Estrogen deprivation and estrogen receptor α antagonism decrease DSS colitis in female mice.

The association of hormonal contraception with increased risk of inflammatory bowel disease (IBD) observed in females suggests involvement of ovarian hormones, such as estradiol, and the estrogen receptors in the progression of intestinal inflammation. Here, we investigated the effects of prophylactic SERM2 and estradiol supplementation in dextran sulfate sodium-induced colitis using mice with intact ovaries and ovariectomized (OVX) female mice. We found that graded colitis score was threefold reduced in the OVX mice, compared to mice with intact ovaries. Estradiol supplementation, however, aggravated the colitis in OVX mice, increasing the colitis score to a similar level than what was observed in the intact mice. Further, we observed that immune infiltration and gene expression of inflammatory interleukins Il1b, Il6, and Il17a were up to 200-fold increased in estradiol supplemented OVX colitis mice, while a mild but consistent decrease was observed by SERM2 treatment in intact animals. Additionally, cyclo-oxygenase 2 induction was increased in the colon of colitis mice, in correlation with increased serum estradiol levels. Measured antagonist properties of SERM2, together with the other results presented here, indicates an exaggerating role of ERα signaling in colitis. Our results contribute to the knowledge of ovarian hormone effects in colitis and encourage further research on the potential use of ER antagonists in the colon, in order to alleviate inflammation.

Colonocyte keratins stabilize mitochondria and contribute to mitochondrial energy metabolism.

Keratin intermediate filaments form dynamic filamentous networks, which provide mechanical stability, scaffolding, and protection against stress to epithelial cells. Keratins and other intermediate filaments have been increasingly linked to the regulation of mitochondrial function and homeostasis in different tissues and cell types. While deletion of keratin 8 (K8-/-) in mouse colon elicits a colitis-like phenotype, epithelial hyperproliferation, and blunted mitochondrial ketogenesis, the role of K8 in colonocyte mitochondrial function and energy metabolism is unknown. We used two K8 knockout mouse models and CRISPR/Cas9 K8-/- colorectal adenocarcinoma Caco-2 cells to answer this question. The results show that K8-/- colonocyte mitochondria in vivo are smaller and rounder and that mitochondrial motility is increased in K8-/- Caco-2 cells. Furthermore, K8-/- Caco-2 cells displayed diminished mitochondrial respiration and decreased mitochondrial membrane potential compared with controls, whereas glycolysis was not affected. The levels of mitochondrial respiratory chain complex proteins and mitochondrial regulatory proteins mitofusin-2 and prohibitin were decreased both in vitro in K8-/- Caco-2 cells and in vivo in K8-/- mouse colonocytes, and reexpression of K8 into K8-/- Caco-2 cells normalizes the mitofusin-2 levels. Mitochondrial Ca2+ is an important regulator of mitochondrial energy metabolism and homeostasis, and Caco-2 cells lacking K8 displayed decreased levels and altered dynamics of mitochondrial matrix and cytoplasmic Ca2+. In summary, these novel findings attribute an important role for colonocyte K8 in stabilizing mitochondrial shape and movement and maintaining mitochondrial respiration and Ca2+ signaling. Further, how these metabolically compromised colonocytes are capable of hyperproliferating presents an intriguing question for future studies.NEW & NOTEWORTHY In this study, we show that colonocyte intermediate filament protein keratin 8 is important for stabilizing mitochondria and maintaining mitochondrial energy metabolism, as keratin 8-deficient colonocytes display smaller, rounder, and more motile mitochondria, diminished mitochondrial respiration, and altered Ca2+ dynamics. Changes in fusion-regulating proteins are rescued with reexpression of keratin 8. These alterations in colonocyte mitochondrial homeostasis contribute to keratin 8-associated colitis pathophysiology.

The keratin-desmosome scaffold of internal epithelia in health and disease - The plot is thickening.

Keratin (K) intermediate filaments are attached to desmosomes and constitute the orchestrators of epithelial cell and tissue architecture. While their relevance in the epidermis is well recognized, our review focuses on their emerging importance in internal epithelia. The significance of keratin-desmosome scaffolds (KDSs) in the intestine is highlighted by transgenic mouse models and individuals with inflammatory bowel disease who display profound KDS alterations. In lung, high K8 expression defines a transitional cell subset during regeneration, and K8 variants are associated with idiopathic pulmonary fibrosis. Inherited variants in desmosomal proteins are overrepresented in idiopathic lung fibrosis, and familiar eosinophilic esophagitis. K18 serum fragments are established hepatocellular injury markers that correlate with the extent of histological inflammation. K17 expression is modified in multiple tumors, and K17 levels might be of prognostic relevance. These data should spur further studies on biological roles of these versatile tissue protectors and efforts on their therapeutic targeting.

Regulation of epithelial transitional states in murine and human pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disease arising from impaired regeneration of the alveolar epithelium after injury. During regeneration, type 2 alveolar epithelial cells (AEC2s) assume a transitional state that upregulates multiple keratins and ultimately differentiate into AEC1s. In IPF, transitional AECs accumulate with ineffectual AEC1 differentiation. However, whether and how transitional cells cause fibrosis, whether keratins regulate transitional cell accumulation and fibrosis, and why transitional AECs and fibrosis resolve in mouse models but accumulate in IPF are unclear. Here, we show that human keratin 8 (KRT8) genetic variants were associated with IPF. Krt8-/- mice were protected from fibrosis and accumulation of the transitional state. Keratin 8 (K8) regulated the expression of macrophage chemokines and macrophage recruitment. Profibrotic macrophages and myofibroblasts promoted the accumulation of transitional AECs, establishing a K8-dependent positive feedback loop driving fibrogenesis. Finally, rare murine transitional AECs were highly senescent and basaloid and may not differentiate into AEC1s, recapitulating the aberrant basaloid state in human IPF. We conclude that transitional AECs induced and were maintained by fibrosis in a K8-dependent manner; in mice, most transitional cells and fibrosis resolved, whereas in human IPF, transitional AECs evolved into an aberrant basaloid state that persisted with progressive fibrosis.

The Effect of a Fish Oil and/or Probiotic Intervention from Early Pregnancy Onwards on Colostrum Immune Mediators: A Randomized, Placebo-Controlled, Double-Blinded Clinical Trial in Overweight/Obese Mothers.

SCOPE: Modifying the composition of colostrum by external factors may provide opportunities to improve the infant's health. Here, we evaluated how fish oil and/or probiotics supplementation modify concentrations of colostrum immune mediators and their associations with perinatal clinical factors on mothers with overweight/obesity. METHODS AND RESULTS: Pregnant women were randomized in a double-blind manner into four intervention groups, and the supplements were consumed daily from early pregnancy onwards. Colostrum samples were collected from 187 mothers, and 16 immune mediators were measured using bead-based immunoassays. Interventions modified colostrum composition; the fish oil+probiotics group had higher concentrations of IL-12p70 than probiotics+placebo and higher FMS-like tyrosine kinase 3 ligand (FLT-3L) than fish oil+placebo and probiotics+placebo (one-way analysis of variance, post-hoc Tukey's test). Although the fish oil+probiotics group had higher levels of IFNα2 compared to the fish oil+placebo group, these differences were not statistically significant after correction for multiple testing. Multivariate linear model revealed significant associations between several immune mediators and the perinatal use of medication. CONCLUSION: Fish oil/probiotics intervention exerted a minor effect on concentrations of colostrum immune mediators. However, medication during the perinatal period modulated the immune mediators. These changes in colostrum's composition may contribute to immune system development in the infant.

Colonocyte keratin 7 is expressed de novo in inflammatory bowel diseases and associated with pathological changes and drug-resistance.

The clinical course of IBD, characterized by relapses and remissions, is difficult to predict. Initial diagnosis can be challenging, and novel disease markers are needed. Keratin 7 (K7) is a cytoskeletal intermediate filament protein not expressed in the colonic epithelium but has been reported in IBD-associated colorectal tumors. Our aim was to analyze whether K7 is expressed in chronic colonic inflammatory diseases and evaluate its potential as a novel biomarker. K7 was analyzed in two patient cohorts using immunohistochemistry-stained colon samples and single-cell quantitative digital pathology methods. K7 was correlated to pathological changes and clinical patient characteristics. Our data shows that K7 is expressed de novo in the colonic epithelium of ulcerative colitis and Crohn's disease IBD patients, but not in collagenous or lymphocytic colitis. K7 mRNA expression was significantly increased in colons of IBD patients compared to controls when assessed in publicly available datasets. While K7 increased in areas with inflammatory activity, it was not expressed in specific crypt compartments and did not correlate with neutrophils or stool calprotectin. K7 was increased in areas proximal to pathological alterations and was most pronounced in drug-resistant ulcerative colitis. In conclusion, colonic epithelial K7 is neo-expressed selectively in IBD patients and could be investigated for its potential as a disease biomarker.

Cytoplasmic keratins couple with and maintain nuclear envelope integrity in colonic epithelial cells.

Keratin intermediate filaments convey mechanical stability and protection against stress to epithelial cells. Keratins are essential for colon health, as seen in keratin 8 knockout (K8-/-) mice exhibiting a colitis phenotype. We hypothesized that keratins support the nuclear envelope and lamina in colonocytes. K8-/- colonocytes in vivo exhibit significantly decreased levels of lamins A/C, B1, and B2 in a colon-specific and cell-intrinsic manner. CRISPR/Cas9- or siRNA-mediated K8 knockdown in Caco-2 cells similarly decreased lamin levels, which recovered after reexpression of K8 following siRNA treatment. Nuclear area was not decreased, and roundness was only marginally increased in cells without K8. Down-regulation of K8 in adult K8flox/flox;Villin-CreERt2 mice following tamoxifen administration significantly decreased lamin levels at day 4 when K8 levels had reduced to 40%. K8 loss also led to reduced levels of plectin, LINC complex, and lamin-associated proteins. While keratins were not seen in the nucleoplasm without or with leptomycin B treatment, keratins were found intimately located at the nuclear envelope and complexed with SUN2 and lamin A. Furthermore, K8 loss in Caco-2 cells compromised nuclear membrane integrity basally and after shear stress. In conclusion, colonocyte K8 helps maintain nuclear envelope and lamina composition and contributes to nuclear integrity.

Vimentin Suppresses Inflammation and Tumorigenesis in the Mouse Intestine.

Vimentin has been implicated in wound healing, inflammation, and cancer, but its functional contribution to intestinal diseases is poorly understood. To study how vimentin is involved during tissue injury and repair of simple epithelium, we induced colonic epithelial cell damage in the vimentin null (Vim-/-) mouse model. Vim-/- mice challenged with dextran sodium sulfate (DSS) had worse colitis manifestations than wild-type (WT) mice. Vim-/- colons also produced more reactive oxygen and nitrogen species, possibly contributing to the pathogenesis of gut inflammation and tumorigenesis than in WT mice. We subsequently describe that CD11b+ macrophages served as the mainly cellular source of reactive oxygen species (ROS) production via vimentin-ROS-pSTAT3-interleukin-6 inflammatory pathways. Further, we demonstrated that Vim-/- mice did not develop colitis-associated cancer model upon DSS treatment spontaneously but increased tumor numbers and size in the distal colon in the azoxymethane/DSS model comparing with WT mice. Thus, vimentin has a crucial role in protection from colitis induction and tumorigenesis of the colon.

Targeted deletion of keratin 8 in intestinal epithelial cells disrupts tissue integrity and predisposes to tumorigenesis in the colon.

Keratin 8 (K8) is the main intestinal epithelial intermediate filament protein with proposed roles for colonic epithelial cell integrity. Here, we used mice lacking K8 in intestinal epithelial cells (floxed K8 and Villin-Cre1000 and Villin-CreERt2) to investigate the cell-specific roles of intestinal epithelial K8 for colonocyte function and pathologies. Intestinal epithelial K8 deletion decreased K8 partner proteins, K18-K20, 75-95%, and the remaining keratin filaments were located at the colonocyte apical regions with type II K7, which decreased 30%. 2-Deoxy-2-[18F]-fluoroglucose positron emission tomography in vivo imaging identified a metabolic phenotype in the lower gut of the conditional K8 knockouts. These mice developed intestinal barrier leakiness, mild diarrhea, and epithelial damage, especially in the proximal colon. Mice exhibited shifted differentiation from enterocytes to goblet cells, displayed longer crypts and an increased number of Ki67 + transit-amplifying cells in the colon. Significant proproliferative and regenerative signaling occurred in the IL-22, STAT3, and pRb pathways, with minor effects on inflammatory parameters, which, however, increased in aging mice. Importantly, colonocyte K8 deletion induced a dramatically increased sensitivity to azoxymethane-induced tumorigenesis. In conclusion, intestinal epithelial K8 plays a significant role in colonocyte epithelial integrity maintenance, proliferation regulation and tumor suppression.

Keratin intermediate filaments in the colon: guardians of epithelial homeostasis.

Keratin intermediate filament proteins are major cytoskeletal components of the mammalian simple layered columnar epithelium in the gastrointestinal tract. Human colon crypt epithelial cells express keratins 18, 19 and 20 as the major type I keratins, and keratin 8 as the type II keratin. Keratin expression patterns vary between species, and mouse colonocytes express keratin 7 as a second type II keratin. Colonic keratin patterns change during cell differentiation, such that K20 increases in the more differentiated crypt cells closer to the central lumen. Keratins provide a structural and mechanical scaffold to support cellular stability, integrity and stress protection in this rapidly regenerating tissue. They participate in central colonocyte processes including barrier function, ion transport, differentiation, proliferation and inflammatory signaling. The cell-specific keratin compositions in different epithelial tissues has allowed for the utilization of keratin-based diagnostic methods. Since the keratin expression pattern in tumors often resembles that in the primary tissue, it can be used to recognize metastases of colonic origin. This review focuses on recent findings on the biological functions of mammalian colon epithelial keratins obtained from pivotal in vivo models. We also discuss the diagnostic value of keratins in chronic colonic disease and known keratin alterations in colon pathologies. This review describes the biochemical properties of keratins and their molecular actions in colonic epithelial cells and highlights diagnostic data in colorectal cancer and inflammatory bowel disease patients, which may facilitate the recognition of disease subtypes and the establishment of personal therapies in the future.

Novel Selective Estrogen Receptor Modulator Ameliorates Murine Colitis.

Estrogen-receptor-mediated signaling has been suggested to decrease the inflammatory response in monocyte macrophages. Previously, we showed that a novel selective estrogen receptor modulator (SERM2) promotes anti-inflammatory phenotype of monocytes in vitro. In this study, we demonstrate the potential of SERM2 in amelioration of colitis. We utilized a dextran sodium sulfate (DSS)-induced colitis model in FVB/n mice to demonstrate the effects of orally administered SERM2 on the clinical status of the mice and the histopathological changes in the colon, as well as proportion of Mrc-1 positive macrophages. SERM2 nuclear receptor affinities were measured by radioligand binding assays. Orally administered, this compound significantly alleviated DSS-induced colitis in male mice and induced local estrogen receptor activation in the inflamed colon, as well as promoting anti-inflammatory cytokine expression and infiltration of anti-inflammatory monocytes. We show that this novel drug candidate has an affinity to estrogen receptors α and ß and progesterone receptors, but not to glucocorticoid receptor, thus expressing unique binding properties compared to other sex steroid receptor ligands. These results indicate that novel drug candidates to alleviate inflammatory conditions of the colon could be found among sex steroid receptor activating compounds.

Keratins regulate colonic epithelial cell differentiation through the Notch1 signalling pathway.

Keratins (K) are intermediate filament proteins important in stress protection and mechanical support of epithelial tissues. K8, K18 and K19 are the main colonic keratins, and K8-knockout (K8-/-) mice display a keratin dose-dependent hyperproliferation of colonic crypts and a colitis-phenotype. However, the impact of the loss of K8 on intestinal cell differentiation has so far been unknown. Here we show that K8 regulates Notch1 signalling activity and differentiation in the epithelium of the large intestine. Proximity ligation and immunoprecipitation assays demonstrate that K8 and Notch1 co-localize and interact in cell cultures, and in vivo in the colonic epithelial cells. K8 with its heteropolymeric partner K18 enhance Notch1 protein levels and activity in a dose dependent manner. The levels of the full-length Notch1 receptor (FLN), the Notch1 intracellular domain (NICD) and expression of Notch1 downstream target genes are reduced in the absence of K8, and the K8-dependent loss of Notch1 activity can be rescued with re-expression of K8/K18 in K8-knockout CRISPR/Cas9 Caco-2 cells protein levels. In vivo, K8 deletion with subsequent Notch1 downregulation leads to a shift in differentiation towards a goblet cell and enteroendocrine phenotype from an enterocyte cell fate. Furthermore, the K8-/- colonic hyperproliferation results from an increased number of transit amplifying progenitor cells in these mice. K8/K18 thus interact with Notch1 and regulate Notch1 signalling activity during differentiation of the colonic epithelium.

Keratins Are Altered in Intestinal Disease-Related Stress Responses.

Keratin (K) intermediate filaments can be divided into type I/type II proteins, which form obligate heteropolymers. Epithelial cells express type I-type II keratin pairs, and K7, K8 (type II) and K18, K19 and K20 (type I) are the primary keratins found in the single-layered intestinal epithelium. Keratins are upregulated during stress in liver, pancreas, lung, kidney and skin, however, little is known about their dynamics in the intestinal stress response. Here, keratin mRNA, protein and phosphorylation levels were studied in response to murine colonic stresses modeling human conditions, and in colorectal cancer HT29 cells. Dextran sulphate sodium (DSS)-colitis was used as a model for intestinal inflammatory stress, which elicited a strong upregulation and widened crypt distribution of K7 and K20. K8 levels were slightly downregulated in acute DSS, while stress-responsive K8 serine-74 phosphorylation (K8 pS74) was increased. By eliminating colonic microflora using antibiotics, K8 pS74 in proliferating cells was significantly increased, together with an upregulation of K8 and K19. In the aging mouse colon, most colonic keratins were upregulated. In vitro, K8, K19 and K8 pS74 levels were increased in response to lipopolysaccharide (LPS)-induced inflammation in HT29 cells. In conclusion, intestinal keratins are differentially and dynamically upregulated and post-translationally modified during stress and recovery.

Keratin 8-deletion induced colitis predisposes to murine colorectal cancer enforced by the inflammasome and IL-22 pathway.

Keratins (K) are intermediate filament proteins important in protection from cellular stress. K8, K18 and K19 are the main components of keratin filaments in colonic epithelia but their role in intestinal diseases remains ambiguous. A function for keratins in intestinal health is supported by the K8-knock-out (K8(-/-)) mouse which manifests an early chronic ulcerative colitis-like inflammatory bowel disease and epithelial hyperproliferation. We tested whether K8(-/-) mice are more susceptible to colorectal cancer (CRC) compared to K8 wild type (K8(+/+)), and K8 heterozygote (K8(+/-)) mice showing increased proliferation but no inflammation. K8(-/-) mice did not develop CRC spontaneously, but had dramatically increased numbers of tumors in the distal colon in the azoxymethane (AOM) and Apc(Min/+) CRC models while neither K8(+/+) nor K8(+/-) mice were susceptible. Upregulation of IL-22 in combination with a complete loss of its negative regulator IL-22BP, and increased downstream STAT3-signaling in K8(-/-) and K8(-/-)Apc(Min/+) colonic epithelia confirmed that the IL-22 pathway, important in inflammation, proliferation and tissue regeneration, was activated. The nearly total loss of IL-22BP correlated with an activated inflammasome leading to increased cleaved caspase-1, and the putative IL-22BP inhibitor, IL-18, as well as a decrease in ALDH1/2. Ablation of K8 in a colorectal cancer cell line similarly resulted in increased IL-18 and decreased ALDH1/2. K8/K18 co-immunoprecipitated with pro-caspase-1, a component of the inflammasome in the colon, which suggests that keratins modulate inflammasome activity and protect the colon from inflammation and tumorigenesis. The K8-null mouse models also provide novel epithelial-derived robust colon-specific CRC models.

Keratin 8 knockdown leads to loss of the chloride transporter DRA in the colon.

Keratins (K) are intermediate filament proteins important in protection from stress. The roles of keratins in the intestine are not clear, but K8 knockout (K8(-/-)) mice develop a Th2-type colonic inflammation, epithelial hyperproliferation, and mild diarrhea caused by a keratin level-dependent decrease in short-circuit current and net sodium and chloride absorption in the distal colon. The lack of K8 leads to mistargeting or altered levels of membrane proteins in colonocytes; however, the main transporter responsible for the keratin-related ion transport defect is unknown. We here analyzed protein and mRNA levels of candidate ion transporters CFTR, PAT-1, NHE-3, and DRA in ileum, cecum, and proximal and distal colon. Although no differences were observed for CFTR, PAT-1, or NHE-3, DRA mRNA levels were decreased by three- to fourfold and DRA protein was almost entirely lost in K8(-/-) cecum and proximal and distal colon compared with K8(+/+), whereas the levels in ileum were normal. In K8(+/-) mice, DRA mRNA levels were unaltered, while decreased DRA protein levels were detected in the proximal colon. Immunofluorescence staining confirmed the loss of DRA in K8(-/-) distal colon, while K8(+/-) displayed a similar but more patchy apical DRA distribution compared with K8(+/+) DRA was similarly decreased when K8 was knocked down in Caco-2 cells, confirming that K8 levels modulate DRA levels in an inflammation-independent manner. Taken together, the loss of DRA in the K8(-/-) mouse colon and cecum explains the dramatic chloride transport defect and diarrheal phenotype after K8 inactivation and identifies K8 as a novel regulator of DRA.

Targeted modulation of cell differentiation in distinct regions of the gastrointestinal tract via oral administration of differently PEG-PEI functionalized mesoporous silica nanoparticles.

Targeted delivery of drugs is required to efficiently treat intestinal diseases such as colon cancer and inflammation. Nanoparticles could overcome challenges in oral administration caused by drug degradation at low pH and poor permeability through mucus layers, and offer targeted delivery to diseased cells in order to avoid adverse effects. Here, we demonstrate that functionalization of mesoporous silica nanoparticles (MSNs) by polymeric surface grafts facilitates transport through the mucosal barrier and enhances cellular internalization. MSNs functionalized with poly(ethylene glycol) (PEG), poly(ethylene imine) (PEI), and the targeting ligand folic acid in different combinations are internalized by epithelial cells in vitro and in vivo after oral gavage. Functionalized MSNs loaded with γ-secretase inhibitors of the Notch pathway, a key regulator of intestinal progenitor cells, colon cancer, and inflammation, demonstrated enhanced intestinal goblet cell differentiation as compared to free drug. Drug-loaded MSNs thus remained intact in vivo, further confirmed by exposure to simulated gastric and intestinal fluids in vitro. Drug targeting and efficacy in different parts of the intestine could be tuned by MSN surface modifications, with PEI coating exhibiting higher affinity for the small intestine and PEI-PEG coating for the colon. The data highlight the potential of nanomedicines for targeted delivery to distinct regions of the tissue for strict therapeutic control.

Simple Epithelial Keratins.

Simple epithelial keratins (SEKs) are the cytoplasmic intermediate filament proteins of single-layered and glandular epithelial cells as found in the liver, pancreas, intestine, and lung. SEKs have broad cytoprotective functions, which are facilitated by dynamic posttranslational modifications and interaction with associated proteins. SEK filaments are composed of obligate heteropolymers of type II (K7, K8) and type I (K18-K20, K23) keratins. The multifaceted roles of SEKs are increasingly appreciated due to findings obtained from transgenic mouse models and human studies that identified SEK variants in several digestive diseases. Reorganization of the SEK network into aggregates called Mallory-Denk bodies (MDBs) is characteristic for specific liver disorders such as alcoholic and nonalcoholic steatohepatitis. To spur further research on SEKs, we here review the methods and potential caveats of their isolation as well as possibilities to study them in cell culture. The existing transgenic SEK mouse models, their advantages and potential drawbacks are discussed. The tools to induce MDBs, ways of their visualization and quantification, as well as the possibilities to detect SEK variants in humans are summarized.

Keratins in health and disease.

The cytoprotective keratins (K) compose the intermediate filaments of epithelial cells and their inherited and spontaneous mutations give rise to keratinopathies. For example, mutations in K1/K5/K10/K14 cause epidermal skin diseases whereas simple epithelial K8/K18/K19 variants predispose to development of several liver disorders. Due to their abundance, tissue- and context-specific expression, keratins constitute excellent diagnostic markers of both neoplastic and non-neoplastic diseases. During injury and in disease, keratin expression levels, cellular localization or posttranslational modifications are altered. Accumulating evidence suggests that these changes modulate multiple processes including cell migration, tumor growth/metastasis and development of infections. Therefore, our understanding of keratins is shifting from diagnostic markers to active disease modifiers.

Shape engineering vs organic modification of inorganic nanoparticles as a tool for enhancing cellular internalization.

In nanomedicine, physicochemical properties of the nanocarrier affect the nanoparticle's pharmacokinetics and biodistribution, which are also decisive for the passive targeting and nonspecific cellular uptake of nanoparticles. Size and surface charge are, consequently, two main determining factors in nanomedicine applications. Another important parameter which has received much less attention is the morphology (shape) of the nanocarrier. In order to investigate the morphology effect on the extent of cellular internalization, two similarly sized but differently shaped rod-like and spherical mesoporous silica nanoparticles were synthesized, characterized and functionalized to yield different surface charges. The uptake in two different cancer cell lines was investigated as a function of particle shape, coating (organic modification), surface charge and dose. According to the presented results, particle morphology is a decisive property regardless of both the different surface charges and doses tested, whereby rod-like particles internalized more efficiently in both cell lines. At lower doses whereby the shape-induced advantage is less dominant, charge-induced effects can, however, be used to fine-tune the cellular uptake as a prospective 'secondary' uptake regulator for tight dose control in nanoparticle-based drug formulations.

Mesoporous silica nanoparticles as drug delivery systems for targeted inhibition of Notch signaling in cancer.

Notch signaling, a key regulator of stem cells, is frequently overactivated in cancer. It is often linked to aggressive forms of cancer, evading standard treatment highlighting Notch as an exciting therapeutic target. Notch is in principle "druggable" by γ-secretase inhibitors (GSIs), inhibitory peptides and antibodies, but clinical use of Notch inhibitors is restricted by severe side effects and there is a demand for alternative cancer-targeted therapy. Here, we present a novel approach, using imagable mesoporous silica nanoparticles (MSNPs) as vehicles for targeted delivery of GSIs to block Notch signaling. Drug-loaded particles conjugated to targeting ligands induced cell-specific inhibition of Notch activity in vitro and exhibited enhanced tumor retainment with significantly improved Notch inhibition and therapeutic outcome in vivo. Oral administration of GSI-MSNPs controlled Notch activity in intestinal stem cells further supporting the in vivo applicability of MSNPs for GSI delivery. MSNPs showed tumor accumulation and targeting after systemic administration. MSNPs were biocompatible, and particles not retained within the tumors, were degraded and eliminated mainly by renal excretion. The data highlights MSNPs as an attractive platform for targeted drug delivery of anticancer drugs with otherwise restricted clinical application, and as interesting constituents in the quest for more refined Notch therapies.