Deoxyhypusine hydroxylase: A novel therapeutic target differentially expressed in short-term vs long-term survivors of glioblastoma.

Glioblastoma (GB) is the most aggressive neoplasm of the brain. Poor prognosis is mainly attributed to tumor heterogeneity, invasiveness and drug resistance. Only a small fraction of GB patients survives longer than 24 months from the time of diagnosis (ie, long-term survivors [LTS]). In our study, we aimed to identify molecular markers associated with favorable GB prognosis as a basis to develop therapeutic applications to improve patients' outcome. We have recently assembled a proteogenomic dataset of 87 GB clinical samples of varying survival rates. Following RNA-seq and mass spectrometry (MS)-based proteomics analysis, we identified several differentially expressed genes and proteins, including some known cancer-related pathways and some less established that showed higher expression in short-term (<6 months) survivors (STS) compared to LTS. One such target found was deoxyhypusine hydroxylase (DOHH), which is known to be involved in the biosynthesis of hypusine, an unusual amino acid essential for the function of the eukaryotic translation initiation factor 5A (eIF5A), which promotes tumor growth. We consequently validated DOHH overexpression in STS samples by quantitative polymerase chain reaction (qPCR) and immunohistochemistry. We further showed robust inhibition of proliferation, migration and invasion of GB cells following silencing of DOHH with short hairpin RNA (shRNA) or inhibition of its activity with small molecules, ciclopirox and deferiprone. Moreover, DOHH silencing led to significant inhibition of tumor progression and prolonged survival in GB mouse models. Searching for a potential mechanism by which DOHH promotes tumor aggressiveness, we found that it supports the transition of GB cells to a more invasive phenotype via epithelial-mesenchymal transition (EMT)-related pathways.

Nanoparticle impact on innate immune cell pattern-recognition receptors and inflammasomes activation.

Nanotechnology-based strategies can dramatically impact the treatment, prevention and diagnosis of a wide range of diseases. Despite the unprecedented success achieved with the use of nanomaterials to address unmet biomedical needs and their particular suitability for the effective application of a personalized medicine, the clinical translation of those nanoparticulate systems has still been impaired by the limited understanding on their interaction with complex biological systems. As a result, unexpected effects due to unpredicted interactions at biomaterial and biological interfaces have been underlying the biosafety concerns raised by the use of nanomaterials. This review explores the current knowledge on how nanoparticle (NP) physicochemical and surface properties determine their interactions with innate immune cells, with particular attention on the activation of pattern-recognition receptors and inflammasome. A critical perspective will additionally address the impact of biological systems on the effect of NP on immune cell activity at the molecular level. We will discuss how the understanding of the NP-innate immune cell interactions can significantly add into the clinical translation by guiding the design of nanomedicines with particular effect on targeted cells, thus improving their clinical efficacy while minimizing undesired but predictable toxicological effects.

Spontaneous regression of micro-metastases following primary tumor excision: a critical role for primary tumor secretome.

BACKGROUND: Numerous case studies have reported spontaneous regression of recognized metastases following primary tumor excision, but underlying mechanisms are elusive. Here, we present a model of regression and latency of metastases following primary tumor excision and identify potential underlying mechanisms. RESULTS: Using MDA-MB-231HM human breast cancer cells that express highly sensitive luciferase, we monitored early development stages of spontaneous metastases in BALB/c nu/nu mice. Removal of the primary tumor caused marked regression of micro-metastases, but not of larger metastases, and in vivo supplementation of tumor secretome diminished this regression, suggesting that primary tumor-secreted factors promote early metastatic growth. Correspondingly, MDA-MB-231HM-conditioned medium increased in vitro tumor proliferation and adhesion and reduced apoptosis. To identify specific mediating factors, cytokine array and proteomic analysis of MDA-MB-231HM secretome were conducted. The results identified significant enrichment of angiogenesis, growth factor binding and activity, focal adhesion, and metalloprotease and apoptosis regulation processes. Neutralization of MDA-MB-231HM-secreted key mediators of these processes, IL-8, PDGF-AA, Serpin E1 (PAI-1), and MIF, each antagonized secretome-induced proliferation. Moreover, their in vivo simultaneous blockade in the presence of the primary tumor arrested the development of micro-metastases. Interestingly, in the METABRIC cohort of breast cancer patients, elevated expression of Serpin E1, IL-8, or the four factors combined predicted poor survival. CONCLUSIONS: These results demonstrate regression and latency of micro-metastases following primary tumor excision and a crucial role for primary tumor secretome in promoting early metastatic growth in MDA-MB-231HM xenografts. If generalized, such findings can suggest novel approaches to control micro-metastases and minimal residual disease.

Persistent Chemiluminescent Glow of Phenoxy-dioxetane Luminophore Enables Unique CRET-Based Detection of Proteases.

Chemiluminescence is being considered an effective imaging modality as it offers low background and high sensitivity. Recent discovery by our group has led to development of new phenoxy-dioxetane chemiluminescence luminophores, which are highly bright under physiological conditions. However, the current scope of probes based on these luminophores is limited, as they can only be turned on by phenol protecting group removal. Here we present a new chemiluminescence resonance energy transfer (CRET) system, Glow-CRET, in which light emission is triggered by proteolytic cleavage of a peptide substrate that links a dioxetane luminophore and a quencher. In order to compose such system, a new phenoxy-dioxetane luminophore, 7-HC-CL, was developed. This luminophore exhibits intense and persistent glow chemiluminescence; it undergoes very slow chemiexcitation, and it has the highest chemiluminescence quantum yield ever reported under physiological conditions. Based on 7-HC-CL, a Glow-CRET probe for matrix metalloproteinases, MMP-CL, was synthesized. Incubation of MMP-CL with its cognate protease resulted in 160-fold increase in chemiluminescence signal. MMP-CL was also able to detect matrix metalloproteinase activity in cancer cells with significantly higher signal-to-background ratio than an analogous fluorescence resonance energy transfer (FRET)-based probe. This work is expected to open new horizons in chemiluminescence imaging, as it enables to use the dioxetanes in ways that had not been possible. We anticipate that 7-HC-CL and future derivatives will be utilized not only for the construction of further Glow-CRET probes, but also for other applications, such as chemiluminescence tagging of proteins.

Reverting the molecular fingerprint of tumor dormancy as a therapeutic strategy for glioblastoma.

Glioblastoma is an aggressive and invasive brain malignancy with high mortality rates despite current treatment modalities. In this study, we show that a 7-gene signature, previously found to govern the switch of glioblastomas from dormancy to aggressive tumor growth, correlates with improved overall survival of patients with glioblastoma. Using glioblastoma dormancy models, we validated the role of 2 genes from the signature, thrombospondin-1 ( TSP-1) and epidermal growth factor receptor ( EGFR), as regulators of glioblastoma dormancy and explored their therapeutic potential. EGFR up-regulation was reversed using EGFR small interfering RNA polyplex, antibody, or small-molecule inhibitor. The diminished function of TSP-1 was augmented via a peptidomimetic. The combination of EGFR inhibition and TSP-1 restoration led to enhanced therapeutic efficacy in vitro, in 3-dimensional patient-derived spheroids, and in a subcutaneous human glioblastoma model in vivo. Systemic administration of the combination therapy to mice bearing intracranial murine glioblastoma resulted in marginal therapeutic outcomes, probably due to brain delivery challenges, p53 mutation status, and the aggressive nature of the selected cell line. Nevertheless, this study provides a proof of concept for exploiting regulators of tumor dormancy for glioblastoma therapy. This therapeutic strategy can be exploited for future investigations using a variety of therapeutic entities that manipulate the expression of dormancy-associated genes in glioblastoma as well as in other cancer types.-Tiram, G., Ferber, S., Ofek, P., Eldar-Boock, A., Ben-Shushan, D., Yeini, E., Krivitsky, A., Blatt, R., Almog, N., Henkin, J., Amsalem, O., Yavin, E., Cohen, G., Lazarovici, P., Lee, J. S., Ruppin, E., Milyavsky, M., Grossman, R., Ram, Z., Calderón, M., Haag, R., Satchi-Fainaro, R. Reverting the molecular fingerprint of tumor dormancy as a therapeutic strategy for glioblastoma.

Novel Oligo-Guanidyl-PEG Carrier Forming Rod-Shaped Polyplexes.

A novel unconventional supramolecular oligo-cationic structure (Agm6-M-PEG-OCH3) has been synthesized to yield high efficiency therapeutic oligonucleotide (ON) delivery. Agm6-M-PEG-OCH3 was obtained by a multistep protocol that included the conjugation of agmatine (Agm) moieties to maltotriose (M), which was further derivatized with one poly(ethylene glycol) (PEG) chain. Gel electrophoresis analysis showed that the 19 base pairs dsDNA model ON completely associates with Agm6-M-PEG-OCH3 at 3 N/P molar ratio, which is in agreement with the in silico molecular predictions. Isothermal titration calorimetry (ITC) analyses showed that the Agm6-M-PEG-OCH3/ON association occurs through a combination of mechanisms depending on the N/P ratios resulting in different nanostructures. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) revealed that the Agm6-M-PEG-OCH3/ON polyplexes have rod-shape structure with a mean diameter of 50-75 nm and aspect ratio depending on the N/P ratio. The polyplexes were stable over time in buffer, while a slight size increase was observed in the presence of serum proteins. Cell culture studies showed that neither Agm6-M-PEG-OCH3 nor polyplexes displayed cytotoxic effects. Cellular uptake depended on the cell line and polyplex composition: cellular internalization was higher in the case of MCF-7 and KB cells compared to MC3T3-E1 cells and polyplexes with smaller aspect ratio were taken-up by cells more efficiently than polyplexes with higher aspect ratio. Finally, preliminary studies showed that our novel carrier efficiently delivered ONs into cells providing gene silencing.

ortho-Chlorination of phenoxy 1,2-dioxetane yields superior chemiluminescent probes for in vitro and in vivo imaging.

A recent methodology, developed by our group, has enabled a dramatic improvement in the emissive nature of the excited species, formed during the chemiexcitation of dioxetanes under physiological conditions. This approach has resulted in the discovery of distinct phenoxy-dioxetane luminophores that produce a chemiluminescence signal via a direct-mode of emission. Here, we show a significant pKa effect of our new phenoxy-dioxetanes on their chemiexcitation and on their ability to serve as chemiluminescent turn-ON probes for biological applications. Using an appropriate phenoxy-dioxetane probe with a direct-mode of emission, we were able to image ß-galactosidase activity, in cancer cells and in tumor-bearing mice. To the best of our knowledge, this is the first example to demonstrate in vitro and in vivo endogenous enzymatic chemiluminescence images obtained by a single-component phenoxy-dioxetane probe. We anticipate that our strategy, for the design and synthesis of such distinct luminophores, will assist in providing new effective turn-ON probes for non-invasive intravital chemiluminescence imaging techniques.

PEGylated dendritic polyglycerol conjugate targeting NCAM-expressing neuroblastoma: Limitations and challenges.

Neural cell adhesion molecule (NCAM) is found to be a stem-cell marker in several tumor types and its overexpression is known to correlate with increased metastatic capacity. To combine extravasation- and ligand-dependent targeting to NCAM overexpressing-cells in the tumor microenvironment, we developed a PEGylated NCAM-targeted dendritic polyglycerol (PG) conjugate. Here, we describe the synthesis, physico-chemical characterization and biological evaluation of a PG conjugate bearing the mitotic inhibitor paclitaxel (PTX) and an NCAM-targeting peptide (NTP). PG-NTP-PTX-PEG was evaluated for its ability to inhibit neuroblastoma progression in vitro and in vivo as compared to non-targeted derivatives and free drug. NCAM-targeted conjugate inhibited the migration of proliferating endothelial cells, suggesting it would be able to inhibit tumor angiogenesis. The targeting conjugate provided an improved binding and uptake on IMR-32 cells compared to non-targeted control. However, these results did not translate to our in vivo model on orthotopic neuroblastoma bearing mice.

Amphiphilic poly(α)glutamate polymeric micelles for systemic administration of siRNA to tumors.

RNAi therapeutics carried a great promise to the area of personalized medicine: the ability to target "undruggable" oncogenic pathways. Nevertheless, their efficient tumor targeting via systemic administration had not been resolved yet. Amphiphilic alkylated poly(α)glutamate amine (APA) can serve as a cationic carrier to the negatively-charged oligonucleotides. APA polymers complexed with siRNA to form round-shaped, homogenous and reproducible nano-sized polyplexes bearing ~50 nm size and slightly negative charge. In addition, APA:siRNA polyplexes were shown to be potent gene regulators in vitro. In light of these preferred physico-chemical characteristics, their performance as systemically-administered siRNA nanocarriers was investigated. Intravenously-injected APA:siRNA polyplexes accumulated selectively in tumors and did not accumulate in the lungs, heart, liver or spleen. Nevertheless, the polyplexes failed to induce specific mRNA degradation, hence neither reduction in tumor volume nor prolonged mice survival was seen.

Nanoparticulate vaccine inhibits tumor growth via improved T cell recruitment into melanoma and huHER2 breast cancer.

Nanoparticulate vaccines are promising tools to overcome cancer immune evasion. However, a deeper understanding on nanoparticle-immune cell interactions and treatments regime is required for optimal efficacy. We provide a comprehensive study of treatment schedules and mode of antigen-association to nanovaccines on the modulation of T cell immunity in vivo, under steady-state and tumor-bearing mice. The coordinated delivery of antigen and two adjuvants (Monophosphoryl lipid A, oligodeoxynucleotide cytosine-phosphate-guanine motifs (CpG)) by nanoparticles was crucial for dendritic cell activation. A single vaccination dictated a 3-fold increase on cytotoxic memory-T cells and raised antigen-specific immune responses against B16.M05 melanoma. It generated at least a 5-fold increase on IFN-γ cytokine production, and presented over 50% higher lymphocyte count in the tumor microenvironment, compared to the control. The number of lymphocytes at the tumor site doubled with triple immunization. This lymphocyte infiltration pattern was confirmed in mammary huHER2 carcinoma, with significant tumor reduction.

Direct Real-Time Monitoring of Prodrug Activation by Chemiluminescence.

The majority of theranostic prodrugs reported so far relay information through a fluorogenic response generated upon release of the active chemotherapeutic agent. A chemiluminescence detection mode offers significant advantages over fluorescence, mainly due to the superior signal-to-noise ratio of chemiluminescence. Here we report the design and synthesis of the first theranostic prodrug monitored by a chemiluminescence diagnostic mode. As a representative model, we prepared a prodrug from the chemotherapeutic monomethyl auristatin E, which was modified for activation by ß-galactosidase. The activation of the prodrug in the presence of ß-galactosidase is accompanied by emission of a green photon. Light emission intensities, which increase with increasing concentration of the prodrug, were linearly correlated with a decrease in the viability of a human cell line that stably expresses ß-galactosidase. We obtained sharp intravital chemiluminescent images of endogenous enzymatic activity in ß-galactosidase-overexpressing tumor-bearing mice. The exceptional sensitivity achieved with the chemiluminescence diagnostic mode should allow the exploitation of theranostic prodrugs for personalized cancer treatment.

Near-Infrared Dioxetane Luminophores with Direct Chemiluminescence Emission Mode.

Chemiluminescent luminophores are considered as one of the most sensitive families of probes for detection and imaging applications. Due to their high signal-to-noise ratios, luminophores with near-infrared (NIR) emission are particularly important for in vivo use. In addition, light with such long wavelength has significantly greater capability for penetration through organic tissue. So far, only a few reports have described the use of chemiluminescence systems for in vivo imaging. Such systems are always based on an energy-transfer process from a chemiluminescent precursor to a nearby emissive fluorescent dye. Here, we describe the development of the first chemiluminescent luminophores with a direct mode of NIR light emission that are suitable for use under physiological conditions. Our strategy is based on incorporation of a substituent with an extended π-electron system on the excited species obtained during the chemiexcitation pathway of Schaap's adamantylidene-dioxetane probe. In this manner, we designed and synthesized two new luminophores with direct light emission wavelength in the NIR region. Masking of the luminophores with analyte-responsive groups has resulted in turn-ON probes for detection and imaging of ß-galactosidase and hydrogen peroxide. The probes' ability to image their corresponding analyte/enzyme was effectively demonstrated in vitro for ß-galactosidase activity and in vivo in a mouse model of inflammation. We anticipate that our strategy for obtaining NIR luminophores will open new doors for further exploration of complex biomolecular systems using non-invasive intravital chemiluminescence imaging techniques.

Are nanotheranostics and nanodiagnostics-guided drug delivery stepping stones towards precision medicine?

The progress in medical research has led to the understanding that cancer is a large group of heterogeneous diseases, with high variability between and within individuals. This variability sprouted the ambitious goal to improve therapeutic outcomes, while minimizing drug adverse effects through stratification of patients by the differences in their disease markers, in a personalized manner, as opposed to the strategy of "one therapy fits all". Nanotheranostics, composed of nanoparticles (NPs) carrying therapeutic and/or diagnostics probes, have the potential to revolutionize personalized medicine. There are different modalities to combine these two distinct fields into one system for a synergistic outcome. The addition of a nanocarrier to a theranostic system holds great promise. Nanocarriers possess high surface area, enabling sophisticated functionalization with imaging agents, thus gaining enhanced diagnostic ability in real-time. Yet, most of the FDA-approved theranostic approaches are based on small molecules. The theranostic approaches that are reviewed herein are paving the road towards personalized medicine through all stages of patient care: starting from screening and diagnostics, proceeding to treatment and ending with treatment follow-up. Our current review provides a broad background and highlights new insights for the rational design of theranostic nanosystems for desired therapeutic niches, while summoning the hurdles on their way to become first-line diagnostics and therapeutics for cancer patients.

A Highly Efficient Chemiluminescence Probe for the Detection of Singlet Oxygen in Living Cells.

Singlet oxygen is among the reactive oxygen species (ROS) with the shortest life-times in aqueous media because of its extremely high reactivity. Therefore, designing sensors for detection of 1 O2 is perhaps one of the most challenging tasks in the field of molecular probes. Herein, we report a highly selective and sensitive chemiluminescence probe (SOCL-CPP) for the detection of 1 O2 in living cells. The probe reacts with 1 O2 to form a dioxetane that spontaneously decomposes under physiological conditions through a chemiexcitation pathway to emit green light with extraordinary intensity. SOCL-CPP demonstrated promising ability to detect and image intracellular 1 O2 produced by a photosensitizer in HeLa cells during photodynamic therapy (PDT) mode of action. Our findings make SOCL-CPP the most effective known chemiluminescence probe for the detection of 1 O2 . We anticipate that our chemiluminescence probe for 1 O2 imaging would be useful in PDT-related applications and for monitoring 1 O2 endogenously generated by cells in response to different stimuli.

Remarkable Enhancement of Chemiluminescent Signal by Dioxetane-Fluorophore Conjugates: Turn-ON Chemiluminescence Probes with Color Modulation for Sensing and Imaging.

Chemiluminescence is among the most sensitive methods for achieving a high signal-to-noise ratio in various chemical and biological applications. We have developed a modular practical synthetic route for preparation of turn-ON fluorophore-tethered dioxetane chemiluminescent probes. The chemiluminescent emission of the probes was significantly amplified through an energy-transfer mechanism under physiological conditions. Two probes were composed with green and near-infrared (NIR) fluorescent dyes tethered to Schaap's dioxetane. While both probes were able to provide chemiluminescence in vivo images following subcutaneous injection, only the NIR probe could provide a chemiluminescence image following intraperitoneal injection. These are the first in vivo images produced by Schaap's dioxetane chemiluminescence probes with no need of an enhancer. Previously, chemiluminescence cell images could only be obtained with a luciferin-based probe. Our NIR probe was able to image cells transfected with ß-galactosidase gene by chemiluminescence microscopy. We also report, for the first time, the instability of dioxetane-fluorophore conjugates to ambient light. Our synthetic route effectively overcomes this limitation through a late-stage functionalization of the dioxetane intermediate. We anticipate that our practical synthetic methodology will be useful for preparation of various chemiluminescent probes for numerous applications.

Inhibition of Gene Expression and Cancer Cell Migration by CD44v3/6-Targeted Polyion Complexes.

In recent years, siRNA technology has emerged as a promising strategy for gene silencing in cancer therapy. We have designed novel CD44-targeted polyion complexes (PICs) composed of poly(ethylene glycol)-block-polyethylenimine (PEG-b-PEI) and laminin-derived peptides (mA5G27D or mA5G27F) for in vivo siRNA delivery and gene silencing in tumors. The full-length A5G27 peptide (RLVSYNGIIFFLK), from which mA5G27D and mA5G27F are derived, binds to CD44v3 and CD44v6 and inhibits tumor cell migration, invasion, and angiogenesis. Thus, when attached to the surface of PICs, A5G27-based peptides can serve both as targeting ligands to navigate siRNA molecules directly to CD44-overexpressing tumors, and as anti-migratory agents to inhibit tumor progression. The mA5G27D- or mA5G27F-harboring PEG-b-PEI copolymers strongly condensed siRNA molecules into nanosized PICs presenting positive surface charges, low in vitro cytotoxicity, and high serum stability. mA5G27D- or mA5G27F-bearing PICs demonstrated high efficacy and selectivity in delivering siRAC1 into CD44-overexpressing cells, thereby silencing RAC1 mRNA and protein levels in such cells. These PICs presented substantial anti-migratory features in vitro and accumulated significantly in SK-OV-3 tumor-bearing mice, following 3 sequential intraperitoneal (i.p.) injections. Treatment of mice with 8 or 9 sequential parenteral (intravenous, (i.v.) or i.p.) injections of mA5G27F-PEG-b-PEI/siRNA efficiently inhibited tumor growth in two different CD44-overexpressing tumor mouse models (A549 and SK-OV-3), regardless of the type of siRNA (siPLK1 or siLUC) used. The results thus reveal the potential utility of this system for targeted delivery of siRNA molecules into solid tumors to prolong the survival time of mice, while at the same time reducing potential toxicity.

Tagging the Untaggable: A Difluoroalkyl-Sulfinate Ketone-Based Reagent for Direct C-H Functionalization of Bioactive Heteroarenes.

We have developed a new difluoroalkyl ketal sulfinate salt reagent suitable for direct derivatization of heteroarene C-H bonds. The reagent is capable of introducing a ketone functional group on heteroarene bioactive compounds via a one-pot reaction. Remarkably, in three examples the ketone analog and its parent drug had almost identical cytotoxicity. In a representative example, the ketone analog was bioconjugated with a delivery vehicle via an acid-labile semicarbazone linkage and with a photolabile protecting group to produce the corresponding prodrug. Controlled release of the drug-ketone analog was demonstrated in vitro for both systems. This study provides a general approach to obtain taggable ketone analogs directly from bioactive heteroarene compounds with limited options for conjugation. We anticipate that this sodium ketal-sulfinate reagent will be useful for derivatization of other heteroarene-based drugs to obtain ketone-taggable analogs with retained efficacy.

Structure-Function Correlation of Aminated Poly(α)glutamate as siRNA Nanocarriers.

It has been two decades since cationic polymers were introduced to the world of oligonucleotides delivery. However, the optimal physicochemical properties to make them a successful delivery vehicle are yet unknown. An ideal system became particularly interesting and necessary with the introduction of RNA interference as a promising therapeutic approach. Such nanocarrier should overcome challenges such as low plasma stability, poor cellular internalization and endosomal escape to induce gene silencing. To that end, we synthesized a library of biodegradable aminated poly(α)glutamate varied by amine moieties. In an attempt to elucidate the structure-function relationship, our polyplexes were physicochemically characterized and their silencing activity and cytotoxicity were evaluated. We found several structures that demonstrated improved cellular internalization. These candidates silenced gene expression to less than 50% of their initial levels, while being safe to cells and mice. Based on our research, an improved and promising tailor-designed siRNA delivery platform can be developed.

Lomustine Nanoparticles Enable Both Bone Marrow Sparing and High Brain Drug Levels - A Strategy for Brain Cancer Treatments.

PURPOSE: The blood brain barrier compromises glioblastoma chemotherapy. However high blood concentrations of lipophilic, alkylating drugs result in brain uptake, but cause myelosuppression. We hypothesised that nanoparticles could achieve therapeutic brain concentrations without dose-limiting myelosuppression. METHODS: Mice were dosed with either intravenous lomustine Molecular Envelope Technology (MET) nanoparticles (13 mg kg(-1)) or ethanolic lomustine (6.5 mg kg(-1)) and tissues analysed. Efficacy was assessed in an orthotopic U-87 MG glioblastoma model, following intravenous MET lomustine (daily 13 mg kg(-1)) or ethanolic lomustine (daily 1.2 mg kg(-1) - the highest repeated dose possible). Myelosuppression and MET particle macrophage uptake were also investigated. RESULTS: The MET formulation resulted in modest brain targeting (brain/ bone AUC0-4h ratios for MET and ethanolic lomustine = 0.90 and 0.53 respectively and brain/ liver AUC0-4h ratios for MET and ethanolic lomustine = 0.24 and 0.15 respectively). The MET formulation significantly increased mice (U-87 MG tumours) survival times; with MET lomustine, ethanolic lomustine and untreated mean survival times of 33.2, 22.5 and 21.3 days respectively and there were no material treatment-related differences in blood and femoral cell counts. Macrophage uptake is slower for MET nanoparticles than for liposomes. CONCLUSIONS: Particulate drug formulations improved brain tumour therapy without major bone marrow toxicity.

Restoring the oncosuppressor activity of microRNA-34a in glioblastoma using a polyglycerol-based polyplex.

Glioblastoma multiforme (GBM) is the most common and aggressive primary neoplasm of the brain. Poor prognosis is mainly attributed to tumor heterogeneity, invasiveness, and drug resistance. microRNA-based therapeutics represent a promising approach due to their ability to inhibit multiple targets. In this work, we aim to restore the oncosuppressor activity of microRNA-34a (miR-34a) in GBM. We developed a cationic carrier system, dendritic polyglycerolamine (dPG-NH2), which remarkably improves miRNA stability, intracellular trafficking, and activity. dPG-NH2 carrying mature miR-34a targets C-MET, CDK6, Notch1 and BCL-2, consequently inhibiting cell cycle progression, proliferation and migration of GBM cells. Following complexation with dPG-NH2, miRNA is stable in plasma and able to cross the blood-brain barrier. We further show inhibition of tumor growth following treatment with dPG-NH2-miR-34a in a human glioblastoma mouse model. We hereby present a promising technology using dPG-NH2-miR-34a polyplex for brain-tumor treatment, with enhanced efficacy and no apparent signs of toxicity.