Engineering supramolecular dynamics of self-assembly and turnover of oncogenic microRNAs to drive their synergistic destruction in tumor models.

Rationally-engineered functional biomaterials offer the opportunity to interface with complex biology in a predictive, precise, yet dynamic way to reprogram their behaviour and correct shortcomings. Success here may lead to a desired therapeutic effect against life-threatening diseases, such as cancer. Here, we engineered "Crab"-like artificial ribonucleases through coupling of peptide and nucleic acid building blocks, capable of operating alongside and synergistically with intracellular enzymes (RNase H and AGO2) for potent destruction of oncogenic microRNAs. "Crab"-like configuration of two catalytic peptides ("pincers") flanking the recognition oligonucleotide was instrumental here in providing increased catalytic turnover, leading to ≈30-fold decrease in miRNA half-life as compared with that for "single-pincer" conjugates. Dynamic modeling of miRNA cleavage illustrated how such design enabled "Crabs" to drive catalytic turnover through simultaneous attacks at different locations of the RNA-DNA heteroduplex, presumably by producing smaller cleavage products and by providing toeholds for competitive displacement by intact miRNA strands. miRNA cleavage at the 5'-site, spreading further into double-stranded region, likely provided a synergy for RNase H1 through demolition of its loading region, thus facilitating enzyme turnover. Such synergy was critical for sustaining persistent disposal of continually-emerging oncogenic miRNAs. A single exposure to the best structural variant (Crab-p-21) prior to transplantation into mice suppressed their malignant properties and reduced primary tumor volume (by 85 %) in MCF-7 murine xenograft models.

Engineering a New IFN-ApoA-I Fusion Protein with Low Toxicity and Prolonged Action.

Recombinant human interferon alpha-2b (rIFN) is widely used in antiviral and anticancer immunotherapy. However, the high efficiency of interferon therapy is accompanied by a number of side effects; this problem requires the design of a new class of interferon molecules with reduced cytotoxicity. In this work, IFN was modified via genetic engineering methods by merging it with the blood plasma protein apolipoprotein A-I in order to reduce acute toxicity and improve the pharmacokinetics of IFN. The chimeric protein was obtained via biosynthesis in the yeast P. pastoris. The yield of ryIFN-ApoA-I protein when cultivated on a shaker in flasks was 30 mg/L; protein purification was carried out using reverse-phase chromatography to a purity of 95-97%. The chimeric protein demonstrated complete preservation of the biological activity of IFN in the model of vesicular stomatitis virus and SARS-CoV-2. In addition, the chimeric form had reduced cytotoxicity towards Vero cells and increased cell viability under viral load conditions compared with commercial IFN-a2b preparations. Analysis of the pharmacokinetic profile of ryIFN-ApoA-I after a single subcutaneous injection in mice showed a 1.8-fold increased half-life of the chimeric protein compared with ryIFN.

Construction of Recombinant Human GM-CSF and GM-CSF-ApoA-I Fusion Protein and Evaluation of Their Biological Activity.

In this study, two strains of the yeast P. pastoris were constructed, one of which produced authentic recombinant human granulocyte-macrophage colony-stimulating factor (ryGM-CSF), and the other was a chimera consisting of ryGM-CSF genetically fused with mature human apolipoprotein A-I (ApoA-I) (ryGM-CSF-ApoA-I). Both forms of the cytokine were secreted into the culture medium. The proteins' yield during cultivation in flasks was 100 and 60 mg/L for ryGM-CSF and ryGM-CSF-ApoA-I, respectively. Both forms of recombinant GM-CSF stimulated the proliferation of human TF-1 erythroleukemia cells; however, the amount of chimera required was 10-fold that of authentic GM-CSF to induce a similar proliferative effect. RyGM-CSF exhibited a 2-fold proliferative effect on BFU-E (burst-forming units-erythroid) at a concentration 1.7 fold less than non-glycosylated E. coli-derived GM-CSF. The chimera together with authentic ryGM-CSF increased the number of both erythroid precursors and BMC granulocytes after 48 h of incubation of human bone marrow cells (BMCs). In addition, the chimeric form of ryGM-CSF was more effective at increasing the viability of the total amount of BMCs, decreasing apoptosis compared to the authentic form. ryGM-CSF-ApoA-I normalized the proliferation, maturation, and segmentation of neutrophils within the physiological norm, preserving the pool of blast cells under conditions of impaired granulopoiesis. The chimera form of GM-CSF exhibited the properties of a multilinear growth factor, modulating the activity of GM-CSF and, perhaps, it may be more suitable for the normalization of granulopoiesis.

Mesyl phosphoramidate backbone modified antisense oligonucleotides targeting miR-21 with enhanced in vivo therapeutic potency.

The design of modified oligonucleotides that combine in one molecule several therapeutically beneficial properties still poses a major challenge. Recently a new type of modified mesyl phosphoramidate (or µ-) oligonucleotide was described that demonstrates high affinity to RNA, exceptional nuclease resistance, efficient recruitment of RNase H, and potent inhibition of key carcinogenesis processes in vitro. Herein, using a xenograft mouse tumor model, it was demonstrated that microRNA miR-21-targeted µ-oligonucleotides administered in complex with folate-containing liposomes dramatically inhibit primary tumor growth via long-term down-regulation of miR-21 in tumors and increase in biosynthesis of miR-21-regulated tumor suppressor proteins. This antitumoral effect is superior to the effect of the corresponding phosphorothioate. Peritumoral administration of µ-oligonucleotide results in its rapid distribution and efficient accumulation in the tumor. Blood biochemistry and morphometric studies of internal organs revealed no pronounced toxicity of µ-oligonucleotides. This new oligonucleotide class provides a powerful tool for antisense technology.

Apolipoprotein A-I Supports MSCs Survival under Stress Conditions.

Clinical trials have shown the safety of mesenchymal stem/stromal cells (MSCs) transplantation, but the effectiveness of these treatments is limited. Since, transplanted MSCs will undergo metabolic disturbances in the bloodstream, we investigated the influence of blood plasmas of type 2 diabetes (T2D) patients on MSCs viability and examined whether apolipoprotein A-I (apoA-I) could protect cells from stressful conditions of serum deprivation (SD), hypoxia, and elevated concentrations of reactive oxygen species (ROS). ApoA-I exhibits anti-inflammatory, immune activities, improves glycemic control, and is suitable for T2D patients but its influence on MSCs remains unknown. For the first time we have shown that apoA-I decreases intracellular ROS and supports proliferative rate of MSCs, thereby increasing cell count in oxidation conditions. ApoA-I did not influence cell cycle when MSCs were predominantly in the G0/G1 phases under conditions of SD/hypoxia, activated proliferation rapidly, and reduced apoptosis during MSCs transition to the oxygenation or oxidation conditions. Finally, it was found that the blood plasma of T2D individuals had a cytotoxic effect on MSСs in 39% of cases and had a wide variability of antioxidant properties. ApoA-I protects cells under all adverse conditions and can increase the efficiency of MSCs transplantation in T2D patients.

XPS Modeling of Immobilized Recombinant Angiogenin and Apoliprotein A1 on Biodegradable Nanofibers.

The immobilization of viable proteins is an important step in engineering efficient scaffolds for regenerative medicine. For example, angiogenin, a vascular growth factor, can be considered a neurotrophic factor, influencing the neurogenesis, viability, and migration of neurons. Angiogenin shows an exceptional combination of angiogenic, neurotrophic, neuroprotective, antibacterial, and antioxidant activities. Therefore, this protein is a promising molecule that can be immobilized on carriers used for tissue engineering, particularly for diseases that are complicated by neurotrophic and vascular disorders. Another highly important and viable protein is apoliprotein A1. Nevertheless, the immobilization of these proteins onto promising biodegradable nanofibers has not been tested before. In this work, we carefully studied the immobilization of human recombinant angiogenin and apoliprotein A1 onto plasma-coated nanofibers. We developed a new methodology for the quantification of the protein density of these proteins using X-ray photoelectron spectroscopy (XPS) and modeled the XPS data for angiogenin and apoliprotein A1 (Apo-A1). These findings were also confirmed by the analysis of immobilized Apo-A1 using fluorescent microscopy. The presented methodology was validated by the analysis of fibronectin on the surface of plasma-coated poly(ε-caprolactone) (PCL) nanofibers. This methodology can be expanded for other proteins and it should help to quantify the density of proteins on surfaces using routine XPS data treatment.

Catalytic Knockdown of miR-21 by Artificial Ribonuclease: Biological Performance in Tumor Model.

Control of the expression of oncogenic small non-coding RNAs, notably microRNAs (miRNAs), is an attractive therapeutic approach. We report a design platform for catalytic knockdown of miRNA targets with artificial, sequence-specific ribonucleases. miRNases comprise a peptide [(LeuArg)2Gly]2 capable of RNA cleavage conjugated to the miRNA-targeted oligodeoxyribonucleotide, which becomes nuclease-resistant within the conjugate design, without resort to chemically modified nucleotides. Our data presented here showed for the first time a truly catalytic character of our miR-21-miRNase and its ability to cleave miR-21 in a multiple catalytic turnover mode. We demonstrate that miRNase targeted to miR-21 (miR-21-miRNase) knocked down malignant behavior of tumor cells, including induction of apoptosis, inhibition of cell invasiveness, and retardation of tumor growth, which persisted on transplantation into mice of tumor cells treated once with miR-21-miRNase. Crucially, we discover that the high biological activity of miR-21-miRNase can be directly related not only to its truly catalytic sequence-specific cleavage of miRNA but also to its ability to recruit the non-sequence specific RNase H found in most cells to elevate catalytic turnover further. miR-21-miRNase worked synergistically even with low levels of RNase H. Estimated degradation in the presence of RNase H exceeded 103 miRNA target molecules per hour for each miR-21-miRNase molecule, which provides the potency to minimize delivery requirements to a few molecules per cell. In contrast to the comparatively high doses required for the simple steric block of antisense oligonucleotides, truly catalytic inactivation of miRNA offers more effective, irreversible, and persistent suppression of many copy target sequences. miRNase design can be readily adapted to target other pathogenic microRNAs overexpressed in many disease states.

Enhanced Inhibition of Tumorigenesis Using Combinations of miRNA-Targeted Therapeutics.

The search for effective strategies to inhibit tumorigenesis remains one of the most relevant scientific challenges. Among the most promising approaches is the direct modulation of the function of short non-coding RNAs, particularly miRNAs. These molecules are propitious targets for anticancer therapy, since they perform key regulatory roles in a variety of signaling cascades related to cell proliferation, apoptosis, migration, and invasion. The development of pathological states is often associated with deregulation of miRNA expression. The present review describes in detail the strategies aimed at modulating miRNA activity that invoke antisense oligonucleotide construction, such as small RNA zippers, miRNases (miRNA-targeted artificial ribonucleases), miRNA sponges, miRNA masks, anti-miRNA oligonucleotides, and synthetic miRNA mimics. The broad impact of developed miRNA-based therapeutics on the various events of tumorigenesis is also discussed. Above all, the focus of this review is to evaluate the results of the combined application of different miRNA-based agents and chemotherapeutic drugs for the inhibition of tumor development. Many studies indicate a considerable increase in the efficacy of anticancer therapy as a result of additive or synergistic effects of simultaneously applied therapies. Different drug combinations, such as a cocktail of antisense oligonucleotides or multipotent miRNA sponges directed at several oncogenic microRNAs belonging to the same/different miRNA families, a mixture of anti-miRNA oligonucleotides and cytostatic drugs, and a combination of synthetic miRNA mimics, have a more complex and profound effect on the various events of tumorigenesis as compared with treatment with a single miRNA-based agent or chemotherapeutic drug. These data provide strong evidence that the simultaneous application of several distinct strategies aimed at suppressing different cellular processes linked to tumorigenesis is a promising approach for cancer therapy.

Plasma-Coated Polycaprolactone Nanofibers with Covalently Bonded Platelet-Rich Plasma Enhance Adhesion and Growth of Human Fibroblasts.

Biodegradable nanofibers are extensively employed in different areas of biology and medicine, particularly in tissue engineering. The electrospun polycaprolactone (PCL) nanofibers are attracting growing interest due to their good mechanical properties and a low-cost structure similar to the extracellular matrix. However, the unmodified PCL nanofibers exhibit an inert surface, hindering cell adhesion and negatively affecting their further fate. The employment of PCL nanofibrous scaffolds for wound healing requires a certain modification of the PCL surface. In this work, the morphology of PCL nanofibers is optimized by the careful tuning of electrospinning parameters. It is shown that the modification of the PCL nanofibers with the COOH plasma polymers and the subsequent binding of NH2 groups of protein molecules is a rather simple and technologically accessible procedure allowing the adhesion, early spreading, and growth of human fibroblasts to be boosted. The behavior of fibroblasts on the modified PCL surface was found to be very different when compared to the previously studied cultivation of mesenchymal stem cells on the PCL nanofibrous meshes. It is demonstrated by X-ray photoelectron spectroscopy (XPS) that the freeze-thawed platelet-rich plasma (PRP) immobilization can be performed via covalent and non-covalent bonding and that it does not affect biological activity. The covalently bound components of PRP considerably reduce the fibroblast apoptosis and increase the cell proliferation in comparison to the unmodified PCL nanofibers or the PCL nanofibers with non-covalent bonding of PRP. The reported research findings reveal the potential of PCL matrices for application in tissue engineering, while the plasma modification with COOH groups and their subsequent covalent binding with proteins expand this potential even further. The use of such matrices with covalently immobilized PRP for wound healing leads to prolonged biological activity of the immobilized molecules and protects these biomolecules from the aggressive media of the wound.

Luminescent silica mesoparticles for protein transduction.

Unlike silica nanoparticles, the potential of silica mesoparticles (SMPs) (i.e. particles of submicron size) for biological applications in particular the in vitro (let alone in vivo) cellular delivery of biological cargo has so far not been sufficiently studied. Here we examine the potential of luminescent (namely, octahedral molybdenum cluster doped) SMPs synthesised by a simple one-pot reaction for the labelling of cells and for protein transduction into larynx carcinoma (Hep-2) cells using GFP as a model protein. Our data demonstrates that the SMPs internalise into the cells within half an hour. This results in cells that detectably luminesce via conventional methods. In addition, the particles are non-toxic both in darkness and upon photo-irradiation. The SMPs were modified to allow their functionalisation by a protein, which then delivered the protein (GFP) efficiently into the cells. Thus, the luminescent SMPs offer a cheap and trackable alternative to existing materials for cellular internalisation of proteins, such as the HIV TAT protein and commercial protein delivery agents (e.g. Pierce™).

Polyelectrolyte-coated ultra-small nanoparticles with Tb(III)-centered luminescence as cell labels with unusual charge effect on their cell internalization.

The present work reports ultra-small polyelectrolyte-coated water insoluble Tb(III) complex species with bright Tb(III)-centered luminescence resulted from efficient ligand-to-metal energy transfer as efficient labels for Hep-2 cells. The flow cytometry data revealed the enhanced cellular uptake of negatively charged nanoparticles coated by the polystyrenesulfonate (PSS)-monolayer versus the positively charged nanoparticles. The latter are obtained by layer-by-layer deposition of polyethyleneimine (PEI) onto PSS-coated ones. Confocal and TEM images of Hep-2 cells exposed by the colloids confirm favorable cell internalization of the PSS- compared to PEI-PSS-coated colloids illustrating unusual charge-effect. Dynamic light scattering data indicate significant effect of the biological background exemplified by serum bovine albumin and phosphatidylcholine-based bilayers on the exterior charge and aggregation behavior of the colloids. The obtained results reveal the PSS-coated nanoparticles based on water insoluble Tb(III) complex as promising cell labels.

From Photoinduced to Dark Cytotoxicity through an Octahedral Cluster Hydrolysis.

Octahedral molybdenum and tungsten clusters have potential biological applications in photodynamic therapy and bioimaging. However, poor solubility and hydrolysis stability of these compounds hinder their application. The first water-soluble photoluminescent octahedral tungsten cluster [{W6 I8 }(DMSO)6 ](NO3 )4 was synthesised and demonstrated to be at least one order of magnitude more stable towards hydrolysis than its molybdenum analogue. Biological studies of the compound on larynx carcinoma cells suggest that it has a significant photoinduced toxicity, while the dark toxicity increases with the increase of the degree of hydrolysis. The increase of the dark toxicity is associated with the in situ generation of nanoparticles that clog up the cisternae of rough endoplasmic reticulum.

Silica nanoparticles with Tb(III)-centered luminescence decorated by Ag0 as efficient cellular contrast agent with anticancer effect.

The present work introduces composite luminescent nanoparticles (Ag0-Tb3+-SNs), where ultra-small nanosilver (4 ±â€¯2 nm) is deposited onto amino-modified silica nanoparticles (35±6 nm) doped by green luminescent Tb(III) complexes. Ag0-Tb3+-SNs are able to image cancer (Hep-2) cells in confocal microscopy measurements due to efficient cell internalization, which is confirmed by TEM images of the Hep-2 cells exposed by Ag0-Tb3+-SNs. Comparative analysis of the cytotoxicity of normal fibroblasts (DK-4) and cancer cells (Hep-2) incubated with various concentrations of Ag0-Tb3+-SNs revealed the concentration range where the toxic effect on the cancer cells is significant, while it is insignificant towards the nonmalignant fibroblasts cells. The obtained results reveal Ag0-Tb3+-SNs as good cellular contrast agent able to induce the cancer cells death, which makes them promising theranostic in cancer diagnostics and therapy.

Singlet Oxygen Production and Biological Activity of Hexanuclear Chalcocyanide Rhenium Cluster Complexes [{Re6Q8}(CN)6]4- (Q = S, Se, Te).

Octahedral rhenium cluster complexes have recently emerged as relevant building blocks for the design of singlet oxygen photosensitizing materials toward biological applications such as blue-light photodynamic therapy. However, their singlet oxygen generation ability as well as biological properties have been studied only superficially. Herein we investigate in detail the singlet oxygen photogeneration, dark and photoinduced cytotoxicity, cellular uptake kinetics, cellular localization and in vitro photoinduced oxidative stress, and photodynamic cytotoxicity of the series of octahedral rhenium cluster complexes [{Re6Q8}(CN)6]4-, where Q = S, Se, Te. Our results demonstrate that the selenium-containing complex possesses optimal properties in terms of absorption and singlet oxygen productivity. These features coupled with the cellular internalization and low dark toxicity lead to the first photoinduced cytotoxic effect observed for a molecular [{M6Q8}L6] complex, making it a promising object for further study in terms of blue-light PDT.

miRNases: Novel peptide-oligonucleotide bioconjugates that silence miR-21 in lymphosarcoma cells.

MicroRNAs (miRNAs) are active regulators in malignant growth and constitute potential targets for anticancer therapy. Consequently, considerable effort has focused on identifying effective ways to modulate aberrant miRNA expression. Here we introduce and assess a novel type of chemically engineered biomaterial capable of cleaving specific miRNA sequences, i.e. miRNA-specific artificial ribonucleases (hereafter 'miRNase'). The miRNase template presented here consists of the catalytic peptide Acetyl-[(LeuArg)2Gly]2 covalently attached to a miRNA-targeting oligonucleotide, which can be linear or hairpin. The peptide C-terminus is conjugated to an aminohexyl linker located at either the 3'- or 5'-end of the oligonucleotide. The cleavage efficacy, structural aspects of cleavage and biological relevance of a set of these designed miRNases was assayed with respect to highly oncogenic miR-21. Several miRNases demonstrated effective site-selective cleavage of miR-21 exclusively at G-X bonds. One of the most efficient miRNase was shown to specifically inhibit miR-21 in lymphosarcoma cells and lead to a reduction in their proliferative activity. This report provides the first experimental evidence that metallo-independent peptide-oligonucleotide chemical ribonucleases are able to effectively and selectively down-regulate oncogenic miRNA in tumour cells, thus suggesting their potential in development of novel therapeutics aimed at overcoming overexpression of disease-related miRNAs.

Immobilization of Platelet-Rich Plasma onto COOH Plasma-Coated PCL Nanofibers Boost Viability and Proliferation of Human Mesenchymal Stem Cells.

The scaffolds made of polycaprolactone (PCL) are actively employed in different areas of biology and medicine, especially in tissue engineering. However, the usage of unmodified PCL is significantly restricted by the hydrophobicity of its surface, due to the fact that its inert surface hinders the adhesion of cells and the cell interactions on PCL surface. In this work, the surface of PCL nanofibers is modified by Ar/CO2/C2H4 plasma depositing active COOH groups in the amount of 0.57 at % that were later used for the immobilization of platelet-rich plasma (PRP). The modification of PCL nanofibers significantly enhances the viability and proliferation (by hundred times) of human mesenchymal stem cells, and decreases apoptotic cell death to a normal level. According to X-ray photoelectron spectroscopy (XPS), after immobilization of PRP, up to 10.7 at % of nitrogen was incorporated into the nanofibers surface confirming the grafting of proteins. Active proliferation and sustaining the cell viability on nanofibers with immobilized PRP led to an average number of cells of 258 ± 12.9 and 364 ± 34.5 for nanofibers with ionic and covalent bonding of PRP, respectively. Hence, our new method for the modification of PCL nanofibers with PRP opens new possibilities for its application in tissue engineering.

Risk factors for keratoconus in Israel: a case-control study.

PURPOSE: Keratoconus (KC) is a progressive corneal thinning disorder with an uncertain aetiology. Environmental and genetic factors, including consanguinity, eye rubbing and possibly sun exposure, play a role in the aetiology of KC. Here we test for risk factors for KC in an Israeli population with particular emphasis on sun exposure. METHODS: This case-control study included KC patients who were diagnosed at Care Laser Medical Group, a refractive surgery clinic with branches throughout Israel. The control group included age, sex and ethnicity matched individuals who were randomly selected from patients presenting at the clinic for refractive surgery, but without KC. Study subjects were asked to fill out a self-administered questionnaire that included demographic and geographic details, questions on ocular and general health and sun exposure. Conditional logistic regression was used to analyse univariable and multivariable data to identify risk factors for KC. RESULTS: Seventy-three KC patients and 146 controls participated in the study. Univariable analyses demonstrated that eye rubbing [odds ratio (OR) = 3.76], positive family history of KC (OR = 6.10) and parents' education (<12 years, OR = 0.27, 0.23 for father's and mother's education respectively) were significant risk factors for KC. Univariable analyses of sun exposure behaviour during teenage years proved equivocal with some behaviours emerging as protective for KC (wearing a hat outdoors, OR = 3.13) or as risk factors (spending time in the shade, OR = 0.45), while others showed no association [limiting time in the sun (p = 0.51), and wearing sunglasses (p = 0.20)]. Most of the factors that were significant in the univariable analyses, also emerged as statistically significant in the multivariable model (OR = 3.37, 9.68, 0.35, 5.51 for eye rubbing, family history, parental education, wearing a hat outdoors, with the exception of spending time in the shade (p = 0.88). CONCLUSIONS: Eye rubbing, parents' education (as a measure of socio-economic status) and having family members with KC emerged as significant risk factors for KC. The role of sun exposure in KC remains equivocal and warrants further research.