Analysis of tripartite motif (TRIM) family gene expression in prostate cancer bone metastases.

Tripartite motif (TRIM) family proteins are post-translational protein modifiers with E3-ubiquitin ligase activity, thereby involved in various biological processes. The molecular mechanisms driving prostate cancer (PCa) bone metastasis (BM) are incompletely understood, and targetable genetic alterations are lacking in the majority of cases. Therefore, we aimed to explore the expression and potential functional relevance of 71 TRIM members in bone metastatic PCa. We performed transcriptome analysis of all human TRIM family members and 770 cancer-related genes in 29 localized PCa and 30 PCa BM using Nanostring. KEGG, STRING and Ubibrowser were used for further bioinformatic gene correlation and pathway enrichment analyses. Compared to localized tumors, six TRIMs are under-expressed while nine TRIMs are over-expressed in BM. The differentially expressed TRIM proteins are linked to TNF-, TGFß-, PI3K/AKT- and HIF-1-signaling, and to features such as proteoglycans, platelet activation, adhesion and ECM-interaction based on correlation to cancer-related genes. The identification of TRIM-specific E3-ligase-substrates revealed insight into functional connections to oncogenes, tumor suppressors and cancer-related pathways including androgen receptor- and TGFß signaling, cell cycle regulation and splicing. In summary, this is the first study that comprehensively and systematically characterizes the expression of all TRIM members in PCa BM. Our results describe post-translational protein modification as an important regulatory mechanism of oncogenes, tumor suppressors, and pathway molecules in PCa progression. Therefore, this study may provide evidence for novel therapeutic targets, in particular for the treatment or prevention of BM.

Tyrosine-modified linear PEIs for highly efficacious and biocompatible siRNA delivery in vitro and in vivo.

Therapeutic gene silencing by RNA interference relies on the safe and efficient in vivo delivery of small interfering RNAs (siRNAs). Polyethylenimines are among the most studied cationic polymers for gene delivery. For several reasons including superior tolerability, small linear PEIs would be preferable over branched PEIs, but they show poor siRNA complexation. Their chemical modification for siRNA formulation has not been extensively explored so far. We generated a set of small linear PEIs bearing tyrosine modifications (LPxY), leading to substantially enhanced siRNA delivery and knockdown efficacy in vitro in various cell lines, including hard-to-transfect cells. The tyrosine-modified linear 10 kDa PEI (LP10Y) is particularly powerful, associated with favorable physicochemical properties and very high biocompatibility. Systemically administered LP10Y/siRNA complexes reveal antitumor effects in mouse xenograft and patient-derived xenograft (PDX) models, and their direct application into the brain achieves therapeutic inhibition of orthotopic glioma xenografts. LP10Y is particularly interesting for therapeutic siRNA delivery.

Detection of doxorubicin, cisplatin and therapeutic antibodies in formalin-fixed paraffin-embedded human cancer cells.

A major limitation in the pharmacological treatment of clinically detectable primary cancers and their metastases is their limited accessibility to anti-cancer drugs (cytostatics, inhibitory antibodies, small-molecule inhibitors) critically impairing therapeutic efficacies. Investigations on the tissue distribution of such drugs are rare and have only been based on fresh frozen material or methanol-fixed cell culture cells so far. In this paper, we expand the detection of cisplatin-induced DNA adducts and anthracyclines as well as therapeutic antibodies to routinely prepared formalin-fixed, paraffin-embedded sections (FFPE). Using pre-treated cell lines prepared as FFPE samples comparable to tissues from routine analysis, we demonstrate that our method allows for the detection of chemotherapeutics (anthracyclines by autofluorescence, cisplatin by immune detection of DNA adducts) as well as therapeutic antibodies. This methodology thus allows for analyzing archival FFPE tissues, as demonstrated here for the detection of cisplatin, doxorubicin and trastuzumab in FFPE sections of tumor xenografts from drug-treated mice. Analyzing human tumor samples, this will lead to new insights into the tissue penetration of drugs.

Nanoparticle-complexed antimiRs for inhibiting tumor growth and metastasis in prostate carcinoma and melanoma.

BACKGROUND: MiRNAs act as negative regulators of gene expression through target mRNA degradation or inhibition of its translation. In cancer, several miRNAs are upregulated and play crucial roles in tumorigenesis, making the inhibition of these oncomiRs an interesting therapeutic approach. This can be achieved by directly complementary single-stranded anti-miRNA oligonucleotides (antimiRs). A major bottleneck in antimiR therapy, however, is their efficient delivery. The nanoparticle formation with polyethylenimine (PEI) may be particularly promising, based on the PEI's ability to electrostatically interact with oligonucleotides. This leads to their protection and supports delivery. In the present study, we explore for the first time PEI for antimiR formulation and delivery. We use the branched low molecular weight PEI F25-LMW for the complexation of different antimiRs, and analyse tumor- and metastasis-inhibitory effects of PEI/antimiR complexes in different tumor models. RESULTS: In prostate carcinoma, transfection of antimiRs against miR-375 and miR-141 leads to tumor cell inhibition in 2D- and 3D-models. More importantly, an in vivo tumor therapy study in prostate carcinoma xenografts reveals anti-tumor effects of the PEI/antimiR complexes. In advanced melanoma and metastasis, we identify by a microRNA screen miR-150 as a particularly relevant oncomiR candidate, and validate this result in vitro and in vivo. Again, the systemic application of PEI/antimiR complexes inhibiting this miRNA, or the previously described antimiR-638, leads to profound tumor growth inhibition. These effects are associated with the upregulation of direct miRNA target genes. In a melanoma metastasis mouse model, anti-metastatic effects of PEI/antimiR treatment are observed as well. CONCLUSIONS: We thus describe PEI-based complexes as efficient platform for antimiR therapy, as determined in two different tumor entities using in vivo models of tumor growth or metastasis. Our study also highlights the therapeutic relevance of miR-375, miR-141, miR-150 and miR-638 as target miRNAs for antimiR-mediated inhibition.

Determination of atomoxetine or escitalopram in human plasma by HPLC: Applications in neuroscience research studies
.

BACKGROUND: Atomoxetine and escitalopram are potent and selective drugs approved for noradrenergic or serotonergic modulation of neuronal networks in attention-deficit hyperactivity disorder (ADHD) or depression, respectively. High-performance liquid chromatography (HPLC) methods still play an important role in the therapeutic drug monitoring (TDM) of psychopharmacological drugs, and coupled with tandem mass spectrometry are the gold standard for the quantification of drugs in biological matrices, but not available everywhere. The aim of this work was to develop and validate a HPLC method for neuroscientific studies using atomoxetine or escitalopram as a test drug. MATERIALS AND METHODS: A HPLC method from routine TDM determination of atomoxetine or citalopram in plasma was adapted and validated for use in neuroscientific research. Using photo diode array detection with UV absorption at 205 nm, the variation of internal standard within one chromatographic method enables separate drug monitoring for concentration-controlled explorative studies in healthy humans and patients with Parkinson's disease. RESULTS: The method described here was found to be linear in the range of 0.002 - 1.4 mg/L for atomoxetine and 0.0012 - 0.197 mg/L for escitalopram, with overall mean intra-day and inter-day imprecision and accuracy bias < 10% for both drugs. The method was successfully applied in concentration-controlled neuroimaging studies in populations of healthy humans and patients with Parkinson's disease. CONCLUSION: A simple, sensitive, robust HPLC method capable of monitoring escitalopram and atomoxetine is presented and validated, as a useful tool for drug monitoring and the study of pharmacokinetics in neuroscientific study applications.

SATB1-Mediated Upregulation of the Oncogenic Receptor Tyrosine Kinase HER3 Antagonizes MET Inhibition in Gastric Cancer Cells.

MET-amplified gastric cancer cells are extremely sensitive to MET inhibition in vitro, whereas clinical efficacy of MET inhibitors is disappointing. The compensatory activation of other oncogenic growth factor receptors may serve as an underlying mechanism of resistance. In this study, we analyzed the role of HER receptors, in particular HER3 and its ligand heregulin, in this respect. This also included the chromatin-organizer protein SATB1, as an established regulator of HER expression in other tumor entities. In a panel of MET-amplified gastric carcinoma cell lines, cell growth under anchorage-dependent and independent conditions was studied upon inhibitor treatment or siRNA-mediated knockdown. Expression analyses were performed using RT-qPCR, FACS, and immunoblots. Signal transduction was monitored via antibody arrays and immunoblots. As expected, MET inhibition led to a growth arrest and inhibition of MAPK signaling. Strikingly, however, this was accompanied by a rapid and profound upregulation of the oncogenic receptor HER3. This finding was determined as functionally relevant, since HER3 activation by HRG led to partial MET inhibitor resistance, and MAPK/Akt signaling was even found enhanced upon HRG+MET inhibitor treatment compared to HRG alone. SATB1 was identified as mediator of HER3 upregulation. Concomitantly, SATB1 knockdown prevented upregulation of HER3, thus abrogating the HRG-promoted rescue from MET inhibition. Taken together, our results introduce the combined HER3/MET inhibition as strategy to overcome resistance towards MET inhibitors.

Mono- and Polyassociation Processes of Pentavalent Biotinylated PEI Glycopolymers for the Fabrication of Biohybrid Structures with Targeting Properties.

This study describes new mechanistic insights in the sequential polyassociation of streptavidin with biotinylated poly(ethyleneimine) glycopolymers and biotinylated PEGylated folic acid components for the preparation of biohybrid structures (BHS) for controlled targeting experiments. Characterization of the BHS revealed that during the formation and postfunctionalization of BHS, reversible dissociation and reassociation processes occur. The BHS are stable over weeks after finalizing the equilibrium-driven polyassociation process. Cellular uptake studies showed that this sequential polyassociation involving biotinylated PEGylated folic acid components does not lead to enhanced cellular uptake of the resulting BHS. In contrast, polyplexes, containing small interfering RNA and bioconjugates (1:1 molar ratio between biotinylated glycopolymer and monomeric streptavidin-lectin fusion protein), enabled us to control the targeting of tumor cells as revealed by knockdown of the tumor-associated protein survivin. Overall, this study demonstrates the high potential of (networklike) streptavidin-biotin interactions with a dynamic character in the formation of complex BHS and extracellular matrix materials.

Spray-Dried Nanoparticle-in-Microparticle Delivery Systems (NiMDS) for Gene Delivery, Comprising Polyethylenimine (PEI)-Based Nanoparticles in a Poly(Vinyl Alcohol) Matrix.

Nucleic acid-based therapies rely on efficient formulations for nucleic acid protection and delivery. As nonviral strategies, polymeric and lipid-based nanoparticles have been introduced; however, biological efficacy and biocompatibility as well as poor storage properties due to colloidal instability and their unavailability as ready-to-use systems are still major issues. Polyethylenimine is the most widely explored and promising candidate for gene delivery. Polyethylenimine-based polyplexes and their combination with liposomes, lipopolyplexes, are efficient for DNA or siRNA delivery in vitro and in vivo. In this study, a highly potent spray-dried nanoparticle-in-microparticle delivery system is presented for the encapsulation of polyethylenimine-based polyplexes and lipopolyplexes into poly(vinyl alcohol) microparticles, without requiring additional stabilizing agents. This easy-to-handle gene delivery device allows prolonged nanoparticle storage and protection at ambient temperature. Biological analyses reveal further advantages regarding profoundly reduced cytotoxicity and enhanced transfection efficacies of polyethylenimine-based nanoparticles from the nanoparticle-in-microparticle delivery system over their freshly prepared counterparts, as determined in various cell lines. Importantly, this nanoparticle-in-microparticle delivery system is demonstrated as ready-to-use dry powder to be an efficient device for the inhalative delivery of polyethylenimine-based lipopolyplexes in vivo, as shown by transgene expression in mice after only one administration.

Transfection of primary brain capillary endothelial cells for protein synthesis and secretion of recombinant erythropoietin: a strategy to enable protein delivery to the brain.

Treatment of chronic disorders affecting the central nervous system (CNS) is complicated by the inability of drugs to cross the blood-brain barrier (BBB). Non-viral gene therapy applied to brain capillary endothelial cells (BCECs) denotes a novel approach to overcome the restraints in this passage, as turning BCECs into recombinant protein factories by transfection could result in protein secretion further into the brain. The present study aims to investigate the possibility of transfecting primary rat brain endothelial cells (RBECs) for recombinant protein synthesis and secretion of the neuroprotective protein erythropoietin (EPO). We previously showed that 4% of RBECs with BBB properties can be transfected without disrupting the BBB integrity in vitro, but it can be questioned whether this is sufficient to enable protein secretion at therapeutic levels. The present study examined various transfection vectors, with regard to increasing the transfection efficiency without disrupting the BBB integrity. Lipofectamine 3000™ was the most potent vector compared to polyethylenimine (PEI) and Turbofect. When co-cultured with astrocytes, the genetically modified RBECs secreted recombinant EPO into the cell culture medium both luminally and abluminally, and despite lower levels of EPO reaching the abluminal chamber, the amount of recombinant EPO was sufficient to evolve a biological effect on astrocytes cultured at the abluminal side in terms of upregulated gene expression of brain-derived neurotropic factor (BDNF). In conclusion, non-viral gene therapy to RBECs leads to protein secretion and signifies a method for therapeutic proteins to target cells inside the CNS otherwise omitted due to the BBB.

A Poly(Propyleneimine) Dendrimer-Based Polyplex-System for Single-Chain Antibody-Mediated Targeted Delivery and Cellular Uptake of SiRNA.

Therapeutics based on small interfering RNAs (siRNAs) offer a great potential to treat so far incurable diseases or metastatic cancer. However, the broad application of siRNAs using various nonviral carrier systems is hampered by unspecific toxic side effects, poor pharmacokinetics due to unwanted delivery of siRNA-loaded nanoparticles into nontarget organs, or rapid renal excretion. In order to overcome these obstacles, several targeting strategies using chemically linked antibodies and ligands have emerged. This study reports a new modular polyplex carrier system for targeted delivery of siRNA, which is based on transfection-disabled maltose-modified poly(propyleneimine)-dendrimers (mal-PPI) bioconjugated to single chain fragment variables (scFvs). To achieve targeted delivery into tumor cells expressing the epidermal growth factor receptor variant III (EGFRvIII), monobiotinylated anti-EGFRvIII scFv fused to a Propionibacterium shermanii transcarboxylase-derived biotinylation acceptor (P-BAP) is bioconjugated to mal-PPI through a novel coupling strategy solely based on biotin-neutravidin bridging. In contrast to polyplexes containing an unspecific control scFv-P-BAP, the generated EGFRvIII-specific polyplexes are able to exclusively deliver siRNA to tumor cells and tumors by receptor-mediated endocytosis. These results suggest that receptor-mediated uptake of otherwise noninternalized mal-PPI-based polyplexes is a promising avenue to improve siRNA therapy of cancer, and introduce a novel strategy for modular bioconjugation of protein ligands to nanoparticles.

Analysis of cellular and molecular antitumor effects upon inhibition of SATB1 in glioblastoma cells.

BACKGROUND: The Special AT-rich Sequence Binding Protein 1 (SATB1) regulates the expression of many genes by acting as a global chromatin organizer. While in many tumor entities SATB1 overexpression has been observed and connected to pro-tumorigenic processes, somewhat contradictory evidence exists in brain tumors with regard to SATB1 overexpression in glioblastoma and its association with poorer prognosis and tumor progression. On the functional side, initial data indicate that SATB1 may be involved in several tumor cell-relevant processes. METHODS: For the detailed analysis of the functional relevance and possible therapeutic potential of SATB1 inhibition, we employ transient siRNA-mediated knockdown and comprehensively analyze the cellular and molecular role of SATB1 in glioblastoma. RESULTS: In various cell lines with different SATB1 expression levels, a SATB1 gene dose-dependent inhibition of anchorage-dependent and -independent proliferation is observed. This is due to cell cycle-inhibitory and pro-apoptotic effects of SATB1 knockdown. Molecular analyses reveal SATB1 knockdown effects on multiple important (proto-) oncogenes, including Myc, Bcl-2, Pim-1, EGFR, ß-catenin and Survivin. Molecules involved in cell cycle, EMT and cell adhesion are affected as well. The putative therapeutic relevance of SATB1 inhibition is further supported in an in vivo tumor xenograft mouse model, where the treatment with polymeric nanoparticles containing SATB1-specific siRNAs exerts antitumor effects. CONCLUSION: Our results demonstrate that SATB1 may represent a promising target molecule in glioblastoma therapy whose inhibition or knockdown affects multiple crucial pathways.

Liposome-polyethylenimine complexes (DPPC-PEI lipopolyplexes) for therapeutic siRNA delivery in vivo.

Therapeutic applications of RNA interference (RNAi) require efficient siRNA delivery strategies in vivo. Combining lipid-based carriers with polymeric nanoparticles offers the favorable properties of both systems. This is the first study to explore polyethylenimine-based lipopolyplexes comprising a low-molecular weight PEI and the phospholipid DPPC for therapeutic siRNA use. Lipopolyplex structures are analyzed by electron microscopy. Biological efficacies are demonstrated in vitro by cellular uptake, knockdown of the target oncogene survivin, and concomitant cell growth inhibition. Upon systemic administration in tumor-bearing mice, here performed by intraperitoneal (i.p.) injection, radioactive biodistribution assays show lipopolyplex-mediated delivery of intact siRNAs. Absence of blood serum parameter alterations, erythrocyte aggregation or immunostimulation, and the observation of animal well-being and stable body weight confirm biocompatibility. Exploring therapeutic efficacies in a preclinical model, a considerable inhibition of prostate carcinoma xenograft growth is achieved, paralleled by an ~65% survivin knockdown in the tumors. We, thus, demonstrate that PEI-based lipopolyplexes represent an efficient platform for therapeutic use of small RNAs.

Advancing the use of noncoding RNA in regulatory toxicology: Report of an ECETOC workshop.

The European Centre for the Ecotoxicology and Toxicology of Chemicals (ECETOC) organised a workshop to discuss the state-of-the-art research on noncoding RNAs (ncRNAs) as biomarkers in regulatory toxicology and as analytical and therapeutic agents. There was agreement that ncRNA expression profiling data requires careful evaluation to determine the utility of specific ncRNAs as biomarkers. To advance the use of ncRNA in regulatory toxicology, the following research priorities were identified: (1) Conduct comprehensive literature reviews to identify possibly suitable ncRNAs and areas of toxicology where ncRNA expression profiling could address prevailing scientific deficiencies. (2) Develop consensus on how to conduct ncRNA expression profiling in a toxicological context. (3) Conduct experimental projects, including, e.g., rat (90-day) oral toxicity studies, to evaluate the toxicological relevance of the expression profiles of selected ncRNAs. Thereby, physiological ncRNA expression profiles should be established, including the biological variability of healthy individuals. To substantiate the relevance of key ncRNAs for cell homeostasis or pathogenesis, molecular events should be dose-dependently linked with substance-induced apical effects. Applying a holistic approach, knowledge on ncRNAs, 'omics and epigenetics technologies should be integrated into adverse outcome pathways to improve the understanding of the functional roles of ncRNAs within a regulatory context.

Regioselective Ring-Opening Polymerization of a Polyhydroxycarboxylic Acid for the Synthesis of a Nanoscale Carrier Material with pH-Dependent Stability and Sustained Drug Release.

Synthetic polyesters are usually composed of monohydroxycarboxylic acids to avoid the problem of regioselectivity during ring-opening polymerization. In contrast, the linear polyester BICpoly contains four secondary OH groups and is nevertheless esterified regioselectively at only one of these positions. Neither the synthesis of the tricyclic monomers nor the ring-opening polymerization requires protecting groups, making BICpoly an attractive novel and biocompatible polymer. BICpoly nanoparticles can be loaded with low-molecular weight drugs or coated onto surfaces as thin films. The release of loaded compounds makes BICpoly an attractive depot for drug release, as shown herein by loading BICpoly with dyes or the cytostatic drug doxorubicin. BICpoly is distinguishable from other polymers by its characteristic pH-dependent degradation.

Multiple effects of the special AT-rich binding protein 1 (SATB1) in colon carcinoma.

SATB1 (special AT-rich binding protein 1) is a global chromatin organizer regulating the expression of a large number of genes. Overexpression has been found in various solid tumors and positively correlated with prognostic and clinicopathological properties. In colorectal cancer (CRC), SATB1 overexpression and its correlation with poor differentiation, invasive depth, TNM (tumor, nodes, metastases) stage and prognosis have been demonstrated. However, more detailed studies on the SATB1 functions in CRC are warranted. In this article, we comprehensively analyze the cellular and molecular role of SATB1 in CRC cell lines with different SATB1 expression levels by using RNAi-mediated knockdown. Using siRNAs with different knockdown efficacies, we demonstrate antiproliferative, cell cycle-inhibitory and proapoptotic effects of SATB1 knockdown in a SATB1 gene dose-dependent manner. Tumor growth inhibition is confirmed in vivo in a subcutaneous tumor xenograft mouse model using stable knockdown cells. The in-depth analysis of cellular effects reveals increased activities of caspases-3, -7, -8, -9 and other mediators of apoptotic pathways. Similarly, the analysis of E- and N-cadherin, slug, twist, ß-catenin and MMP7 indicates SATB1 effects on epithelial-mesenchymal transition (EMT) and matrix breakdown. Our results also establish SATB1 effects on receptor tyrosine kinases and (proto-)oncogenes such as HER receptors and Pim-1. Taken together, this suggests a more complex molecular interplay between tumor-promoting and possible inhibitory effects in CRC by affecting multiple pathways and molecules involved in proliferation, cell cycle, EMT, invasion and cell survival.

A novel method for the assessment of targeted PEI-based nanoparticle binding based on a static surface plasmon resonance system.

The delivery of nucleic acids is a major hurdle in gene therapy or therapeutic gene knockdown, and the development of intelligent and safe nanoparticles as carrier systems is thus under intense investigation. The introduction of ligands for their targeted delivery is of major interest. Here, we describe a novel approach for the analysis of the binding properties of antibody-functionalized nanoparticles, using surface plasmon resonance (SPR) in a static cuvette system. By chemical coupling of the Epidermal Growth Factor Receptor (EGFR)-specific antibody cetuximab to poly(ethylene imine) (PEI) via a PEG-spacer and subsequent DNA or siRNA complexation, we generated targeted nanoplexes with low surface charge. Antibody-mediated uptake into EGFR overexpressing cells was observed. SPR measurements with use of a novel, protein A-based sandwich system for the immobilization of the target receptor in its correct steric orientation allowed the analysis of the specific PEI-PEG-cetuximab binding to EGFR and the determination of binding affinities. Importantly, our cuvette-based SPR assay system was also suitable for the monitoring of ligand-mediated nanoparticle binding, without convection or shear stress. We conclude that our SPR sandwich system allows the precise analysis of the binding of ligand-functionalized nanoparticles in real-time, and we thus establish SPR for the in vitro evaluation of ligand modifications for generating targeted nanoparticles.

Efficient polymeric nanoparticles for RNAi in macrophage reveal complex effects on polarization markers upon knockdown of STAT3/STAT6.

Tumor associated macrophages (TAMs) are the most abundant immune cell type in the tissue microenvironment, affecting tumor progression, metastasis and therapeutic response. Different macrophage activation ("polarization") states can be distinguished: resting (M0; non-activated), pro-inflammatory/anti-tumorigenic (M1) and anti-inflammatory/pro-tumorigenic (M2). When exploring macrophages as targets in novel cancer immunotherapy approaches, TAM repolarization from the M2 into the M1 phenotype is an intriguing strategy to block their pro-tumoral and enhance their anti-tumoral properties. In the context of RNAi-based gene knockdown of M2 promoting genes, major bottlenecks include cellular siRNA delivery and correct intracellular processing. This is particularly true in case of macrophages as a cell type well-known to be notoriously hard-to-transfect. Among polymeric nanocarriers, the cationic polymer polyethylenimine (PEI) is widely explored for delivering nucleic acids. Further advanced nanocarriers are tyrosine-modified polymers based on PEI or polypropylenimine dendrimers (PPI) for highly efficient siRNA delivery in vitro and in vivo. In this paper, we explored a panel of PEI- or PPI-based nanoparticle systems for siRNA-mediated gene knockdown efficacy in macrophages and subsequent TAM repolarization. The tyrosine-modified linear 10 kDa PEI (LP10Y) or branched 5 kDa PEI (P5Y) as well as a tyrosine-modified PPI (PPI-Y) were found most efficient for gene knockdown in macrophage cell lines or primary macrophages, independent of their polarization. Knockdown of STAT6 or STAT3 led to repolarization of M2 macrophages, as indicated by alterations in various M2 and M1 marker levels. This highly specific approach also demonstrated non-redundant functions of STAT3 and STAT6. Importantly, macrophage re-polarization from M2 to M1 upon PPI-Y/siRNA-mediated STAT6 knockdown increased tumor cell phagocytosis in a co-culture model. In conclusion, we identify certain tyrosine-modified PEI- or PPI-based nanoparticles as particularly efficient for macrophage transfection, and the specific, siRNA-mediated STAT6 knockdown as a promising approach for macrophage repolarization and enhancement of their tumor cell suppressive role.

Spray-drying of PEI-/PPI-based nanoparticles for DNA or siRNA delivery.

Spray-drying of nucleic acid-based drugs designed for gene therapy or gene knockdown is associated with many advantages including storage stability and handling as well as the possibility of pulmonary application. The encapsulation of nucleic acids in nanoparticles prior to spray-drying is one strategy for obtaining efficient formulations. This, however, strongly relies on the definition of optimal nanoparticles, excipients and spray-drying conditions. Among polymeric nanoparticles, polyethylenimine (PEI)-based complexes with or without chemical modifications have been described previously as very efficient for gene or oligonucleotide delivery. The tyrosine-modification of linear or branched low molecular weight PEIs, or of polypropylenimine (PPI) dendrimers, has led to high complex stability, improved cell uptake and transfection efficacy as well as high biocompatibility. In this study, we identify optimal spray-drying conditions for PEI-based nanoparticles containing large plasmid DNA or small siRNAs, and further explore the spray-drying of nanoparticles containing chemically modified polymers. Poly(vinyl alcohol) (PVA), but not trehalose or lactose, is particularly well-suited as excipient, retaining or even enhancing transfection efficacies compared to fresh complexes. A big mesh size is critically important as well, while the variation of the spray-drying temperature plays a minor role. Upon spray-drying, microparticles in a âˆ¼ 3.3 - 8.5 µm size range (laser granulometry) are obtained, dependent on the polymers. Upon their release from the spray-dried material, the nanoparticles show increased sizes and markedly altered zeta potentials as compared to their fresh counterparts. This may contribute to their high efficacy that is seen also after prolonged storage of the spray-dried material. We conclude that these spray-dried systems offer a great potential for the preparation of nucleic acid drug storage forms with facile reconstitution, as well as for their direct pulmonary application as dry powder.

HDAC inhibitors activate lipid peroxidation and ferroptosis in gastric cancer.

Gastric cancer remains among the deadliest neoplasms worldwide, with limited therapeutic options. Since efficacies of targeted therapies are unsatisfactory, drugs with broader mechanisms of action rather than a single oncogene inhibition are needed. Preclinical studies have identified histone deacetylases (HDAC) as potential therapeutic targets in gastric cancer. However, the mechanism(s) of action of HDAC inhibitors (HDACi) are only partially understood. This is particularly true with regard to ferroptosis as an emerging concept of cell death. In a panel of gastric cancer cell lines with different molecular characteristics, tumor cell inhibitory effects of different HDACi were studied. Lipid peroxidation levels were measured and proteome analysis was performed for the in-depth characterization of molecular alterations upon HDAC inhibition. HDACi effects on important ferroptosis genes were validated on the mRNA and protein level. Upon HDACi treatment, lipid peroxidation was found increased in all cell lines. Class I HDACi (VK1, entinostat) showed the same toxicity profile as the pan-HDACi vorinostat. Proteome analysis revealed significant and concordant alterations in the expression of proteins related to ferroptosis induction. Key enzymes like ACSL4, POR or SLC7A11 showed distinct alterations in their expression patterns, providing an explanation for the increased lipid peroxidation. Results were also confirmed in primary human gastric cancer tissue cultures as a relevant ex vivo model. We identify the induction of ferroptosis as new mechanism of action of class I HDACi in gastric cancer. Notably, these findings were independent of the genetic background of the cell lines, thus introducing HDAC inhibition as a more general therapeutic principle.

Glucose-oxygen deprivation constrains HMGCR function and Rac1 prenylation and activates the NLRP3 inflammasome in human monocytes.

Hypoxia and low glucose abundance often occur simultaneously at sites of inflammation. In monocytes and macrophages, glucose-oxygen deprivation stimulates the assembly of the NLRP3 inflammasome to generate the proinflammatory cytokine IL-1ß. We found that concomitant glucose deprivation and hypoxia activated the NLRP3 inflammasome by constraining the function of HMG-CoA reductase (HMGCR), the rate-limiting enzyme of the mevalonate kinase pathway. HMGCR is involved in the synthesis of geranylgeranyl pyrophosphate (GGPP), which is required for the prenylation and lipid membrane integration of proteins. Under glucose-oxygen deprivation, GGPP synthesis was decreased, leading to reduced prenylation of the small GTPase Rac1, increased binding of nonprenylated Rac1 to the scaffolding protein IQGAP1, and enhanced activation of the NLRP3 inflammasome. In response to restricted oxygen and glucose supply, patient monocytes with a compromised mevalonate pathway due to mevalonate kinase deficiency or Muckle-Wells syndrome released more IL-1ß than did control monocytes. Thus, reduced GGPP synthesis due to inhibition of HMGCR under glucose-oxygen deprivation results in proinflammatory innate responses, which are normally kept in check by the prenylation of Rac1. We suggest that this mechanism is also active in inflammatory autoimmune conditions.