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.

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.

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.

Amide linkage isomerism as an activity switch for organometallic osmium and ruthenium anticancer complexes.

We show that the binding mode adopted by picolinamide derivatives in organometallic Os(II) and Ru(II) half-sandwich complexes can lead to contrasting cancer cell cytotoxicity. N-Phenyl picolinamide derivatives (XY) in Os(II) (1, 3-5, 7, 9) and Ru(II) (2, 6, 8, 10) complexes [(eta(6)-arene)(Os/Ru)(XY)Cl](n+), where arene = p-cymene (1-8, 10) or biphenyl (9), can act as N,N- or N,O-donors. Electron-withdrawing substituents on the phenyl ring resulted in N,N-coordination and electron-donating substituents in N,O-coordination. Dynamic interconversion between N,O and N,N configurations can occur in solution and is time- and temperature- (irreversible) as well as pH-dependent (reversible). The neutral N,N-coordinated compounds (1-5 and 9) hydrolyzed rapidly (t(1/2) > 4 > 1 > 9). In contrast, N,O-coordinated complexes 7 and 8 hydrolyzed slowly, did not bind to guanine or adenine, and were nontoxic.

Current applications and future potential for bioinorganic chemistry in the development of anticancer drugs.

This review illustrates notable recent progress in the field of medicinal bioinorganic chemistry as many new approaches to the design of innovative metal-based anticancer drugs are emerging. Current research addressing the problems associated with platinum drugs has focused on other metal-based therapeutics that have different modes of action and on prodrug and targeting strategies in an effort to diminish the side-effects of cisplatin chemotherapy.

Organometallic osmium(II) arene anticancer complexes containing picolinate derivatives.

Chlorido osmium(II) arene [(eta(6)-biphenyl)Os(II)(X-pico)Cl] complexes containing X = Br (1), OH (2), and Me (3) as ortho, or X = Cl (4), CO(2)H (5), and Me (6) as para substituents on the picolinate (pico) ring have been synthesized and characterized. The X-ray crystal structures of 1 and 6 show typical "piano-stool" geometry with intermolecular pi-pi stacking of the biphenyl outer rings of 6. At 288 K the hydrolysis rates follow the order 2 >> 6 > 4 > 3 > 5 >> 1 with half-lives ranging from minutes to 4.4 h illustrating the influence of both electronic and steric effects of the substituents. The pK(a) values of the aqua adducts 3A, 4A, 5A, and 6A were all in the range of 6.3-6.6. The para-substituted pico complexes 4-6 readily formed adducts with both 9-ethyl guanine (9EtG) and 9-ethyl adenine (9EtA), but these were less favored for the ortho-substituted complexes 1 and 3 showing little reaction with 9EtG and 9EtA, respectively. Density-functional theory calculations confirmed the observed preferences for nucleobase binding for complex 1. In cytotoxicity assays with A2780, cisplatin-resistant A2780cis human ovarian, A549 human lung, and HCT116 colon cancer cells, only complexes 4 (p-Cl) and 6 (p-Me) exhibited significant activity (IC(50) values < 25 microM). Both of these complexes were as active as cisplatin in A2780 (ovarian) and HCT116 (colon) cell lines, and even overcome cisplatin resistance in the A2780cis (ovarian) cell line. The inactivity of 5 is attributed to the negative charge on its para carboxylate substituent. These data illustrate how the chemical reactivity and cancer cell cytotoxicity of osmium arene complexes can be controlled and "fine-tuned" by the use of steric and electronic effects of substituents on a chelating ligand to give osmium(II) arene complexes which are as active as cisplatin but have a different mechanism of action.