A Nanoprobe Based on Gated Mesoporous Silica Nanoparticles for The Selective and Sensitive Detection of Benzene Metabolite t,t-Muconic Acid in Urine.

Benzene is a highly toxic aromatic hydrocarbon. Inhaling benzene can cause dizziness, vertigo, headaches, aplasia, mutations and, in the most extreme cases, cancer. Trans,trans-muconic acid (t,t-MA) is one of the metabolization products of benzene. Although different analytical methods have been reported for the determination of t,t-MA, these are often expensive, require trained personnel, are not suitable for on-site measurements, and use hazardous organic solvents. For these reasons, the development of reliable, selective and sensitive methods for rapid and in situ detection of t,t-MA are of importance. Addressing this challenge, a nanodevice for the selective and sensitive quantification of t,t-MA in urine is reported. The nanodevice used is achieved using mesoporous silica nanoparticles loaded with a dye reporter and capped with a dicopper(II) azacryptand. Pore opening and payload release is induced rapidly (10 min) and selectively with t,t-MA in urine, using a simple fluorimeter without sample pretreatment.

Highly Sensitive and Selective Molecular Probes for Chromo-Fluorogenic Sensing of Carbon Monoxide in Air, Aqueous Solution and Cells.

Optical sensing offers a low-cost and effective means to sense carbon monoxide in air and in solution. This contribution reports the synthesis of a new series of vinyl complexes [Ru(CH=CHR)Cl(CO)(TBTD)(PPh3 )2 ] (R=aryl, TBTD=5-(3-thienyl)-2,1,3-benzothiadiazole) and shows them to be highly sensitive and selective probes for carbon monoxide in both solution and air. Depending on the vinyl substituent, chromogenic and fluorogenic responses signalled the presence of this invisible, odourless, tasteless and toxic gas. Adsorbing the complexes on silica produced colorimetric probes for the 'naked eye' detection of CO in the gas phase with a limit of detection as low as 8 ppm in some cases, while the release of the TBTD fluorophore allowed detection at much lower concentrations through the fluorescence response. Structural data were obtained by single-crystal X-ray diffraction techniques, while the photophysical behaviour was explored computationally using TD-DFT experiments. The systems were also shown to be selective for CO over all other gases tested, including water vapour and common organic solvents. By introducing a poly(ethylene)glycol chain to the vinyl functionality, water compatibility was achieved and these non-cytotoxic complexes were employed in the sensing of CO in HeLa cells, offering a simple and rapid system for sensing this gasotransmitter in this challenging medium.

ϵ-Polylysine-Capped Mesoporous Silica Nanoparticles as Carrier of the C9h Peptide to Induce Apoptosis in Cancer Cells.

Apoptotic signaling pathways are altered in numerous pathologies such as cancer. In this scenario, caspase-9/PP2Acα interaction constitutes a key target with pharmacological interest to re-establish apoptosis in tumor cells. Very recently, a short peptide (C9h) known to disrupt caspase-9/PP2Acα interaction with subsequent apoptosis induction was described. Here, we prepared two sets of mesoporous silica nanoparticles loaded with safranin O (S2) or with C9h peptide (S4) and functionalized with ϵ-polylysine as capping unit. Aqueous suspensions of both nanoparticles showed negligible cargo release whereas in the presence of pronase, a marked delivery of safranin O or C9h was observed. Confocal microscopy studies carried out with HeLa cells indicated that both materials were internalized and were able to release their entrapped cargos. Besides, a marked decrease in HeLa cell viability (ca. 50 %) was observed when treated with C9h-loaded S4 nanoparticles. Moreover, S4 provides peptide protection from degradation additionally allowing for a dose reduction to observe an apoptotic effect when compared with C9h alone or in combination with a cell-penetrating peptide (i.e., Mut3DPT-C9h). Flow cytometry studies, by means of Annexin V-FITC staining, showed the activation of apoptotic pathways in HeLa as a consequence of S4 internalization, release of C9h peptide and disruption of caspase-9/PP2Acα interaction.

VSL#3 probiotic differently influences IEC-6 intestinal epithelial cell status and function.

The data here reported introduce the wound-healing assay as a tool for testing probiotics aimed at protecting gastrointestinal mucosal surfaces and to verify the consistency of their manufacturing. At the scope, we compared the in vitro effects of two multi-strain high concentration formulations both commercialized under the same brand VSL#3 but sourced from different production sites (USA and Italy) on a non-transformed small-intestinal epithelial cell line, IEC-6. The effects on cellular morphology, viability, migration, and H2 O2 -induced damage, were assessed before and after the treatment with both VSL#3 formulations. While the USA-sourced product ("USA-made") VSL#3 did not affect monolayer morphology and cellular density, the addition of bacteria from the Italy-derived product ("Italy-made") VSL#3 caused clear morphological cell damage and strongly reduced cellularity. The treatment with "USA-made" lysate led to a higher rate of wounded monolayer healing, while the addition of "Italy-made" bacterial lysate did not influence the closure rate as compared to untreated cells. While lysates from "USA-made" VSL#3 clearly enhanced the formation of elongated and aligned stress fibers, "Italy-made" lysates had not similar effect. "USA-made" lysate was able to cause a total inhibition of H2 O2 -induced cytotoxic effect whereas "Italy-made" VSL#3 lysate was unable to protect IEC-6 cells from H2 O2 -induced damage. ROS generation was also differently influenced, thus supporting the hypotesis of a protective action of "USA-made" VSL#3 lysates, as well as the idea that "Italy-made" formulation was unable to prevent significantly the H2 O2 -induced oxidative stress.

Enzyme-Controlled Nanodevice for Acetylcholine-Triggered Cargo Delivery Based on Janus Au-Mesoporous Silica Nanoparticles.

This work reports a new gated nanodevice for acetylcholine-triggered cargo delivery. We prepared and characterized Janus Au-mesoporous silica nanoparticles functionalized with acetylcholinesterase on the Au face and with supramolecular ß-cyclodextrin:benzimidazole inclusion complexes as caps on the mesoporous silica face. The nanodevice is able to selectively deliver the cargo in the presence of acetylcholine via enzyme-mediated acetylcholine hydrolysis, locally lowering the pH and opening the supramolecular gate. Given the key role played by ACh and its relation with Parkinson's disease and other nervous system diseases, we believe that these findings could help design new therapeutic strategies.

Nitric Oxide Chemical Donor Affects the Early Phases of In Vitro Wound Healing Process.

An artificial wound in a confluent monolayer of human keratinocyte HaCaT cells or mouse embryo fibroblast Swiss NIH 3T3 cells was used to analyze the effects of the nitric oxide (NO) chemical donor, S-nitroso-N-acetylpenicillamine (SNAP). SNAP exposure promoted an enhanced rate of wound closure and accelerated motility of both keratinocytes and fibroblasts compared to control cells. The wounded monolayer cultures of HaCaT and NIH 3T3 cells, treated with or without SNAP, were monitored under a phase contrast microscope. Structural and ultrastructural modifications were analyzed by scanning electron microscopy (SEM). The images were captured by a digital camera at different time points (0-28 h) and the wound area was analyzed through software included in Matlab®. As early as 15 min, SNAP induced significant cytoskeletal remodeling, as shown by immunostaining (phalloidin-labelling), which in turn was associated with increased filopodium number and length rise. NO donor treatment also induced overexpression of Ki-67 protein, a typical marker of cell proliferation, as shown by immunostaining. Both SNAP-induced migration and proliferation were antagonized by the NO-sensitive GC inhibitor 1H-[1,2,4]oxadiazolo[-4,3-a]quinoxalin-1-one (ODQ), which suggests activation of the NO/cGMP signalling cascade in the observed SNAP-induced effects in the early stages of the healing process. Moreover, we provide evidence that PPAR-ß antagonist (GSK0660) may interfere with NO-mediated wound healing process. J. Cell. Physiol. 231: 2185-2195, 2016. © 2016 Wiley Periodicals, Inc.

Polyglutamic Acid-Gated Mesoporous Silica Nanoparticles for Enzyme-Controlled Drug Delivery.

Mesoporous silica nanoparticles (MSNs) are highly attractive as supports in the design of controlled delivery systems that can act as containers for the encapsulation of therapeutic agents, overcoming common issues such as poor water solubility and poor stability of some drugs and also enhancing their bioavailability. In this context, we describe herein the development of polyglutamic acid (PGA)-capped MSNs that can selectively deliver rhodamine B and doxorubicin. PGA-capped MSNs remain closed in an aqueous environment, yet they are able to deliver the cargo in the presence of pronase because of the hydrolysis of the peptide bonds in PGA. The prepared solids released less than 20% of the cargo in 1 day in water, whereas they were able to reach 90% of the maximum release of the entrapped guest in ca. 5 h in the presence of pronase. Studies of the PGA-capped nanoparticles with SK-BR-3 breast cancer cells were also undertaken. Rhodamine-loaded nanoparticles were not toxic, whereas doxorubicin-loaded nanoparticles were able to efficiently kill more than 90% of the cancer cells at a concentration of 100 µg/mL.

Improving the outcome of fractional CO2 laser resurfacing using a probiotic skin cream: Preliminary clinical evaluation.

As known, fractional CO2 resurfacing treatments are more effective than non-ablative ones against aging signs, but post-operative redness and swelling prolong the overall downtime requiring up to steroid administration in order to reduce these local systems. In the last years, an increasing interest has been focused on the possible use of probiotics for treating inflammatory and allergic conditions suggesting that they can exert profound beneficial effects on skin homeostasis. In this work, the Authors report their experience on fractional CO2 laser resurfacing and provide the results of a new post-operative topical treatment with an experimental cream containing probiotic-derived active principles potentially able to modulate the inflammatory reaction associated to laser-treatment. The cream containing DermaACB (CERABEST™) was administered post-operatively to 42 consecutive patients who were treated with fractional CO2 laser. All patients adopted the cream twice a day for 2 weeks. Grades were given according to outcome scale. The efficacy of the cream containing DermaACB was evaluated comparing the rate of post-operative signs vanishing with a control group of 20 patients topically treated with an antibiotic cream and a hyaluronic acid based cream. Results registered with the experimental treatment were good in 22 patients, moderate in 17, and poor in 3 cases. Patients using the study cream took an average time of 14.3 days for erythema resolution and 9.3 days for swelling vanishing. The post-operative administration of the cream containing DermaACB induces a quicker reduction of post-operative erythema and swelling when compared to a standard treatment.

In vitro evaluation of different methods of handling human liposuction aspirate and their effect on adipocytes and adipose derived stem cells.

Nowadays, fat tissue transplantation is widely used in regenerative and reconstructive surgery. However, a shared method of lipoaspirate handling for ensuring a good quality fat transplant has not yet been established. The study was to identify a method to recover from the lipoaspirate samples the highest number of human viable adipose tissue-derived stem cells (hADSCs) included in stromal vascular fraction (SVF) cells and of adipocytes suitable for transplantation, avoiding an extreme handling. We compared the lipoaspirate spontaneous stratification (10-20-30 min) with the centrifugation technique at different speeds (90-400-1500 × g). After each procedure, lipoaspirate was separated into top oily lipid layer, liquid fraction, "middle layer", and bottom layer. We assessed the number of both adipocytes in the middle layer and SVF cells in all layers. The histology of middle layer and the surface phenotype of SVF cells by stemness markers (CD105+, CD90+, CD45-) was analyzed as well. The results showed a normal architecture in all conditions except for samples centrifuged at 1500 × g. In both methods, the flow cytometry analysis showed that greater number of ADSCs was in middle layer; in the fluid portion and in bottom layer was not revealed significant expression levels of stemness markers. Our findings indicate that spontaneous stratification at 20 min and centrifugation at 400 × g are efficient approaches to obtain highly viable ADSCs cells and adipocytes, ensuring a good thickness of lipoaspirate for autologous fat transfer. Since an important aspect of surgery practice consists of gain time, the 400 × g centrifugation could be the recommended method when the necessary instrumentation is available.

Immunophenotypic characterization of human glioblastoma stem cells: correlation with clinical outcome.

Recently, glioma stem cells have been identified as the main cause of glioma propagation and recurrence and a number of several cell markers have been indicated as putative GSC markers. In the present work, a retrospective study to evaluate the prognostic potential of ability to generate GSCs in our series of 15 glioblastoma patients is described. ß-tubulin III, nestin, CD133, GFAP, and SOX-2 marker expression, both in primary GBM cultures and in respective glioblastoma stem cells (GSCs), was evaluated by flow cytometric analysis. Our results demonstrated various expression levels of these markers in both cell cultures; of note, only those cells expressing SOX-2 at greater than 30% levels were able to produce in vitro neurospheres. Moreover, statistical analysis revealed that the GSCs generation negatively affected overall survival (OS) (P = 0.000) and progression-free survival (PFS) (P = 0.001). In addition, a very poor OS (P = 0.000) and PFS (P = 0.000) were observed among patients whose tumors expressed Ki67, evaluated by immunohistochemistry, and showed the ability to generate in vitro GSCs. Overall, the results suggest that in vitro GSCs generation associated to the expression of Ki67 and SOX-2 may be useful to identify patients at risk of disease progression.

Caspase 3 Targeted Cargo Delivery in Apoptotic Cells Using Capped Mesoporous Silica Nanoparticles.

Excessive apoptotic cell death is at the origin of several pathologies, such as degenerative disorders, stroke or ischemia-reperfusion damage. In this context, strategies to improve inhibition of apoptosis and other types of cell death are of interest and may represent a pharmacological opportunity for the treatment of cell-death-related disorders. In this scenario new peptide-containing delivery systems (solids S1 -P1 and S1 -P2 ) are described based on mesoporous silica nanoparticles (MSNs) loaded with a dye and capped with the KKGDEVDKKARDEVDK (P1 ) peptide that contains two repeats of the DEVD target sequence that are selectively hydrolyzed by caspase 3 (C3). This enzyme plays a central role in the execution-phase of apoptosis. HeLa cells electroporated with S1 -P1 are able to deliver the cargo in the presence of staurosporin (STS), which induces apoptosis with the consequent activation of the cytoplasmic C3 enzyme. Moreover, the nanoparticles S1 -P2 , containing both a cell-penetrating TAT peptide and P1 also entered in HeLa cells and delivered the cargo preferentially in cells treated with the apoptosis inducer cisplatin.

Gated mesoporous silica nanoparticles for the controlled delivery of drugs in cancer cells.

In recent years, mesoporous silica nanoparticles (MSNs) have been used as effective supports for the development of controlled-release nanodevices that are able to act as multifunctional delivery platforms for the encapsulation of therapeutic agents, enhancing their bioavailability and overcoming common issues such as poor water solubility and poor stability of some drugs. In particular, redox-responsive delivery systems have attracted the attention of scientists because of the intracellular reductive environment related to a high concentration of glutathione (GSH). In this context, we describe herein the development of a GSH-responsive delivery system based on poly(ethylene glycol)- (PEG-) capped MSNs that are able to deliver safranin O and doxorubicin in a controlled manner. The results showed that the PEG-capped systems designed in this work can be maintained closed at low GSH concentrations, yet the cargo can be delivered when the concentration of GSH is increased. Moreover, the efficacy of the PEG-capped system in delivering the cytotoxic agent doxorubicin in cells was also demonstrated.

Toward the design of smart delivery systems controlled by integrated enzyme-based biocomputing ensembles.

We report herein the design of a smart delivery system in which cargo delivery from capped mesoporous silica (MS) nanoparticles is controlled by an integrated enzyme-based "control unit". The system consists of Janus-type nanoparticles having opposing Au and MS faces, functionalized with a pH-responsive ß-cyclodextrin-based supramolecular nanovalve on the MS surface and two effectors, glucose oxidase and esterase, immobilized on the Au face. The nanodevice behaves as an enzymatic logical OR operator which is selectively fueled by the presence of D-glucose and ethyl butyrate.

Cathepsin-B induced controlled release from peptide-capped mesoporous silica nanoparticles.

New capped silica mesoporous nanoparticles for intracellular controlled cargo release within cathepsin B expressing cells are described. Nanometric mesoporous MCM-41 supports loaded with safranin O (S1-P) or doxorubicin (S2-P) containing a molecular gate based on a cathepsin B target peptidic sequence were synthesized. Solids were designed to show "zero delivery" and to display cargo release in the presence of cathepsin B enzyme, which selectively hydrolyzed in vitro the capping peptide sequence. Controlled delivery in HeLa, MEFs WT, and MEFs lacking cathepsin B cell lines were also tested. Release of safranin O and doxorubicin in these cells took place when cathepsin B was active or present. Cells treated with S2-P showed a fall in cell viability due to nanoparticles internalization, cathepsin B hydrolysis of the capping peptide, and cytotoxic agent delivery, proving the possible use of these nanodevices as new therapeutic tools for cancer treatment.

Enzyme-responsive intracellular-controlled release using silica mesoporous nanoparticles capped with ε-poly-L-lysine.

The synthesis and characterization of two new capped silica mesoporous nanoparticles for controlled delivery purposes are described. Capped hybrid systems consist of MCM-41 nanoparticles functionalized on the outer surface with polymer ε-poly-L-lysine by two different anchoring strategies. In both cases, nanoparticles were loaded with model dye molecule [Ru(bipy)3](2+). An anchoring strategy involved the random formation of urea bonds by the treatment of propyl isocyanate-functionalized MCM-41 nanoparticles with the lysine amino groups located on the ε-poly-L-lysine backbone (solid Ru-rLys-S1). The second strategy involved a specific attachment through the carboxyl terminus of the polypeptide with azidopropyl-functionalized MCM-41 nanoparticles (solid Ru-tLys-S1). Once synthesized, both nanoparticles showed a nearly zero cargo release in water due to the coverage of the nanoparticle surface by polymer ε-poly-L-lysine. In contrast, a remarkable payload delivery was observed in the presence of proteases due to the hydrolysis of the polymer's amide bonds. Once chemically characterized, studies of the viability and the lysosomal enzyme-controlled release of the dye in intracellular media were carried out. Finally, the possibility of using these materials as drug-delivery systems was tested by preparing the corresponding ε-poly-L-lysine capped mesoporous silica nanoparticles loaded with cytotoxic drug camptothecin (CPT), CPT-rLys-S1 and CPT-tLys-S1. Cellular uptake and cell-death induction were studied. The efficiency of both nanoparticles as new potential platforms for cancer treatment was demonstrated.

Self-propelled enzyme-controlled IR-mesoporous silica Janus nanomotor for smart delivery.

Here, we report the preparation of a novel Janus nanoparticle with opposite Ir and mesoporous silica nanoparticles through a partial surface masking with toposelective modification method. This nanomaterial was employed to construct an enzyme-powered nanomachine with self-propulsion properties for on-command delivery. The cargo-loaded nanoparticle was provided with a pH-sensitive gate and unit control at the mesoporous face by first attaching boronic acid residues and further immobilization of glucose oxidase through reversible boronic acid esters with the carbohydrate residues of the glycoenzyme. Addition of glucose leads to the enzymatic production of H2O2 and gluconic acid, being the first compound catalytically decomposed at the Ir nanoparticle face producing O2 and causing the nanomachine propulsion. Gluconic acid leads to a pH reduction at the nanomachine microenvironment causing the disruption of the gating mechanism with the subsequent cargo release. This work demonstrates that enzyme-mediated self-propulsion improved release efficiency being this nanomotor successfully employed for the smart release of Doxorubicin in HeLa cancer cells.

In Situ-Forming Gels Loaded with Stimuli-Responsive Gated Mesoporous Silica Nanoparticles for Local Sustained Drug Delivery.

A novel combination of in situ-forming hydrogels of hyaluronic acid with gated mesoporous materials was developed to design depots for local sustained release of chemotherapeutics. The depot consists of a hyaluronic-based gel loaded with redox-responsive mesoporous silica nanoparticles loaded with safranin O or doxorubicin and capped with polyethylene glycol chains containing a disulfide bond. The nanoparticles are able to deliver the payload in the presence of the reducing agent, glutathione (GSH), that promotes the cleavage of the disulfide bonds and the consequent pore opening and cargo delivery. Release studies and cellular assays demonstrated that the depot can successfully liberate the nanoparticles to the media and, subsequently, that the nanoparticles are internalized into the cells where the high concentration of GSH induces cargo delivery. When the nanoparticles were loaded with doxorubicin, a significant reduction in cell viability was observed. Our research opens the way to the development of new depots that enhance the local controlled release of chemotherapeutics by combining the tunable properties of hyaluronic gels with a wide range of gated materials.

Azobenzene polyesters used as gate-like scaffolds in nanoscopic hybrid systems.

The synthesis and characterisation of new capped silica mesoporous nanoparticles for on-command delivery applications is reported. Functional capped hybrid systems consist of MCM-41 nanoparticles functionalised on the external surface with polyesters bearing azobenzene derivatives and rhodamine B inside the mesopores. Two solid materials, Rh-PAzo8-S and Rh-PAzo6-S, containing two closely related polymers, PAzo8 and PAzo6, in the pore outlets have been prepared. Materials Rh-PAzo8-S and Rh-PAzo6-S showed an almost zero release in water due to steric hindrance imposed by the presence of anchored bulky polyesters, whereas a large delivery of the cargo was observed in the presence of an esterase enzyme due to the progressive hydrolysis of polyester chains. Moreover, nanoparticles Rh-PAzo8-S and Rh-PAzo6-S were used to study the controlled release of the dye in intracellular media. Nanoparticles were not toxic for HeLa cells and endocytosis-mediated cell internalisation was confirmed by confocal microscopy. Furthermore, the possible use of capped materials as a drug-delivery system was demonstrated by the preparation of a new mesoporous silica nanoparticle functionalised with PAzo6 and loaded with the cytotoxic drug camptothecin (CPT-PAzo6-S). Following cell internalisation and lysosome resident enzyme-dependent gate opening, CPT-PAzo6-S induced CPT-dependent cell death in HeLa cells.

A ß-Cyclodextrin-Based Nanoparticle with Very High Transfection Efficiency Unveils siRNA-Activated TLR3 Responses in Human Prostate Cancer Cells.

Synthetic double-stranded small interfering RNAs (siRNAs) mimic interference RNAs (RNAi) and can bind target mRNAs with a high degree of specificity, leading to selective knockdown of the proteins they encode. However, siRNAs are very labile and must be both protected and transported by nanoparticles to be efficiently delivered into cells. In this work, we used a Janus-type polycationic amphiphilic ß-cyclodextrin derivative to efficiently transfect siRNAs targeting mRNAs encoding mitogen-activated protein kinase (p42-MAPK) or Ras homolog enriched in brain (Rheb) into different cancer cell lines as well as astrocytes. We took advantage of this high transfection efficiency to simultaneously knock down p42-MAPK and Rheb to boost docetaxel (DTX)-mediated toxicity in two human prostate cancer cell lines (LNCaP and PC3). We found that double knockdown of p42-MAPK and Rheb increased DTX-toxicity in LNCaP but not in PC3 cells. However, we also observed the same effect when scramble siRNA was used, therefore pointing to an off-target effect. Indeed, we found that the siRNA we used in this work induced toll-like receptor 3 activation, leading to ß-interferon production and caspase activation. We believe that this mechanism could be very useful as a general strategy to elicit an immune response against prostate cancer cells.

Enhancement of photoactivity and cellular uptake of (Bu4N)2[Mo6I8(CH3COO)6] complex by loading on porous MCM-41 support. Photodynamic studies as an anticancer agent.

The incorporation by ionic assembly of the hexanuclear molybdenum cluster (Bu4N)2[Mo6I8(CH3CO2)6] (1) in amino-decorated mesoporous silica nanoparticles MCM-41, has yielded the new molybdenum-based hybrid photosensitizer 1@MCM-41. The new photoactive material presents a high porosity, due to the intrinsic high specific surface area of MCM-41 nanoparticles (989 m2 g-1) which is responsible for the good dispersion of the hexamolybdenum clusters on the nanoparticles surface, as observed by STEM analysis. The hybrid photosensitizer can generate efficiently singlet oxygen, which was demonstrated by using the benchmark photooxygenation reaction of 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA) in water. The photodynamic therapy activity has been tested using LED light as an irradiation source (λirr ~ 400-700 nm; 15.6 mW/cm2). The results show a good activity of the hybrid photosensitizer against human cervical cancer (HeLa) cells, reducing up to 70 % their viability after 20 min of irradiation, whereas low cytotoxicity is detected in the darkness. The main finding of this research is that the incorporation of molybdenum complexes at porous MCM-41 supports enhances their photoactivity and improves cellular uptake, compared to free clusters.