Stability In Biological Fluids Research Articles

2034-P: ß Cell miRNAs Function as Molecular Hubs of Type 1 Diabetes Pathogenesis and Risk

Biomarkers capable of monitoring β cell stress during the evolution of type 1 diabetes (T1D) are currently lacking. MicroRNAs (miRNAs) are a class of small non-coding RNAs ∼22 nucleotides in length that modulate gene expression by binding to the 3’untranslated region of target mRNAs. Given their stability in biological fluids and enrichment in cell-derived EVs, we hypothesized that miRNAs from human islet and islet-derived EVs could identify β-cell stress/death and be leveraged in T1D biomarker strategies. To test this, human islets were obtained from 10 cadaveric donors (5 male/5 female) and treated with or without cytokines (IL-1β and IFN-γ) for 24 hrs, as an ex vivo model of T1D. Small RNA sequencing was performed and identified 1110 and 890 miRNAs in total and 20 and 14 differentially expressed (DE) miRNAs (fold change≥1.5 and p<0.05) from islets and EVs, respectively. These findings were validated in an independent set of cytokine-treated islets and islet-derived EVs (7M and 5F donors). Interestingly, miRNA expression pattern was strikingly different between male and female donors at baseline and under cytokine stress with < 10% overlap among the DE miRNAs. miR-155-5p and miR-146a-5p were the only two miRNAs that were upregulated in cytokine-treated islets and EVs in both the sexes. Functional enrichment analysis of DE miRNAs identified pathways such as insulin signaling, ER stress and apoptosis. Taken together, these data suggest that miRNA expression patterns change dynamically in both human islets and islet-derived EVs in response to pro-inflammatory cytokine stress. EV miRNAs were largely distinct from those in the islet fraction, suggesting that miRNAs are selectively packaged into EVs in response to extrinsic cues. Finally, these data highlight the importance of considering sex as a biological variable when defining T1D biomarkers. Disclosure F. Syed: None. P. Krishnan: None. I. Restrepo: None. R. Mirmira: Advisory Panel; Self; Hibercell, Sigilon Therapeutics, Veralox Therapeutics. Employee; Spouse/Partner; Eli Lilly and Company. C. Evans-Molina: Consultant; Self; Bristol-Myers Squibb. Funding National Institute of Diabetes and Digestive and Kidney Diseases (R01DK093954, UC4-DK104166 to C.E-M., R.M.); U.S. Department of Veterans Affairs (I01BX001733 to C.E-M.); JDRF (3-SRA-2014-41-Q-R); Sigma Beta Sorority; Ball Brothers Foundation; George and Frances Ball Foundation (to C.E-M.); Indiana Diabetes Research Center (P30DK097512)

Strategies for the Controlled Covalent Double Functionalization of Graphene Oxide.

Graphene oxide (GO) is a versatile platform with unique properties that have found broad applications in the biomedical field. Double functionalization is a key aspect in the design of multifunctional GO with combined imaging, targeting, and therapeutic properties. Compared to noncovalent functionalization, covalent strategies lead to GO conjugates with a higher stability in biological fluids. However, only a few double covalent functionalization approaches have been developed so far. The complexity of GO makes the derivatization of the oxygenated groups difficult to control. The combination of a nucleophilic epoxide ring opening with the derivatization of the hydroxyl groups through esterification or Williamson reaction was investigated. The conditions were selective and mild, thus preserving the structure of GO. Our strategy of double functionalization holds great potential for different applications in which the derivatization of GO with different molecules is needed, especially in the biomedical field.

Circulating microRNAs as potential biomarkers for genetic generalized epilepsies: a three microRNA panel.

Genetic generalized epilepsies (GGEs) encompass a group of syndromes of mainly genetic causes, characterized by the involvement of both hemispheres. MicroRNAs (miRNAs) are small non-coding RNAs with a critical role in the regulation of neuronal biological processes through gene expression modulation. Dysregulated miRNA expression has been shown in epilepsy. Due to their stability in biological fluids like serum, miRNAs have assumed a prominent role in biomarker research. Our aim was to evaluate circulating levels of three miRNAs in GGE patients and assess their putative diagnostic value. MiR-146a, miR-155 and miR-132 were quantified by real-time polymerase chain reaction in the serum of 79 GGE patients (47 women, 32 men, 35.1±12.4years) and 67 healthy individuals (41 women, 26 men, 42.4±10.1years). Relative expression values were calculated using the 2-ΔΔCt method. Receiver operating characteristic curve analysis was performed to assess diagnostic value. MiRNA expression was correlated with clinicopathological features. Serum levels of miR-146a and miR-155 were significantly upregulated in GGE patients relative to controls (3.13 and 6.05, respectively). Combined miR-146a, miR-155 and miR-132 serum levels performed well as a diagnostic biomarker, discriminating GGE patients from controls with an area under the curve of 0.85, 80% specificity and 73% sensitivity. Our results indicate that miR-146a, miR-155 and miR-132 may partake in GGE epileptogenesis. A panel of three circulating miRNAs with potential value as a GGE biomarker is reported for the first time. Novel biomarkers may help to identify new treatment targets and contribute to improved patients' quality of life through earlier diagnosis and a more precise prognosis.

Barium Ferrite Magnetic Nanoparticles Labeled with 223Ra: A New Potential Radiobioconjugate for Targeted Alpha Therapy and Magnetic Hyperthermia

Among all alpha particle emitters, only a few nuclides are of considerable interest for targeted radionuclide therapy because of their properties, such as half-live, high cytotoxicity and short path length. One of the most important issues, which affects wider use of targeted α therapy in nuclear medicine, is the availability and the price of the radionuclides. 223Ra, as radium chloride, is the first commercially and widely used α-radiopharmaceutic. It is easily obtained from the 227Ac/223Ra generator. However, 223Ra is used mostly for treatment of bone metastases derived from primary prostate cancer, because 223Ra, as a member of alkaline earth metals, forms very weak complexes. There is a lack of chelators which can effectively bind 223Ra for the purposes of receptor targeted therapy. In our studies we propose to use barium ferrite (BaFe12O19) nanoparticles as multifunctional carriers for 223Ra radionuclide for targeted α therapy and magnetic hyperthermia. Barium hexaferrite nanoparticles labeled with 223Ra were synthesized by a modified autoclave method described by Drofenik et al [1]. The reaction mixture of FeCl3, BaCl2 and 223RaCl2 was alkalized with NaOH solution. Next, the reaction mixture was stirred in autoclave at 210 Celsius degrees for 5 h. Obtained radioactive, magnetic [223Ra]BaFe12O19 nanoparticles were washed with distilled water and hydrochloric acid (0.001 mol/L HCl). Yield of labeling was about 70% (for 100kBq 223Ra). Stability of the obtained radioactive nanoparticles was tested in various biological solutions: 0.01M PBS,0.9% NaCl and in human blood serum. It is confirmed that 223Ra was highly retained inside nanoparticles in every tested solution. Only about 20% of 211Pb (decay product of 223Ra) was released to the solution. Obtained magnetic BaFe12O19 nanoparticles were characterized by transmission emission microscopy and dynamic light scattering. The diameter of synthesized nanoparticles was ∼20 nm and the determined magnetization of nanoparticles in room temperature was about 42 emu/g. In order to synthesize a radiobioconjugate having affinity to HER2 receptors, the monoclonal antibody trastuzumab was conjugated to the obtained barium ferrite nanoparticles. Firstly, the surface of barium ferrite nanoparticles was modified with 3-phosphonopropionic acid (CEPA) linker using a method described by Mohapatra et al [2], and then, the monoclonal antibodies were coupled to the barium ferrite nanoparticles using the carbodiimide chemistry. Synthesized bioconjugate was characterized by thermogravimetric analysis, dynamic light scattering and were tested for stability in biological fluids. The obtained [223Ra]BaFe12O19CEPA-trastuzumab radiobioconjugate almost quantitatively retains 223Ra and majority of the daughter products. Radiobioconjugate has high receptor affinity towards HER2 receptors expressing on ovarian cancer cells and exhibits high cytotoxic effect in vitro. This work was supported by National Science Centre of Poland (Grant NCN Preludium 2015/17/N/ST4/03943). [1]M.Drofenik et al Mat.Chem.Phys. 2011; 127: 415–419.[2]S.Mohapatra et al Nanotechnology 2007; 18: 385102–11.

Abstract SY15-03: Coding and noncoding genome for blood biopsy

Abstract The development of effective diagnostic and prognostic biomarkers is crucial for early detection of cancer, prediction of clinical outcome, detection and monitoring minimal residual disease, and identification of patients who can benefit from therapy. Furthermore, they can provide important information of biological characteristics of tumor. Liquid biopsy, an alternative and non-invasive technique to surgical biopsy, enables the investigation of tumor markers in biological fluids, such as serum/plasma, urine, saliva, and pleural effusion. Increasing interest on liquid biopsy has grown over the past decade with particular focus on circulating non-coding RNAs (ncRNAs) and tumor-derived cell-free DNA (cfDNA) as cancer biomarkers. Owing to their high abundance and stability in biological fluids, circulating ncRNAs and tumor-derived cell-free DNA have potential utility to provide information on tumor biology (e.g.: EGFR and PIK3CA mutational status) which can help both diagnosis and prediction of effects of treatments, including standard treatment, targeted therapies, and immunotherapies. Recent advances in the field have highlighted the role of circulating ncRNAs in cell-to-cell communication. Particularly, ncRNAs can be transported through extracellular vesicle-mediated transfer to recipient cells, where they exert their functions of gene expression regulation, with implications for cancer progression and therapy resistance. This characteristic of circulating ncRNAs introduces an additional step in the information on tumor biology that can be obtained from liquid biopsy. Besides cancer, ncRNAs can also serve as biomarkers for microbial infection. Indeed, miRNAs originated from non-human genomes (xeno-miRNAs), such as viral miRNAs (Epstein-Barr virus, Kaposi sarcoma-associated herpesvirus), have been shown to be present in human body fluids. They can be used as biomarkers for diagnosis, prediction of clinical outcome, and the can also mediate cell-to-cell communication. Liquid biopsy has the potential to serve as additional tool to improve diagnosis, prognosis, prediction of treatment responses, and can provide information of biological characteristics of tumor. Citation Format: Daniel F. Hayes, Catherine Alix-Panabières, George A. Calin. Coding and noncoding genome for blood biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr SY15-03.

Shielding and stealth effects of zwitterion moieties in double-functionalized silica nanoparticles

Surface functionalization of silica nanoparticles (SiO2NPs) has been considered as a promising strategy to develop target-specific nanostructures. However, finding a chemical functionalization that can be used as an active targeting moiety while preserving the nanoparticles colloidal stability in biological fluids is still challenging. We present here a dual surface modification strategy for SiO2NPs where a zwitterion (ZW) and a biologically active group (BAG) (amino, mercapto or carboxylic functionalities) are simultaneously grafted on the nanoparticles’ surface. The rationale behind this strategy is to generate colloidally stable nanoparticles and avoid the nonspecific protein adsorption due to ZW groups insertion, while the effective interaction with biosystems is guaranteed by the BAGs presence. The biological efficacy was tested against VERO cells, E. coli bacteria and Zika viruses and a similar trend was observed for all tested particles. The desirable “stealth property” to prevent nonspecific protein adhesion also generated a ZW shielding effect of the BAG functionality hindering their proper interaction and activity in cells, bacteria and viruses.

MicroRNA Biomarkers in Cerebrospinal Fluid and Serum Reflect Injury Severity in Human Acute Traumatic Spinal Cord Injury.

Spinal cord injury (SCI) is a devastating condition with variability in injury mechanisms and neurologic recovery. Spinal cord impairment after SCI is measured and classified by a widely accepted standard neurological examination. In the very acute stages post-injury, however, this examination is extremely challenging (and often impossible) to conduct and has modest prognostic value in terms of neurological recovery. The lack of objective tools to classify injury severity and predict outcome is a barrier for clinical trials and thwarts development of therapies for those with SCI. Biological markers (biomarkers) represent a promising, complementary approach to these challenges because they represent an unbiased approach to classify injury severity and predict neurological outcome. Identification of a suitable panel of molecular biomarkers would comprise a fundamental shift in how patients with acute SCI are evaluated, stratified, and treated in clinical trials. MicroRNA are attractive biomarker candidates in neurological disorders for several reasons, including their stability in biological fluids, their conservation between humans and model mammals, and their tissue specificity. In this study, we used next-generation sequencing to identify microRNA associated with injury severity within the cerebrospinal fluid (CSF) and serum of human patients with acute SCI. The CSF and serum samples were obtained 1-5 days post-injury from 39 patients with acute SCI (24 American Spinal Injury Association Impairment Scale [AIS] A, 8 AIS B, 7 AIS C) and from five non-SCI controls. We identified a severity-dependent pattern of change in microRNA expression in CSF and identified a set of microRNA that are diagnostic of baseline AIS classification and prognostic of neurological outcome six months post-injury. The data presented here provide a comprehensive description of the CSF and serum microRNA expression changes that occur after acute human SCI. This data set reveals microRNA candidates that warrant further evaluation as biomarkers of injury severity after SCI and as key regulators in other neurological disorders.

Surface Modifications of Nanoparticles for Stability in Biological Fluids.

Due to the high surface: volume ratio and the extraordinary properties arising from the nanoscale (optical, electric, magnetic, etc.), nanoparticles (NPs) are excellent candidates for multiple applications. In this context, nanoscience is opening a wide range of modern technologies in biological and biomedical fields, among others. However, one of the main drawbacks that still delays its fast evolution and effectiveness is related to the behavior of nanomaterials in the presence of biological fluids. Unfortunately, biological fluids are characterized by high ionic strengths which usually induce NP aggregation. Besides this problem, the high content in biomacromolecules—such as lipids, sugars, nucleic acids and, especially, proteins—also affects NP stability and its viability for some applications due to, for example, the formation of the protein corona around the NPs. Here, we will review the most common strategies to achieve stable NPs dispersions in high ionic strength fluids and, also, antifouling strategies to avoid the protein adsorption.

In vitro/in vivo investigation on the potential of Pluronic® mixed micelles for pulmonary drug delivery

In this paper, we shed light on the potential of Pluronic® mixed micelles in lung delivery of poorly water-soluble drugs. To this purpose, Pluronic® P123/F127 mixed micelles (PMM), exhibiting superior stability in biological fluids, were loaded with budesonide (BUD), a model hydrophobic corticosteroid, and fully investigated focusing on their stability in pulmonary-relevant media, transport through the mucus barrier and aerodynamic behaviour in vitro. Then, lung bio-distribution and efficacy were evaluated in vivo, after intra-tracheal administration in rats. PMM showed excellent stability in saline, mucin, artificial airway mucus and simulated interstitial lung fluid. Likely due to their small size coupled with the hydrophilic biofouling shell, PMM did not interact with mucin and consequently diffused through artificial mucus. BUD was loaded with high efficiency in PMM and released at sustained rate in artificial mucus. BUD-PMM dispersion in saline was efficiently delivered through a common jet nebulizer without aggregation. After intratracheal administration in rats, PMM labelled with Rhodamine B persisted in the lung up to 24 h, while serum levels rapidly dropped. Finally, the effects of BUD-PMM in a rat model of lung inflammation induced by intra-tracheal aerosolization of lipopolysaccharide (LPS) from E. coli were investigated. Of note, a single intra-tracheal aerosolization of BUD-PMM significantly reduced bronchoalveolar neutrophil infiltration and the expression of protein/enzymes derived from the arachidonic acid cascade induced by LPS, whereas a control BUD aqueous suspension showed a weaker effect. Overall, this study demonstrates that inhalable formulations of PMM can be considered as a platform for local delivery of hydrophobic drugs at lungs worth of further consideration.

Biochemical Properties of a Decoy Oligodeoxynucleotide Inhibitor of STAT3 Transcription Factor.

Cyclic STAT3 decoy (CS3D) is a second-generation, double-stranded oligodeoxynucleotide (ODN) that mimics a genomic response element for signal transducer and activator of transcription 3 (STAT3), an oncogenic transcription factor. CS3D competitively inhibits STAT3 binding to target gene promoters, resulting in decreased expression of proteins that promote cellular proliferation and survival. Previous studies have demonstrated antitumor activity of CS3D in preclinical models of solid tumors. However, prior to entering human clinical trials, the efficiency of generating the CS3D molecule and its stability in biological fluids should be determined. CS3D is synthesized as a single-stranded ODN and must have its free ends ligated to generate the final cyclic form. In this study, we report a ligation efficiency of nearly 95 percent. The ligated CS3D demonstrated a half-life of 7.9 h in human serum, indicating adequate stability for intravenous delivery. These results provide requisite biochemical characterization of CS3D that will inform upcoming clinical trials.

Turn-on fluorescent probe-encapsulated micelle as colloidally stable nano-chemosensor for highly selective detection of Al3+ in aqueous solution and living cell imaging

It is well known that high amount of aluminum ion are not only detrimental to plant growth but also threat to our health. Most of fluorescent chemosensors for the detection Al3+ suffer from some restrictions, such as poor selectivity, indifferent sensitivity, inferior stability in biological fluids, and being unsuitable for the applications in aqueous environment and biological systems. To address the above limitations, we used DSPE-PEG as a carrier of fluorescent sensor (Dye) to obtain a new type of fluorescent nanochemosensor (DSPE-PEG-Dye). The hydrophobic fluorescent schiff base (Dye), synthesized by simple two-step reaction, exhibited excellent sensitivity for determination of Al3+ in the mixture of tetrahydrofuran and water (2:3, v/v). Subsequently, the Dye was successfully encapsulated in DSPE-PEG micelles to prepare the nanochemosensor (DSPE-PEG-Dye) by the solvent evaporation method, which remained the properties of Dye and displayed high selectivity and sensitivity towards Al3+ ions even in the presence of other commonly coexisting metal ions in aqueous solution. Furthermore, DSPE-PEG-Dye can act as a reversible sensor for Al3+. Sequential addition of EDTA and Al3+ into DSPE-PEG-Dye-Al3+ complex resulted in quench and recovery of fluorescence, respectively. Moreover, DSPE-PEG-Dye with low detection limit of 2.3 × 10−8 M is colloidally stable in water, PBS buffer (pH 7.4) and cell culture medium even after storage for one month. Interestingly, the nano-chemosensor exerted superior cell internalization and could be used as a nontoxic bioimaging reagent for visualizing Al3+ in living cells.

In vitro and in vivo activity of cyclopeptide Dmt-c[d-Lys-Phe-Asp]NH2, a mu opioid receptor agonist biased toward β-arrestin

Morphine and related drugs, which are the most effective analgesics for the relief of severe pain, act through activating opioid receptors. The endogenous ligands of these receptors are opioid peptides which cannot be used as antinociceptive agents due to their low bioactivity and stability in biological fluids. The major goal of opioid research is to understand the mechanism of action of opioid receptor agonists in order to improve therapeutic utility of opioids. Analgesic effects of morphine are mediated mostly through activation of the mu opioid receptor. However, in the search for safer and more effective drug candidates, analogs with mixed opioid receptor profile gained a lot of interest. Recently, the concept of biased agonists able to differentially activate GPCR downstream pathways, became a new approach in the design of novel drug candidates. It is hypothesized that compounds promoting G-protein signaling may produce analgesia while β-arrestin recruitment may be responsible for opioid side effects. In this report we showed that replacement of the tyrosine residue in the mu-selective ligand Tyr-c[d-Lys-Phe-Asp]NH2 with 2′,6′-dimethyltyrosine (Dmt) produced a cyclopeptide Dmt-c[d-Lys-Phe-Asp]NH2 with mu/delta opioid receptor agonist profile. This analog showed improved antinociception in the hot-plate test, probably due to the simultaneous activation of mu and delta receptors but also significantly inhibited the gastrointestinal transit. Using the bioluminescence resonance energy transfer (BRET) assay it was shown that this analog was a mu receptor agonist biased toward β-arrestin. β-Arrestin-dependent signaling is most likely responsible for the observed inhibition of gastrointestinal motility exerted by the novel cyclopeptide.

Biodistribution of colloidal gold nanoparticles after intravenous injection: Effects of PEGylation at the same particlesize.

Recently, polyethylene glycol (PEG) modified gold nanoparticles have been studied to maintaining long-term stability in biological fluids. Its biodistribution was also reported, however, comparison of bare gold nanoparticles and PEGylated gold nanoparticles with equal particle size is not sufficient. We prepared bare gold nanoparticles and PEGylated gold nanoparticles with diameters of 20-30-nm or 50-nm to avoid the influence of particle diameter, and studied their biodistribution in the mouse. Gold concentrations in brain, heart, lungs, liver, stomach, pancreas, spleen, kidneys, blood, urine, and feces were measured at 0.5, 1, 2, 3, 6, 12, 18, 24, and 48 h after administration of gold nanoparticles using inductively coupled plasma atomic emission spectrometry. At 48 h after intravenous administration, accumulation in the liver and spleen was significantly reduced by PEGylation, and the gold amounts of PEGylated gold nanoparticles with diameters of 20-30 nm and 50-nm in the brain were 3.6 times and 2.7 times higher than those of bare gold nanoparticles, respectively. These results indicated that the usefulness of PEGylated gold nanoparticles with small particle size for a drug carrier.

Biomarkers in Spinal Cord Injury: from Prognosis to Treatment.

Spinal cord injury (SCI) is considered an incurable condition, having a heterogenous recovery and uncertain prognosis. Therefore, a reliable prediction of the improvement in the acute phase could benefit patients. Physicians are unanimous in insisting that at the initial damage of the spinal cord (SC), the patient should be carefully evaluated in order to help selecting an appropriate neuroprotective treatment. However, currently, neurologic impairment after SCI is measured and classified by functional examination. The identification of prognostic biomarkers of SCI would help to designate SC injured patients and correlate to diagnosis and correct treatment. Some proteins have already been identified as good potential biomarkers of central nervous system injury, both in cerebrospinal fluid (CSF) and blood serum. However, the problem for using them as biomarkers is the way they should be collected, as acquiring CSF through a lumbar puncture is significantly invasive. Remarkably, microRNAs (miRNAs) have emerged as interesting biomarker candidates because of their stability in biological fluids and their tissue specificity. Several miRNAs have been identified to have their expressions altered in SCI in many animal models, making them promising candidates as biomarkers after SCI. Moreover, there are yet no effective therapies for SCI. It is already known that altered lysophospholipids (LPs) signaling are involved in the biology of disorders, such as inflammation. Reports have demonstrated that LPs when locally distributed can regulate SCI repair and key secondary injury processes such as apoptosis and inflammation, and so could become in the future new therapeutic approaches for treating SCI.

Photocrosslinkable electrospun fiber meshes for tissue engineering applications

Electrospun polymeric meshes are known to exhibit promising properties for the regeneration of several soft tissues. Herein, electrospun polymeric meshes were prepared from blends of polycaprolactone and functionalized gelatin. The meshes were then photocrosslinked under UV light using Irgacure® 2959 as the photoinitiator, aiming to improve membranes’ stability in biological fluids. Moreover, meshes suitability to be used as vascular grafts was evaluated by characterizing their chemical/physical properties as well as their haemo and biocompatibility in vitro. The obtained results show that the blended polymeric meshes are biodegradable and those with a higher content of gelatin display a lower water contact angle. Blood compatibility studies showed that the photocrosslinked membranes are haemocompatible, i.e. they display low values of thrombogenicity and do not trigger any haemolytic effect. Also, Normal Human Dermal Fibroblasts cells were incubated in the presence of the produced membranes and they were able to adhere and proliferate, thus revealing the biocompatibility of the photocrosslinked meshes.

MicroRNAs as biomarkers for clinical studies.

The development of better diagnostic and prognostic non-invasive biomarkers holds an enormous potential to improve the ability to diagnose and individualize treatment of a great number of human diseases and substantially reduce health care cost. The discovery of a fundamental role of microRNAs in the disease pathogenesis and their presence and stability in biological fluids has led to extensive investigation of the role of microRNAs as potential non-invasive biomarkers for disease diagnosis and prognosis. The result of this research has suggested that alterations of microRNAs may be sensitive indicators of various pathologies; however, despite the indisputable progress in this field, the diagnostic promise of microRNAs has remained a work in progress, and circulating microRNAs have not entered the field of clinical medicine yet. Commonly reported microRNAs as disease biomarkers are largely not disease-specific and the results are often contradicting in independent studies. This review summarizes the current knowledge on the role of microRNAs as disease indicators and emphasizes the current gaps, challenges, and questions that need to be addressed in future well-designed and well-controlled studies for a successful translation of microRNA profiling into clinically meaningful tests. Impact statement This review summarizes the current knowledge on the role of circulating miRNAs as clinical diagnostic biomarkers and highlights the challenges that need to be addressed in future studies for a successful translation of circulating miRNAs into a novel diagnostic tool.

Biological Fate of Fe₃O₄ Core-Shell Mesoporous Silica Nanoparticles Depending on Particle Surface Chemistry.

The biological fate of nanoparticles (NPs) for biomedical applications is highly dependent of their size and charge, their aggregation state and their surface chemistry. The chemical composition of the NPs surface influences their stability in biological fluids, their interaction with proteins, and their attraction to the cell membranes. In this work, core-shell magnetic mesoporous silica nanoparticles (Fe3O4@MSN), that are considered as potential theranostic candidates, are coated with polyethylene glycol (PEG) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayer. Their biological fate is studied in comparison to the native NPs. The physicochemical properties of these three types of NPs and their suspension behavior in different media are investigated. The attraction to a membrane model is also evaluated using a supported lipid bilayer. The surface composition of NPs strongly influences their dispersion in biological fluids mimics, protein binding and their interaction with cell membrane. While none of these types of NPs is found to be toxic on mice four days after intravenous injection of a dose of 40 mg kg−1 of NPs, their surface coating nature influences the in vivo biodistribution. Importantly, NP coated with DMPC exhibit a strong accumulation in liver and a very low accumulation in lung in comparison with nude or PEG ones.

Serum MicroRNAs Reflect Injury Severity in a Large Animal Model of Thoracic Spinal Cord Injury

Therapeutic development for spinal cord injury is hindered by the difficulty in conducting clinical trials, which to date have relied solely on functional outcome measures for patient enrollment, stratification, and evaluation. Biological biomarkers that accurately classify injury severity and predict neurologic outcome would represent a paradigm shift in the way spinal cord injury clinical trials could be conducted. MicroRNAs have emerged as attractive biomarker candidates due to their stability in biological fluids, their phylogenetic similarities, and their tissue specificity. Here we characterized a porcine model of spinal cord injury using a combined behavioural, histological, and molecular approach. We performed next-generation sequencing on microRNAs in serum samples collected before injury and then at 1, 3, and 5 days post injury. We identified 58, 21, 9, and 7 altered miRNA after severe, moderate, and mild spinal cord injury, and SHAM surgery, respectively. These data were combined with behavioural and histological analysis. Overall miRNA expression at 1 and 3 days post injury strongly correlates with outcome measures at 12 weeks post injury. The data presented here indicate that serum miRNAs are promising candidates as biomarkers for the evaluation of injury severity for spinal cord injury or other forms of traumatic, acute, neurologic injury.

Discovery and molecular interaction studies of a highly stable, tarantula peptide modulator of acid-sensing ion channel 1

Acute pharmacological inhibition of acid-sensing ion channel 1a (ASIC1a) is efficacious in rodent models in alleviating symptoms of neurological diseases such as stroke and multiple sclerosis. Thus, ASIC1a is a promising therapeutic target and selective ligands that modulate it are invaluable research tools and potential therapeutic leads. Spider venoms have provided an abundance of voltage-gated ion channel modulators, however, only one ASIC modulator (PcTx1) has so far been isolated from this source. Here we report the discovery, characterization, and chemical stability of a second spider venom peptide that potently modulates ASIC1a and ASIC1b, and investigate the molecular basis for its subtype selectivity. π-TRTX-Hm3a (Hm3a) is a 37-amino acid peptide isolated from Togo starburst tarantula (Heteroscodra maculata) venom with five amino acid substitutions compared to PcTx1, and is also three residues shorter at the C-terminus. Hm3a pH-dependently inhibited ASIC1a with an IC50 of 1–2 nM and potentiated ASIC1b with an EC50 of 46.5 nM, similar to PcTx1. Using ASIC1a to ASIC1b point mutants in rat ASIC1a revealed that Glu177 and Arg175 in the palm region opposite α-helix 5 play an important role in the Hm3a-ASIC1 interaction and contribute to the subtype-dependent effects of the peptide. Despite its high sequence similarity with PcTx1, Hm3a showed higher levels of stability over 48 h. Overall, Hm3a represents a potent, highly stable tool for the study of ASICs and will be particularly useful when stability in biological fluids is required, for example in long term in vitro cell-based assays and in vivo experiments.This article is part of the Special Issue entitled ‘Venom-derived Peptides as Pharmacological Tools.’

Antisense Oligonucleotides: Treatment Strategies and Cellular Internalization.

The clinical applicaton of antisense oligonucleotides (ASOs) is becoming more of a reality as several drugs have been approved for the treatment of human disorders and many others are in various phases in development and clinical trials. ASOs are short DNA/RNA oligos which are heavily modified to increase their stability in biological fluids and retain the properties of creating RNA-RNA and DNA-RNA duplexes that knock-down or correct genetic expression. This review outlines several strategies that ASOs utilize for the treatment of various congenital diseases and syndromes that develop with aging. In addition, we discuss some of the mechanisms for specific non-targeted ASO internalization within cells.