 10.9% (sd 34.4) and 9.2% (sd 3.9) for patients and donors, respectively. The automated PBSC collection with the new program COM.TEC Fresenius autoMNC demonstrated a very high CD34(+) cell collection efficiency. Moreover, the ability to predict the CD34(+) cell yield permits improved management of the leukapheresis collection, with the only disadvantage of larger leukapheresis volume and higher hematocrit.

Immunomagnetic cell selection performed for HLA haploidentical transplants with the CliniMACS device: effect of additional platelet removal on CD34+ cell recovery.

Immunomagnetic CD34+ cell selection (ICS) is a widely employed technology in autotransplant and allotransplant settings. When an haploidentical transplant is performed, a high dose of purified CD34+ cells together with efficient T and B cell depletion are required to minimize the risks of graft versus host disease (GVHD) and Epstein-Barr virus (EBV)-related lymphoma. To ameliorate the performances of the CliniMACS (Miltenyi Biotec) ICS device, we compared 73 ICS performed following the manufacturer's recommended platelet depletion versus 48 performed after adjunctive centrifugations to increase platelet depletion of the leukapheresis (LKF) product. A total of 121 ICS (from single or fractioned LKF products) were carried out on 93 LKF collected from 47 related healthy donors. A statistical significance in terms of CD34+ cell recovery (81.8% vs. 71.2%) was found in favor of the modified ICS procedure (p=0.0049) with a comparable stem cell purity and viability. The modification of the standard manufacturer's technique for increasing platelet depletion can further improve the recovery of stem cells with no influence on T and B cell depletion. These results demonstrate the negative influence exerted on CD34+ cell recovery by LKF platelet contamination.