Crosstalk between long noncoding RNA and microRNA in Cancer.

miRNAs and lncRNAs play a central role in cancer-associated gene regulations. The dysregulated expression of lncRNAs has been reported as a hallmark of cancer progression, acting as an independent prediction marker for an individual cancer patient. The interplay of miRNA and lncRNA decides the variation of tumorigenesis that could be mediated by acting as sponges for endogenous RNAs, regulating miRNA decay, mediating intra-chromosomal interactions, and modulating epigenetic components. This paper focuses on the influence of crosstalk between lncRNA and miRNA on cancer hallmarks such as epithelial-mesenchymal transition, hijacking cell death, metastasis, and invasion. Other cellular roles of crosstalks, such as neovascularization, vascular mimicry, and angiogenesis were also discussed. Additionally, we reviewed crosstalk mechanism with specific host immune responses and targeting interplay (between lncRNA and miRNA) in cancer diagnosis and management.

Microfluidic-Assisted Fabrication of Dual-Coated pH-Sensitive Mesoporous Silica Nanoparticles for Protein Delivery.

Microfluidics has become a popular method for constructing nanosystems in recent years, but it can also be used to coat other materials with polymeric layers. The polymeric coating may serve as a diffusion barrier against hydrophilic compounds, a responsive layer for controlled release, or a functional layer introduced to a nanocomposite for achieving the desired surface chemistry. In this study, mesoporous silica nanoparticles (MSNs) with enlarged pores were synthesized to achieve high protein loading combined with high protein retention within the MSN system with the aid of a microfluidic coating. Thus, MSNs were first coated with a cationic polyelectrolyte, poly (diallyldimethylammonium chloride) (PDDMA), and to potentially further control the protein release, a second coating of a pH-sensitive polymer (spermine-modified acetylated dextran, SpAcDEX) was deposited by a designed microfluidic device. The protective PDDMA layer was first formed under aqueous conditions, whereby the bioactivity of the protein could be maintained. The second coating polymer, SpAcDEX, was preferred to provide pH-sensitive protein release in the intracellular environment. The optimized formulation was effectively taken up by the cells along with the loaded protein cargo. This proof-of-concept study thus demonstrated that the use of microfluidic technologies for the design of protein delivery systems has great potential in terms of creating multicomponent systems and preserving protein stability.

Circumventing Drug Treatment? Intrinsic Lethal Effects of Polyethyleneimine (PEI)-Functionalized Nanoparticles on Glioblastoma Cells Cultured in Stem Cell Conditions.

Glioblastoma (GB) is the most frequent malignant tumor originating from the central nervous system. Despite breakthroughs in treatment modalities for other cancer types, GB remains largely irremediable due to the high degree of intratumoral heterogeneity, infiltrative growth, and intrinsic resistance towards multiple treatments. A sub-population of GB cells, glioblastoma stem cells (GSCs), act as a reservoir of cancer-initiating cells and consequently, constitute a significant challenge for successful therapy. In this study, we discovered that PEI surface-functionalized mesoporous silica nanoparticles (PEI-MSNs), without any anti-cancer drug, very potently kill multiple GSC lines cultured in stem cell conditions. Very importantly, PEI-MSNs did not affect the survival of established GB cells, nor other types of cancer cells cultured in serum-containing medium, even at 25 times higher doses. PEI-MSNs did not induce any signs of apoptosis or autophagy. Instead, as a potential explanation for their lethality under stem cell culture conditions, we demonstrate that the internalized PEI-MSNs accumulated inside lysosomes, subsequently causing a rupture of the lysosomal membranes. We also demonstrate blood-brain-barrier (BBB) permeability of the PEI-MSNs in vitro and in vivo. Taking together the recent indications for the vulnerability of GSCs for lysosomal targeting and the lethality of the PEI-MSNs on GSCs cultured under stem cell culture conditions, the results enforce in vivo testing of the therapeutic impact of PEI-functionalized nanoparticles in faithful preclinical GB models.

Fluorescent and Electron-Dense Green Color Emitting Nanodiamonds for Single-Cell Correlative Microscopy.

Correlative light and electron microscopy (CLEM) is revolutionizing how cell samples are studied. CLEM provides a combination of the molecular and ultrastructural information about a cell. For the execution of CLEM experiments, multimodal fiducial landmarks are applied to precisely overlay light and electron microscopy images. Currently applied fiducials such as quantum dots and organic dye-labeled nanoparticles can be irreversibly quenched by electron beam exposure during electron microscopy. Generally, the sample is therefore investigated with a light microscope first and later with an electron microscope. A versatile fiducial landmark should offer to switch back from electron microscopy to light microscopy while preserving its fluorescent properties. Here, we evaluated green fluorescent and electron dense nanodiamonds for the execution of CLEM experiments and precisely correlated light microscopy and electron microscopy images. We demonstrated that green color emitting fluorescent nanodiamonds withstand electron beam exposure, harsh chemical treatments, heavy metal straining, and, importantly, their fluorescent properties remained intact for light microscopy.

Cell Volume (3D) Correlative Microscopy Facilitated by Intracellular Fluorescent Nanodiamonds as Multi-Modal Probes.

Three-dimensional correlative light and electron microscopy (3D CLEM) is attaining popularity as a potential technique to explore the functional aspects of a cell together with high-resolution ultrastructural details across the cell volume. To perform such a 3D CLEM experiment, there is an imperative requirement for multi-modal probes that are both fluorescent and electron-dense. These multi-modal probes will serve as landmarks in matching up the large full cell volume datasets acquired by different imaging modalities. Fluorescent nanodiamonds (FNDs) are a unique nanosized, fluorescent, and electron-dense material from the nanocarbon family. We hereby propose a novel and straightforward method for executing 3D CLEM using FNDs as multi-modal landmarks. We demonstrate that FND is biocompatible and is easily identified both in living cell fluorescence imaging and in serial block-face scanning electron microscopy (SB-EM). We illustrate the method by registering multi-modal datasets.

Plant-Derived Natural Biomolecule Picein Attenuates Menadione Induced Oxidative Stress on Neuroblastoma Cell Mitochondria.

Several bioactive compounds are in use for the treatment of neurodegenerative disorders, such as Alzheimer's and Parkinson's disease. Historically, willow (salix sp.) bark has been an important source of salisylic acid and other natural compounds with anti-inflammatory, antipyretic and analgesic properties. Among these, picein isolated from hot water extract of willow bark, has been found to act as a natural secondary metabolite antioxidant. The aim of this study was to investigate the unrevealed pharmacological action of picein. In silico studies were utilized to direct the investigation towards the neuroprotection abilities of picein. Our in vitro studies demonstrate the neuroprotective properties of picein by blocking the oxidative stress effects, induced by free radical generator 2-methyl-1,4-naphthoquinone (menadione, MQ), in neuroblastoma SH-SY5Y cells. Several oxidative stress-related parameters were evaluated to measure the protection for mitochondrial integrity, such as mitochondrial superoxide production, mitochondrial activity (MTT), reactive oxygen species (ROS) and live-cell imaging. A significant increase in the ROS level and mitochondrial superoxide production were measured after MQ treatment, however, a subsequent treatment with picein was able to mitigate this effect by decreasing their levels. Additionally, the mitochondrial activity was significantly decreased by MQ exposure, but a follow-up treatment with picein recovered the normal metabolic activity. In conclusion, the presented results demonstrate that picein can significantly reduce the level of MQ-induced oxidative stress on mitochondria, and thereby plays a role as a potent neuroprotectant.

Brilliant blue, green, yellow, and red fluorescent diamond particles: synthesis, characterization, and multiplex imaging demonstrations.

Until recently, the number of emission colors available from fluorescent diamond particles was primarily limited to red to near-infrared fluorescence from the nitrogen-vacancy color center in type Ib synthetic diamond and green fluorescence associated with the nitrogen-vacancy-nitrogen center in type Ia natural diamond. Using our recently reported rapid thermal annealing technique, we demonstrate the capability of producing fluorescent diamond particles that exhibit distinctive blue, green, yellow, and red fluorescence from the same synthetic diamond starting material. Utilizing these multiple colored diamonds, we analyze their fluorescence characteristics both in-solution as well as on-substrate and additionally evaluate their viability in simple multiplex imaging and cellular bioimaging experiments. While there are still challenges associated with their immediate use in traditional multiplex imaging, this novel approach opens new opportunities to enhance the capability and flexibility of fluorescent diamond particles at the nanoscale.

Green Nanotechnology: Advancement in Phytoformulation Research.

The ultimate goal of any scientific development is to increase well-being and human health. Novel strategies are required for the achievement of safe and effective therapeutic treatments beyond the conventional ones, and society needs new requirements for new technologies, moving towards clean and green technology development. Green nanotechnology is a branch of green technology that utilizes the concepts of green chemistry and green engineering. It reduces the use of energy and fuel by using less material and renewable inputs wherever possible. Green nanotechnology, in phytoformulations, significantly contributes to environmental sustainability through the production of nanomaterials and nanoproducts, without causing harm to human health or the environment. The rationale behind the utilization of plants in nanoparticle formulations is that they are easily available and possess a broad variability of metabolites, such as vitamins, antioxidants, and nucleotides. For instance, gold (Au) nanoparticles have attracted substantial attention for their controllable size, shape, and surface properties. A variety of copper (Cu) and copper oxide (CuO) nanoparticles have also been synthesized from plant extracts. Titanium dioxide and zinc oxide nanoparticles are also important metal oxide nanomaterials that have been synthesized from a number of plant extracts. International and domestic laws, government and private-party programs, regulations and policies are being carefully reviewed and revised to increase their utility and nurture these nanoscale materials for commercialization. Inspiring debates and government initiatives are required to promote the sustainable use of nanoscale products. In this review, we will discuss the potential of the utilization of plant extracts in the advancement of nanotechnology.

CaP coated mesoporous polydopamine nanoparticles with responsive membrane permeation ability for combined photothermal and siRNA therapy.

Combined photothermal and gene therapy provides a promising modality toward cancer treatment, yet facile integration and controlled codelivery of gene payloads and photothermal conversion agents (PTCAs) remains a great challenge. Inspired by the robust wet adhesion of marine mussels, we present a rationally designed nanosystem constructed by using hybrid mesoporous polydopamine nanoparticles (MPDA) with sub-100 nm sizes and a high photothermal conversion efficiency of 37%. The surface of the particles were modified with tertiary amines by the facile Michael addition/Schiff base reactions of PDA to realize high siRNA loading capacity (10 wt%). Moreover, a successful calcium phosphate (CaP) coating via biomineralization was constructed on the cationic nanoparticle to prohibit premature release of siRNA. The CaP coating underwent biodegradation in weakly-acidic subcellular conditions (lysosomes). The synergistic integration of tertiary amines and catechol moieties on the subsequently exposed surfaces was demonstrated to feature the destabilization/disruption ability toward model cellular membranes via the greatly enhanced interfacial adhesion and interactions. Consequently, sufficient permeability of lysosomal membranes, and in turn, a high lysosomal escape efficiency, was realized, which then resulted in high gene silencing efficiencies via sufficient cytosolic delivery of siRNA. When an efficient knocking down (65%) of survivin (an inhibitor of apoptosis proteins) was combined with a subsequent photothermal ablation, remarkably higher therapeutic efficiencies were observed both in vitro and in vivo, as compared with monotherapy. The system may help to pave a new avenue on the utilization of bio-adhesive surfaces for handling the obstacles of combined photothermal and gene therapy. STATEMENT OF SIGNIFICANCE: Polydopamine (PDA) based porous photothermal-conversion agent (PTCA) with sufficiently high conversion efficiency was employed to deliver photothermal/gene therapy modalities towards cancer treatment. CaP coating via PDA-induced biomineralization was constructed to prohibit premature release of siRNA loaded in the pore space of the nanocarriers. Responsive degradation of CaP also led to the exposure of membrane-lytic surfaces built through the synergistic integration of tertiary amines and catechol moieties, and in turn the significantly enhanced lysosomal escape and cytosol siRNA delivery. Therapeutic targeting of survivin was successfully applied for activation of apoptosis and programmed cell death. Combined photothermal and gene therapy improved therapeutic effectiveness.

Factors Affecting Intracellular Delivery and Release of Hydrophilic Versus Hydrophobic Cargo from Mesoporous Silica Nanoparticles on 2D and 3D Cell Cultures.

Intracellular drug delivery by mesoporous silica nanoparticles (MSNs) carrying hydrophilic and hydrophobic fluorophores as model drug cargo is demonstrated on 2D cellular and 3D tumor organoid level. Two different MSN designs, chosen on the basis of the characteristics of the loaded cargo, were used: MSNs with a surface-grown poly(ethylene imine), PEI, coating only for hydrophobic cargo and MSNs with lipid bilayers covalently coupled to the PEI layer as a diffusion barrier for hydrophilic cargo. First, the effect of hydrophobicity corresponding to loading degree (hydrophobic cargo) as well as surface charge (hydrophilic cargo) on intracellular drug release was studied on the cellular level. All incorporated agents were able to release to varying degrees from the endosomes into the cytoplasm in a loading degree (hydrophobic) or surface charge (hydrophilic) dependent manner as detected by live cell imaging. When administered to organotypic 3D tumor models, the hydrophilic versus hydrophobic cargo-carrying MSNs showed remarkable differences in labeling efficiency, which in this case also corresponds to drug delivery efficacy in 3D. The obtained results could thus indicate design aspects to be taken into account for the development of efficacious intracellular drug delivery systems, especially in the translation from standard 2D culture to more biologically relevant organotypic 3D cultures.

Core-Shell Structures of Upconversion Nanocrystals Coated with Silica for Near Infrared Light Enabled Optical Imaging of Cancer Cells.

Optical imaging of cancer cells using near infrared (NIR) light is currently an active area of research, as this spectral region directly corresponds to the therapeutic window of biological tissues. Upconversion nanocrystals are photostable alternatives to conventional fluorophores. In our work, we have prepared upconversion nanocrystals of NaYF4:Yb/Er and encapsulated them in silica to form core-shell structures. The as-prepared core-shell nanostructures have been characterized for their structure, morphology, and optical properties using X-ray diffraction, transmission electron microscopy coupled with elemental mapping, and upconversion luminescence spectroscopy, respectively. The cytotoxicity examined using cell viability assay indicated a low level of toxicity of these core-shell nanostructures. Finally, these core-shell nanostructures have been utilized as photostable probes for NIR light enabled optical imaging of human breast cancer cells. This work paves the way for the development of advanced photostable, biocompatible, low-toxic core-shell nanostructures for potential optical imaging of biological cells and tissues.

Fluorescent single-digit detonation nanodiamond for biomedical applications.

Detonation nanodiamonds (DNDs) have emerged as promising candidates for a variety of biomedical applications, thanks to different physicochemical and biological properties, such as small size and reactive surfaces. In this study, we propose carbon dot decorated single digit (4-5 nm diameter) primary particles of detonation nanodiamond as promising fluorescent probes. Due to their intrinsic fluorescence originating from tiny (1-2 atomic layer thickness) carbonaceous structures on their surfaces, they exhibit brightness suitable for in vitro imaging. Moreover, this material offers a unique, cost effective alternative to sub-10 nm nanodiamonds containing fluorescent nitrogen-vacancy color centers, which have not yet been produced at large scale. In this paper, carbon dot decorated nanodiamonds are characterized by several analytical techniques. In addition, the efficient cellular uptake and fluorescence of these particles are observed in vitro on MDA-MD-231 breast cancer cells by means of confocal imaging. Finally, the in vivo biocompatibility of carbon dot decorated nanodiamonds is demonstrated in zebrafish during the development. Our results indicate the potential of single-digit detonation nanodiamonds as biocompatible fluorescent probes. This unique material will find application in correlative light and electron microscopy, where small sized NDs can be attached to antibodies to act as a suitable dual marker for intracellular correlative microscopy of biomolecules.

A method for optical imaging and monitoring of the excretion of fluorescent nanocomposites from the body using artificial neural networks.

In this study, a new approach to the implementation of optical imaging of fluorescent nanoparticles in a biological medium using artificial neural networks is proposed. The studies were carried out using new synthesized nanocomposites - nanometer graphene oxides, covered by the poly(ethylene imine)-poly(ethylene glycol) copolymer and by the folic acid. We present an example of a successful solution of the problem of monitoring the removal of nanocomposites based on nGO and their components with urine using fluorescent spectroscopy and artificial neural networks. However, the proposed method is applicable for optical imaging of any fluorescent nanoparticles used as theranostic agents in biological tissue.

Chitosan-Cellulose Multifunctional Hydrogel Beads: Design, Characterization and Evaluation of Cytocompatibility with Breast Adenocarcinoma and Osteoblast Cells.

Cytocompatible polysaccharide-based functional scaffolds are potential extracellular matrix candidates for soft and hard tissue engineering. This paper describes a facile approach to design cytocompatible, non-toxic, and multifunctional chitosan-cellulose based hydrogel beads utilising polysaccharide dissolution in sodium hydroxide-urea-water solvent system and coagulation under three different acidic conditions, namely 2 M acetic acid, 2 M hydrochloric acid, and 2 M sulfuric acid. The effect of coagulating medium on the final chemical composition of the hydrogel beads is investigated by spectroscopic techniques (ATR-FTIR, Raman, NMR), and elemental analysis. The beads coagulated in 2 M acetic acid displayed an unchanged chitosan composition with free amino groups, while the beads coagulated in 2 M hydrochloric and sulfuric acid showed protonation of amino groups and ionic interaction with the counterions. The ultrastructural morphological study of lyophilized beads showed that increased chitosan content enhanced the porosity of the hydrogel beads. Furthermore, cytocompatibility evaluation of the hydrogel beads with human breast adenocarcinoma cells (soft tissue) showed that the beads coagulated in 2 M acetic acid are the most suitable for this type of cells in comparison to other coagulating systems. The acetic acid fabricated hydrogel beads also support osteoblast growth and adhesion over 192 h. Thus, in future, these hydrogel beads can be tested in the in vitro studies related to breast cancer and for bone regeneration.

Gold nanoparticle printed coverslips to facilitate fluorescence-TEM correlative microscopy.

Correlative light and electron microscopy (CLEM) allows combining the advantages of fluorescence microscopy and electron microscopy for cell imaging. Rare phenomenon expressing cells can be studied by specifically tagged fluorophores with fluorescence microscopy. Subsequently, cells can be fixed and ultra-structural details can be studied with transmission electron microscopy (TEM) at a higher resolution. However, precise landmarks are necessary to track the same cell throughout the CLEM process. In this technical report, we present a high contrast inkjet-printed gold nanoparticle patterns over commercial glass coverslip to facilitate cell tracking with correlative microscopy. High contrast and strong reflection from nano gold pattern can be used as a fixed landmark for cell identification with fluorescence microscopy. Nano gold printed letters over coverslips are visible in resin blocks, which can be further used to identify the cell of interest for performing sectioning of embedded cell blocks for TEM.

STED-TEM Correlative Microscopy Leveraging Nanodiamonds as Intracellular Dual-Contrast Markers.

Development of fluorescent and electron dense markers is essential for the implementation of correlative light and electron microscopy, as dual-contrast landmarks are required to match the details in the multimodal images. Here, a novel method for correlative microscopy that utilizes fluorescent nanodiamonds (FNDs) as dual-contrast probes is reported. It is demonstrated how the FNDs can be used as dual-contrast labels-and together with automatic image registration tool SuperTomo, for precise image correlation-in high-resolution stimulated emission depletion (STED)/confocal and transmission electron microscopy (TEM) correlative microscopy experiments. It is shown how FNDs can be employed in experiments with both live and fixed cells as well as simple test samples. The fluorescence imaging can be performed either before TEM imaging or after, as the robust FNDs survive the TEM sample preparation and can be imaged with STED and other fluorescence microscopes directly on the TEM grids.

Intracellular Trafficking of Fluorescent Nanodiamonds and Regulation of Their Cellular Toxicity.

In this paper, cellular management of fluorescent nanodiamonds (FNDs) has been studied for better understanding in the design for potential applications of FNDs in biomedicine. The FNDs have shown to be photostable probes for bioimaging and thus are well-suited, for example, long-term tracking purposes. The FNDs also exhibit good biocompatibility and, in general, low toxicity for cell labeling. To demonstrate the underlying mechanism of cells coping the low but potentially toxic effects by nondegradable FNDs, we have studied their temporal intracellular trafficking. The FNDs were observed to be localized as distinct populations inside cells in early endosomes, lysosomes, and in proximity to the plasma membrane. The localization of FNDs in early endosomes suggests the internalization of FNDs, and lysosomal localization, in turn, can be interpreted as a prestate for exocytosis via lysosomal degradation pathway. The endocytosis and exocytosis appear to be occurring simultaneously in our observations. The mechanism of continuous endocytosis and exocytosis of FNDs could be necessary for cells to maintain normal proliferation. Furthermore, 120 h cell growth assay was performed to verify the long-term biocompatibility of FNDs for cellular studies.

Ratiometric Sensing and Imaging of Intracellular pH Using Polyethylenimine-Coated Photon Upconversion Nanoprobes.

Measurement of changes of pH at various intracellular compartments has potential to solve questions concerning the processing of endocytosed material, regulation of the acidification process, and also acidification of vesicles destined for exocytosis. To monitor these events, the nanosized optical pH probes need to provide ratiometric signals in the optically transparent biological window, target to all relevant intracellular compartments, and to facilitate imaging at subcellular resolution without interference from the biological matrix. To meet these criteria we sensitize the surface conjugated pH sensitive indicator via an upconversion process utilizing an energy transfer from the nanoparticle to the indicator. Live cells were imaged with a scanning confocal microscope equipped with a low-energy 980 nm laser excitation, which facilitated high resolution and penetration depth into the specimen, and low phototoxicity needed for long-term imaging. Our upconversion nanoparticle resonance energy transfer based sensor with polyethylenimine-coating provides high colloidal stability, enhanced cellular uptake, and distribution across cellular compartments. This distribution was modulated with membrane integrity perturbing treatment that resulted into total loss of lysosomal compartments and a dramatic pH shift of endosomal compartments. These nanoprobes are well suited for detection of pH changes in in vitro models with high biological background fluorescence and in in vivo applications, e.g., for the bioimaging of small animal models.

Stimuli-responsive hybrid nanocarriers developed by controllable integration of hyperbranched PEI with mesoporous silica nanoparticles for sustained intracellular siRNA delivery.

Small interfering RNA (siRNA) is a highly potent drug in gene-based therapy with the challenge being to deliver it in a sustained manner. The combination of mesoporous silica nanoparticles (MSNs) and polycations in the confined pore space allows for incorporation and controlled release of therapeutic siRNA payloads. We hereby constructed MSNs with expanded mesopores and pore-surface-hyperbranched poly(ethyleneimine) (PEI) tethered with redox-cleavable linkers that could carry a high payload of siRNA (120 mg·g-1). The developed nanocarriers were efficiently taken up by cancer cells and were subsequently able to escape to the cytoplasm from the endosomes, most likely owing to the integrated PEI. Triggered by the intracellular redox conditions, the siRNA was sustainably released inside the cells over a period of several days. Functionality of siRNAs was demonstrated by using cell-killing siRNA as cargo. Despite not being the aim of the developed system, in vitro experiments using cell-killing siRNAs showed that the efficacy of siRNA transfection was comparable to the commercial in vitro transfection agent Lipofectamine. Consequently, the developed MSN-based delivery system offers a potential approach to hybrid nanocarriers for more efficient and long-term siRNA delivery and, in a longer perspective, in vivo gene silencing for RNA interference (RNAi) therapy.

On the intracellular release mechanism of hydrophobic cargo and its relation to the biodegradation behavior of mesoporous silica nanocarriers.

The intracellular release mechanism of hydrophobic molecules from surface-functionalized mesoporous silica nanoparticles was studied in relation to the biodegradation behavior of the nanocarrier, with the purpose of determining the dominant release mechanism for the studied drug delivery system. To be able to follow the real-time intracellular release, a hydrophobic fluorescent dye was used as model drug molecule. The in vitro release of the dye was investigated under varying conditions in terms of pH, polarity, protein and lipid content, presence of hydrophobic structures and ultimately, in live cancer cells. Results of investigating the drug delivery system show that the degradation and drug release mechanisms display a clear interdependency in simple aqueous solvents. In pure aqueous media, the cargo release was primarily dependent on the degradation of the nanocarrier, while in complex media, mimicking intracellular conditions, the physicochemical properties of the cargo molecule itself and its interaction with the carrier and/or surrounding media were found to be the main release-governing factors. Since the material degradation was retarded upon loading with hydrophobic guest molecules, the cargo could be efficiently delivered into live cancer cells and released intracellularly without pronounced premature release under extracellular conditions. From a rational design point of view, pinpointing the interdependency between these two processes can be of paramount importance considering future applications and fundamental understanding of the drug delivery system.