Modifying the Secretome of Mesenchymal Stem Cells Prolongs the Regenerative Treatment Window for Encephalopathy of Prematurity.

Clinical treatment options to combat Encephalopathy of Prematurity (EoP) are still lacking. We, and others, have proposed (intranasal) mesenchymal stem cells (MSCs) as a potent therapeutic strategy to boost white matter repair in the injured preterm brain. Using a double-hit mouse model of diffuse white matter injury, we previously showed that the efficacy of MSC treatment was time dependent, with a significant decrease in functional and histological improvements after the postponement of cell administration. In this follow-up study, we aimed to investigate the mechanisms underlying this loss of therapeutic efficacy. Additionally, we optimized the regenerative potential of MSCs by means of genetic engineering with the transient hypersecretion of beneficial factors, in order to prolong the treatment window. Though the cerebral expression of known chemoattractants was stable over time, the migration of MSCs to the injured brain was partially impaired. Moreover, using a primary oligodendrocyte (OL) culture, we showed that the rescue of injured OLs was reduced after delayed MSC coculture. Cocultures of modified MSCs, hypersecreting IGF1, LIF, IL11, or IL10, with primary microglia and OLs, revealed a superior treatment efficacy over naïve MSCs. Additionally, we showed that the delayed intranasal administration of IGF1-, LIF-, or IL11-hypersecreting MSCs, improved myelination and the functional outcome in EoP mice. In conclusion, the impaired migration and regenerative capacity of intranasally applied MSCs likely underlie the observed loss of efficacy after delayed treatment. The intranasal administration of IGF1-, LIF-, or IL11-hypersecreting MSCs, is a promising optimization strategy to prolong the window for effective MSC treatment in preterm infants with EoP.

Matrix metalloproteinase degradable, in situ photocrosslinked nanocomposite bioinks for bioprinting applications.

The development of suitable bioinks with high printability, mechanical strength, biodegradability, and biocompatibility is a key challenge for the clinical translation of 3D constructs produced with bioprinting technologies. In this work, we developed a new type of nanocomposite bioinks containing thiolated mesoporous silica nanoparticles (MSN) that act as active fillers within norbornene-functionalized hydrogels. The MSNs could rapidly covalently crosslink the hydrogels upon exposure to UV light. The mechanical properties of the gels could be modulated from 9.3 to 19.7 kPa with increasing concentrations of MSN. The ability of the MSN to covalently crosslink polymeric networks was, however, significantly influenced by polymer architecture and the number of functional groups. Modification of the outer surface of MSNs with matrix metalloproteinase (MMP) sensitive peptides (MSN-MMPs) resulted in proteinase K and MMP-9 enzyme responsive biodegradable bioinks. Additional cysteine modified RGD peptide incorporation enhanced cell-matrix interactions and reduced the gelation time for bioprinting. The nanocomposite bioinks could be printed by using extrusion-based bioprinting. Our nanocomposite bioinks preserved their shape during in vitro studies and encapsulated MG63 cells preserved their viability and proliferated within the bioinks. As such, our nanocomposite bioinks are promising bioinks for creating bioprinted constructs with tunable mechanical and degradation properties.

Development of Mesoporous Silica Nanoparticle-Based Films with Tunable Arginine-Glycine-Aspartate Peptide Global Density and Clustering Levels to Study Stem Cell Adhesion and Differentiation.

Stem cell adhesion is mediated via the binding of integrin receptors to adhesion motifs present in the extracellular matrix (ECM). The spatial organization of adhesion ligands plays an important role in stem cell integrin-mediated adhesion. In this study, we developed a series of biointerfaces using arginine-glycine-aspartate (RGD)-functionalized mesoporous silica nanoparticles (MSN-RGD) to study the effect of RGD adhesion ligand global density (ligand coverage over the surface), spacing, and RGD clustering levels on stem cell adhesion and differentiation. To prepare the biointerface, MSNs were chemically functionalized with RGD peptides via an antifouling poly(ethylene glycol) (PEG) linker. The RGD surface functionalization ratio could be controlled to create MSNs with high and low RGD ligand clustering levels. MSN films with varying RGD global densities could be created by blending different ratios of MSN-RGD and non-RGD-functionalized MSNs together. A computational simulation study was performed to analyze nanoparticle distribution and RGD spacing on the resulting surfaces to determine experimental conditions. Enhanced cell adhesion and spreading were observed when RGD global density increased from 1.06 to 5.32 nmol cm-2 using highly clustered RGD-MSN-based films. Higher RGD ligand clustering levels led to larger cell spreading and increased formation of focal adhesions. Moreover, a higher RGD ligand clustering level promoted the expression of alkaline phosphatase in hMSCs. Overall, these findings indicate that both RGD global density and clustering levels are crucial variables in regulating stem cell behaviors. This study provides important information about ligand-integrin interactions, which could be implemented into biomaterial design to achieve optimal performance of adhesive functional peptides.

Selenium-incorporated mesoporous silica nanoparticles for osteosarcoma therapy.

Selenium (Se) compounds are promising chemotherapeutics due to their ability to inhibit cancer cell activity via the generation of reactive oxygen species (ROS). However, to circumvent adverse effects on bone healthy cells, new methods are needed to allow intracellular Se delivery. Mesoporous silica nanoparticles (MSNs) are promising carriers for therapeutic ion delivery due to their biocompability, rapid uptake via endocytosis, and ability to efficiently incorporate ions within their tunable structure. With the aim of selectively inhibiting cancer cells, here we developed three types of MSNs and investigated their ability to deliver Se. Specifically, MSNs containing SeO32- loaded on the surface and in the pores (MSN-SeL), SeO32- doped in the silica matrix (Se-MSNs) and Se nanoparticles (SeNP) coated with mesoporous silica (SeNP-MSNs), were successfully synthesized. All synthesized nanoparticles were stable in neutral conditions but showed rapid Se release in the presence of glutathione (GSH) and nicotinamide adenine dinucleotide phosphate (NADPH). Furthermore, all nanoparticles were cytotoxic towards SaoS-2 cells and showed significantly lower toxicity towards healthy osteoblasts, where Se doped MSNs showed lowest toxicity towards osteoblasts. We further show that the nanoparticles could induce ROS and cell apoptosis. Here we demonstrate MSNs as promising Se delivery carriers for osteosarcoma (OS) therapy.

Hydroxyapatite nanoparticles-cell interaction: New approaches to disclose the fate of membrane-bound and internalised nanoparticles.

Hydroxyapatite nanoparticles are popular tools in bone regeneration, but they have also been used for gene delivery and as anticancer drugs. Understanding their mechanism of action, particularly for the latter application, is crucial to predict their toxicity. To this end, we aimed to elucidate the importance of nanoparticle membrane interactions in the cytotoxicity of MG-63 cells using two different types of nanoparticles. In addition, conventional techniques for studying nanoparticle internalisation were evaluated and compared with newer and less exploited approaches. Hydroxyapatite and magnesium-doped hydroxyapatite nanoparticles were used as suspensions or compacted as specular discs. Comparison between cells seeded on the discs and those supplemented with the nanoparticles allowed direct interaction of the cell membrane with the material to be ruled out as the main mechanism of toxicity. In addition, standard techniques such as flow cytometry were inconclusive when used to assess nanoparticles toxicity. Interestingly, the use of intracellular calcium fluorescent probes revealed the presence of a high number of calcium-rich vesicles after nanoparticle supplementation in cell culture. These structures could not be detected by transmission electron microscopy due to their liquid content. However, by using cryo-soft X-ray imaging, which was used to visualise the cellular ultrastructure without further treatment other than vitrification and to quantify the linear absorption coefficient of each organelle, it was possible to identify them as multivesicular bodies, potentially acting as calcium stores. In the study, an advanced state of degradation of the hydroxyapatite and magnesium-doped hydroxyapatite nanoparticles within MG-63 cells was observed. Overall, we demonstrate that the combination of fluorescent calcium probes together with cryo-SXT is an excellent approach to investigate intracellular calcium, especially when found in its soluble form.

Mesoporous Silica-Coated Gold Nanoparticles for Multimodal Imaging and Reactive Oxygen Species Sensing of Stem Cells.

Stem cell (SC)-based therapies hold the potential to revolutionize therapeutics by enhancing the body's natural repair processes. Currently, there are only three SC therapies with marketing authorization within the European Union. To optimize outcomes, it is important to understand the biodistribution and behavior of transplanted SCs in vivo. A variety of imaging agents have been developed to trace SCs; however, they mostly lack the ability to simultaneously monitor the SC function and biodistribution at high resolutions. Here, we report the synthesis and application of a nanoparticle (NP) construct consisting of a gold NP core coated with rhodamine B isothiocyanate (RITC)-doped mesoporous silica (AuMS). The MS layer further contained a thiol-modified internal surface and an amine-modified external surface for dye conjugation. Highly fluorescent AuMS of three different sizes were successfully synthesized. The NPs were non-toxic and efficiently taken up by limbal epithelial SCs (LESCs). We further showed that we can functionalize AuMS with a reactive oxygen species (ROS)-sensitive fluorescent dye using two methods, loading the probe into the mesopores, with or without additional capping by a lipid bilayer, and by covalent attachment to surface and/or mesoporous-functionalized thiol groups. All four formulations displayed a ROS concentration-dependent increase in fluorescence. Further, in an ex vivo SC transplantation model, a combination of optical coherence tomography and fluorescence microscopy was used to synergistically identify AuMS-labeled LESC distribution at micrometer resolution. Our AuMS constructs allow for multimodal imaging and simultaneous ROS sensing of SCs and represent a promising tool for in vivo SC tracing.

Peptide-Modified Nano-Bioactive Glass for Targeted Immobilization of Native VEGF.

A limiting factor in large bone defect regeneration is the slow and disorganized formation of a functional vascular network in the defect area, often resulting in delayed healing or implant failure. To overcome this, strategies that induce angiogenic processes should be combined with potent bone graft substitutes in new bone regeneration approaches. To this end, we describe a unique approach to immobilize the pro-angiogenic growth factor VEGF165 in its native state on the surface of nanosized bioactive glass particles (nBGs) via a binding peptide (PR1P). We demonstrate that covalent coupling of the peptide to amine functional groups grafted on the nBG surface allows immobilization of VEGF with high efficiency and specificity. The amount of coupled peptide could be controlled by varying amine density, which eventually allows tailoring the amount of bound VEGF within a physiologically effective range. In vitro analysis of endothelial cell tube formation in response to VEGF-carrying nBG confirmed that the biological activity of VEGF is not compromised by the immobilization. Instead, comparable angiogenic stimulation was found for lower doses of immobilized VEGF compared to exogenously added VEGF. The described system, for the first time, employs a binding peptide for growth factor immobilization on bioactive glass nanoparticles and represents a promising strategy to overcome the problem of insufficient neovascularization in large bone defect regeneration.

The shape-effect of calcium phosphate nanoparticle based films on their osteogenic properties.

Calcium phosphates (CaPs) in the form of hydroxyapatite (HA) have been extensively studied in the context of bone regeneration due to their chemical similarity to natural bone mineral. While HA is known to promote osteogenic differentiation, the structural properties of the ceramic have been shown to affect the extent of this effect; several studies have suggested that nanostructured HA can improve the bioactivity. However, the role shape plays in the osteogenic potential is more elusive. Here we studied the effect of HA nanoparticle shape on the ability to induce osteogenesis in human mesenchymal stromal cells (hMSCs) by developing nanoparticle films using needle-, rice- and spherical-shaped HA. We showed that the HA films made from all three shapes of nanoparticles induced increased levels of osteogenic markers (i.e. runt-related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP2), alkaline phosphatase (ALP), osteopontin (OPN), osteocalcin (OCN) on protein and gene level in comparison to hMSCs cultured on cover glass slides. Furthermore, their expression levels and profiles differed significantly as a function of nanoparticle shape. We also showed that nanoparticle films were more efficient in inducing osteogenic gene expression in hMSCs compared to adding nanoparticles to hMSCs in culture media. Finally, we demonstrated that hMSC morphology upon adhesion to the HA nanoparticle films is dependent on nanoparticle shape, with hMSCs exhibiting a more spread morphology on needle-shaped nanoparticle films compared to hMSCs seeded on rice- and spherical-shaped nanoparticle films. Our data suggests that HA nanoparticle films are efficient in inducing hMSC osteogenesis in basic cell culture conditions and that nanoparticle shape plays a vital role in cell adhesion and morphology and extent of induction of osteogenic differentiation.

Intranasal mesenchymal stem cell therapy to boost myelination after encephalopathy of prematurity.

Encephalopathy of prematurity (EoP) is a common cause of long-term neurodevelopmental morbidity in extreme preterm infants. Diffuse white matter injury (dWMI) is currently the most commonly observed form of EoP. Impaired maturation of oligodendrocytes (OLs) is the main underlying pathophysiological mechanism. No therapies are currently available to combat dWMI. Intranasal application of mesenchymal stem cells (MSCs) is a promising therapeutic option to boost neuroregeneration after injury. Here, we developed a double-hit dWMI mouse model and investigated the therapeutic potential of intranasal MSC therapy. Postnatal systemic inflammation and hypoxia-ischemia led to transient deficits in cortical myelination and OL maturation, functional deficits and neuroinflammation. Intranasal MSCs migrated dispersedly into the injured brain and potently improved myelination and functional outcome, dampened cerebral inflammationand rescued OL maturation after dWMI. Cocultures of MSCs with primary microglia or OLs show that MSCs secrete factors that directly promote OL maturation and dampen neuroinflammation. We show that MSCs adapt their secretome after ex vivo exposure to dWMI milieu and identified several factors including IGF1, EGF, LIF, and IL11 that potently boost OL maturation. Additionally, we showed that MSC-treated dWMI brains express different levels of these beneficial secreted factors. In conclusion, the combination of postnatal systemic inflammation and hypoxia-ischemia leads to a pattern of developmental brain abnormalities that mimics the clinical situation. Intranasal delivery of MSCs, that secrete several beneficial factors in situ, is a promising strategy to restore myelination after dWMI and subsequently improve the neurodevelopmental outcome of extreme preterm infants in the future.

Organ-restricted vascular delivery of nanoparticles for lung cancer therapy.

Nanoparticle-based targeted drug delivery holds promise for treatment of cancers. However, most approaches fail to be translated into clinical success due to ineffective tumor targeting in vivo. Here, the delivery potential of mesoporous silica nanoparticles (MSN) functionalized with targeting ligands for EGFR and CCR2 is explored in lung tumors. The addition of active targeting ligands on MSNs enhances their uptake in vitro but fails to promote specific delivery to tumors in vivo, when administered systemically via the blood or locally to the lung into immunocompetent murine lung cancer models. Ineffective tumor targeting is due to efficient clearance of the MSNs by the phagocytic cells of the liver, spleen, and lung. These limitations, however, are successfully overcome using a novel organ-restricted vascular delivery (ORVD) approach. ORVD in isolated and perfused mouse lungs of Kras-mutant mice enables effective nanoparticle extravasation from the tumor vasculature into the core of solid lung tumors. In this study, ORVD promotes tumor cell-specific uptake of nanoparticles at cellular resolution independent of their functionalization with targeting ligands. Organ-restricted vascular delivery thus opens new avenues for optimized nanoparticles for lung cancer therapy and may have broad applications for other vascularized tumor types.

Guiding mesenchymal stem cell differentiation using mesoporous silica nanoparticle-based films.

The development of smart interfaces that can guide tissue formation is of great importance in the field of regenerative medicine. Nanoparticles represent an interesting class of materials that can be used to enhance regenerative treatments by enabling close control over surface properties and directing cellular responses. Moreover, nanoparticles can be used to provide temporally controlled delivery of (multiple) biochemical compounds. Here, we exploited the cargo loading and surface functionalization properties of mesoporous silica nanoparticles (MSNs) to design films that can guide human mesenchymal stem cell (hMSC) differentiation towards the osteogenic lineage. We developed biocompatible MSN-based films that support stem cell adhesion and proliferation and demonstrated that these MSN films simultaneously allowed efficient local delivery of biomolecules without effecting film integrity. Films loaded with the osteogenesis-stimulating drug dexamethasone (Dex) were able to induce osteogenic differentiation of hMSCs in vitro. Dex delivery from the films led to increased alkaline phosphatase levels and matrix mineralization compared to directly supplementing Dex to the medium. Furthermore, we demonstrated that Dex release kinetics can be modulated using surface modifications with supported lipid bilayers. Together, these data demonstrate that MSN films represent an interesting approach to create biomaterial interfaces with controllable biomolecule release and surface properties to improve the bioactivity of biomaterials. STATEMENT OF SIGNIFICANCE: Engineering surfaces that can control cell and tissue responses is one of the major challenges in biomaterials-based regenerative therapies. Here, we demonstrate the potential of mesoporous silica nanoparticles (MSNs) as drug-delivering surface coatings. First, we show differentiation of mesenchymal stem cells towards the bone lineage when in contact with MSN films loaded with dexamethasone. Furthermore, we demonstrate that modification of MSNs with supported lipid bilayer allows control over drug release dynamics and cell shape. Given the range of loadable cargos and the tunability of release kinetics, MSN coatings can be used to mimic the sequential appearance of bioactive factors during tissue regeneration, which will ultimately lead to biomaterials with improved bioactivity.

Lipid surface modifications increase mesoporous silica nanoparticle labeling properties in mesenchymal stem cells.

BACKGROUND: Nanoparticles have emerged as promising cell-labeling tools, as they can be precisely tailored in terms of chemical and physical properties. Mesoporous silica nanoparticles (MSNs), in particular, are easily tunable with regard to surface and core chemistry, and are able to confine dyes and drug molecules efficiently. PURPOSE: The aim of this study was to investigate the effect of lipid and polyethylene glycol (PEG) surface modifications on MSN stem-cell-tracking abilities. METHODS: Lipid and PEG surface functionalized MSNs were synthesized and the effect of surface functionalization on cell internalization, proliferation, differentiation and cell proteomics was investigated in patient derived mesenchymal stem cells (MSCs). RESULTS: MSNs and lipid surface-modified MSNs were internalized by >80% of the MSC population, with the exception of nanoparticles modified with short PEG chains (molecular weight 750 [MSN-PEG750]). Lipid-modified MSNs had higher labeling efficiency with maximum uptake after 2 hours of exposure and were in addition internalized 17 times higher compared to unmodified MSNs, without negatively affecting differentiation capacity. Using a mass-spectrometry-based label-free quantitative proteomics approach, we show that MSN labeling leads to the up- and downregulation of proteins that were unique for the different surface-modified MSNs. In addition, functional enrichments were found in human MSCs labeled with MSNs, MSN-PEG750, and lipid-modified MSNs. SUMMARY: Here we show that organic modifications with lipids and PEGylation can be used as a promising strategy to improve MSN labeling capabilities. In particular, we show that lipid modifications can optimize such probes in three distinct ways: significantly improved signal strength, a barrier for sustained release of additional probes, and improved stem-cell-labeling efficiency.

Development of a shear stress-free microfluidic gradient generator capable of quantitatively analyzing single-cell morphology.

Microfluidics, the science of engineering fluid streams at the micrometer scale, offers unique tools for creating and controlling gradients of soluble compounds. Gradient generation can be used to recreate complex physiological microenvironments, but is also useful for screening purposes. For example, in a single experiment, adherent cells can be exposed to a range of concentrations of the compound of interest, enabling high-content analysis of cell behaviour and enhancing throughput. In this study, we present the development of a microfluidic screening platform where, by means of diffusion, gradients of soluble compounds can be generated and sustained. This platform enables the culture of adherent cells under shear stress-free conditions, and their exposure to a soluble compound in a concentration gradient-wise manner. The platform consists of five serial cell culture chambers, all coupled to two lateral fluid supply channels that are used for gradient generation through a source-sink mechanism. Furthermore, an additional inlet and outlet are used for cell seeding inside the chambers. Finite element modeling was used for the optimization of the design of the platform and for validation of the dynamics of gradient generation. Then, as a proof-of-concept, human osteosarcoma MG-63 cells were cultured inside the platform and exposed to a gradient of Cytochalasin D, an actin polymerization inhibitor. This set-up allowed us to analyze cell morphological changes over time, including cell area and eccentricity measurements, as a function of Cytochalasin D concentration by using fluorescence image-based cytometry.

Enhancing regenerative approaches with nanoparticles.

In this review, we discuss recent developments in the field of nanoparticles and their use in tissue regeneration approaches. Owing to their unique chemical properties and flexibility in design, nanoparticles can be used as drug delivery systems, to create novel features within materials or as bioimaging agents, or indeed these properties can be combined to create smart multifunctional structures. This review aims to provide an overview of this research field where the focus will be on nanoparticle-based strategies to stimulate bone regeneration; however, the same principles can be applied for other tissue and organ regeneration strategies. In the first section, nanoparticle-based methods for the delivery of drugs, growth factors and genetic material to promote tissue regeneration are discussed. The second section deals with the addition of nanoparticles to materials to create nanocomposites. Such materials can improve several material properties, including mechanical stability, biocompatibility and biological activity. The third section will deal with the emergence of a relatively new field of research using nanoparticles in advanced cell imaging and stem cell tracking approaches. As the development of nanoparticles continues, incorporation of this technology in the field of regenerative medicine will ultimately lead to new tools that can diagnose, track and stimulate the growth of new tissues and organs.

Protease-mediated release of chemotherapeutics from mesoporous silica nanoparticles to ex vivo human and mouse lung tumors.

Nanoparticles allow for controlled and targeted drug delivery to diseased tissues and therefore bypass systemic side effects. Spatiotemporal control of drug release can be achieved by nanocarriers that respond to elevated levels of disease-specific enzymes. For example, matrix metalloproteinase 9 (MMP9) is overexpressed in tumors, is known to enhance the metastatic potency of malignant cells, and has been associated with poor prognosis of lung cancer. Here, we report the synthesis of mesoporous silica nanoparticles (MSNs) tightly capped by avidin molecules via MMP9 sequence-specific linkers to allow for site-selective drug delivery in high-expressing MMP9 tumor areas. We provide proof-of-concept evidence for successful MMP9-triggered drug release from MSNs in human tumor cells and in mouse and human lung tumors using the novel technology of ex vivo 3D lung tissue cultures. This technique allows for translational testing of drug delivery strategies in diseased mouse and human tissue. Using this method we show MMP9-mediated release of cisplatin, which induced apoptotic cell death only in lung tumor regions of Kras mutant mice, without causing toxicity in tumor-free areas or in healthy mice. The MMP9-responsive nanoparticles also allowed for effective combinatorial drug delivery of cisplatin and proteasome inhibitor bortezomib, which had a synergistic effect on the (therapeutic) efficiency. Importantly, we demonstrate the feasibility of MMP9-controlled drug release in human lung tumors.

Medical nanoparticles for next generation drug delivery to the lungs.

Respiratory diseases are an increasing burden for the ageing population. Although our understanding of these diseases has improved significantly over the past decades, diagnostic and therapeutic options for treating lung diseases, such as chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis and lung cancer, remain limited. Multidisciplinary approaches that bridge the gap between medicinal and materials sciences will likely contribute to promising new therapeutic and diagnostic solutions. One such multidisciplinary approach is the use of nanoparticles as carriers for the delivery of drugs. The advantages of using nanoparticles to deliver drugs include: increased drug concentration at the disease site; minimised drug degradation and loss; ease of creating inhalable formulations; and the possibility of specific cell targeting. This article gives a brief overview on the emerging field of nanocarriers as drug delivery vehicles for the treatment of lung diseases.

Potent organometallic osmium compounds induce mitochondria-mediated apoptosis and S-phase cell cycle arrest in A549 non-small cell lung cancer cells.

The problems of acquired resistance associated with platinum drugs may be addressed by chemotherapeutics based on other transition metals as they offer the possibility of novel mechanisms of action. In this study, the cellular uptake and induction of apoptosis in A549 human non-small cell lung cancer cells of three promising osmium(II) arene complexes containing azopyridine ligands, [Os(η(6)-arene)(p-R-phenylazopyridine)X]PF6, where arene is p-cymene or biphenyl, R is OH or NMe2, and X is Cl or I, were investigated. These complexes showed time-dependent (4­48 h) potent anticancer activity with highest potency after 24 h (IC50 values ranging from 0.1 to 3.6 µM). Cellular uptake of the three compounds as quantified by ICP-MS, was independent of their logP values (hydrophobicity). Furthermore, maximum cell uptake was observed after 24 h, with evident cell efflux of the osmium after 48 and 72 h of exposure, which correlated with the corresponding IC50 values. The most active compound 2, [Os(η(6)-p-cymene)(NMe2-phenylazopyridine)I]PF6, was taken up by lung cancer cells predominately in a temperature-dependent manner indicating that energy-dependent mechanisms are important in the uptake of 2. Cell fractionation studies showed that all three compounds accumulated mainly in cellular membranes. Furthermore, compound 2 induced apoptosis and caused accumulation in the S-phase of the cell cycle. In addition, 2 induced cytochrome c release and alterations in mitochondrial membrane potential even after short exposure times, indicating that mitochondrial apoptotic pathways are involved. This study represents the first steps towards understanding the mode of action of this promising class of new osmium-based chemotherapeutics.

Acute cigarette smoke exposure impairs proteasome function in the lung.

Cigarette smoke mediates DNA damage, lipid peroxidation, and modification and misfolding of proteins, thereby inducing severe cellular damage. The ubiquitin proteasome system serves as the major disposal system for modified and misfolded proteins and is thus essential for proper cellular function. Its role in cigarette smoke-induced cell damage, however, is largely unknown. We hypothesized that the ubiquitin-proteasome system is involved in the degradation of cigarette smoke-damaged proteins and that cigarette smoke exposure impairs the proteasome itself. Here, we show that treatment of human alveolar epithelial cells with cigarette smoke extract (CSE) induced time- and dose-dependent cell death, a rise in intracellular reactive oxygen species, and increased levels of carbonylated and polyubiquitinated proteins. While high doses of CSE severely impaired all three proteasomal activities, low CSE concentrations significantly inhibited only the trypsin-like activity of the proteasome in alveolar and bronchial epithelial cells. Moreover, acute exposure of mice to cigarette smoke significantly impaired the trypsin-like activity by 25% in the lungs. Reduced proteasome activity was not due to transcriptional regulation of the proteasome. Notably, cigarette smoke exposure induced accumulation of polyubiquitinated proteins in the soluble and insoluble protein fraction of the lung. We show for the first time that acute exposure to cigarette smoke directly impairs proteasome activity in the lungs of mice and in human epithelial cells at low doses without affecting proteasome expression. Our results indicate that defective proteasomal protein quality control may exacerbate the detrimental effects of cigarette smoke in the lung.

Organometallic osmium arene complexes with potent cancer cell cytotoxicity.

Iodido osmium(II) complexes [Os(η(6)-arene)(XY)I](+) (XY = p-hydroxy or p-dimethylaminophenylazopyridine, arene = p-cymene or biphenyl) are potently cytotoxic at nanomolar concentrations toward a panel of human cancer cell lines; e.g., IC(50) = 140 nM for [Os(η(6)-bip)(azpy-NMe(2))I](+) toward A2780 ovarian cancer cells. They exhibit low toxicity and negligible deleterious effects in a colon cancer xenograft model, giving rise to the possibility of a broad therapeutic window. The most active complexes are stable and inert toward aquation. Their cytotoxic activity appears to involve redox mechanisms.

Cytotoxicity, hydrophobicity, uptake, and distribution of osmium(II) anticancer complexes in ovarian cancer cells.

The cytotoxicity, hydrophobicity (log P), cellular uptake, aqueous reactivity, and extent of DNA adduct formation in the A2780 ovarian carcinoma cells for four osmium(II) arene complexes [(eta(6)-arene)Os(4-methyl-picolinate)Cl] that differ only in their arene ligands as benzene (1), p-cymene (2), biphenyl (3), or tetrahydroanthracene (4) are reported. There is a correlation between hydrophobicity (log P), cellular uptake, nucleus uptake, and cytotoxicity of the complexes, following the order 3 approximately 4 > 2 > 1, suggesting that the arene plays an important role in the biological activity of these types of compounds. Cell distribution studies using fractionation showed that all four compounds distribute similarly within cells. DNA binding of osmium did not correlate with cytotoxicity, indicating that the nature of the DNA lesion may also be crucial to activity. TEM images of ovarian cells treated with 3 revealed morphological changes associated with apoptosis with possible involvement of mitochondria.