Synthesis and Characterization of Novel Plasmid-Layered Double Hydroxides Nanobiohybrids for Gene Delivery

One of the hindering problems faced by conventional gene delivery systems into cells is their efficiency in its delivery and integration. DNA and other genetic materials are easily degraded in both the extracellular as well as intracellular matrix by both endonuclease activities and physiological conditions of the cellular environment. Therefore, research insights have focused on utilizing the emerging field of nanotechnology to overcome this problem. For this reason , a layered nanomaterial, Mg/AI-LD H based on the layered double hydroxide (LDH) system was synthesized at pH 1 0.0 at different Mg to AI ratios, to determine whether it can be used as a vector for gene delivery. A plasmid DNA, encodi ng the green fluorescent protein reporter gene, was intercalated into the LDH intergallery region; previously occupied by nitrate anions. Successful occupation of the circular DNA was confirmed by expansions within the intergallery spacing of the LDH from powder x-ray diffraction analysis. Fourier-transform infrared spectroscopy further revealed the presence of exclusive functional groups belonging to both DNA and LDH in the nanobiohybrid product, and by both CHNS as well as gel electrophoresis data, the plasmid DNA was confirmed to be successfully intercalated within the LDH host. The effects of the host on cells were then evaluated using MTT assay on two cell lines, and the synthesized LDH hosts were found to have no significant lethal effects on cell viability. Microscopic studies using S EM and TEM later revealed the nanobiohybrid size to be withi n the nano-meter range, which was found to enhance its uptake b y cells. Cells transfected with the nanobiohybrid showed successful expression of the GFP gene compared to controls, as observed using fluorescence microscopy. The nanobiohybrid was found to not only deliver the gene into cells, but gene expression efficiency using the host for transfection was even comparable to a commercially available, high cost transfection vector in the market. Compared to the commercial vector, the LDH host was also shown to provide sufficient protection of the intercalated plasmid from degradation by the DNase I and XhollKpnl restriction enzymes, showing the potential of using the LDH host as an alternative delivery vector for gene delivery

Intracellular trafficking and drug release from fluorescently-labeled chitosan nanoparticle systems for development of innovative drug delivery systems

The increased bioavailability of essential biomolecules such as drugs, DNA and peptides is pre-requisite for efficient intracellular efficacy on drug delivery systems. Nanotechnological-based approaches for drug delivery applications potentially promotes a better distribution of energy in vivo, increasing the intracellular uptake of biomolecules for enhanced therapeutic uptake. Realising the ubiquitous utilization of nanoparticles in an increasing myriad of research fields, investigations into nanoparticle uptake, cargo release, as well as nanoparticle carrier persistence are pertinent towards their consequent optimization and development. We describe in this work, the elucidation of nanoparticle uptake and sustained release of its encapsulated cargo in colon cancer cells to model a nanoparticle-mediated drug delivery system. Chitosan nanoparticles were synthesized through ionic gelation routes and characterized by means of light scattering, electron microscopy, and infrared spectroscopic analysis. The nanoparticles were encapsulated with a fluorescently-modified amino acid for in vitro tracking, and its intracellular release was quantitated in a time-dependent study using flow cytometry and fluorescent microscopy. Cytotoxic analysis was subsequently performed to evaluate any inherent efficacy of the nanoparticle for use as a candidate delivery system. Findings arising from our analyses showed that intracellular uptake of nanoparticles occurred within 30 mins of cell treatment; and continually took place up to 48 hours post treatment. Interestingly, release of cargo only occurred 6 hours post treatment and a controlled release system was exhibited up to 48 hours without extracellular leakage. MTT assay showed very low toxicity of the 60-180nm size particles; demonstrating a potential of the chitosan nanoparticle system for use as a systemic, slow release system for drug delivery. Conclusions derived from this study is hoped to provide sufficient data towards more critical developments of nanoparticle delivery systems for targeted and enhanced drug delivery parameters, most clinically relevant in the pharmaceutical and medical fields

Preparation and characterization of carboxymethyl sago starch hydrogel

Hydrogel is a three-dimensional network of polymer chains that receives high attention in scientific research due to their potential in drug delivery, biomedical field and waste water treatment. In this study, carboxymethyl sago starch (CMS) hydrogel was prepared via crosslinking technique where CMS was dissolved in HCl solution under vigorous stirring to form gel. The effect of the percentage amount of CMS, concentration of the acid solution, reaction time and reaction temperature were the parameters that have been studied to identify the optimum condition of CMS hydrogel. It was found that 60% amount of CMS in 2.0M acid solution for 12 hours at room temperature were the optimum conditions for CMS hydrogel. The CMS hydrogel was characterized by using Fourier Transform Infrared (FT-IR), thermogravimetric analysis (TGA) and scanning electron microscopy (SEM). FTIR spectrum of CMS hydrogel shows an additional sharp absorption peak at 1723 cm-1 denote that the Na in CMSS being exchanged to H from hydrochloric acid solution. SEM image of CMS hydrogel shows a spongy surface with empty space called pores in structure and connected to each other to form networks. TGA curve shows that the maximum rate of thermal decomposition of CMS hydrogel is higher than CMS at 311.89 ºC with 60.22 % major weight loss. This could be due to the presence of the cross-linkages in the CMS hydrogel

Encapsulation of miRNA in chitosan nanoparticles as a candidate for an anti-metastatic agent in cancer therapy

MicroRNAs (miRNA) have been utilised as a repressor molecule for metastasis of tumours, as it inhibits fundamental processes related to cellular and physiological pathway of the tumour at the mRNA level. However, therapeutic application of miRNAs is impaired by premature degradation in the extracellular environment by endonucleases. This research describes the optimisation, chemical, and morphological characterisation of nanoparticles for effective encapsulation of miRNA-186 and evaluate its efficiency as anti-metastatic agent in non-small cell lung carcinoma monolayer. Through ionic gelation methods, the miRNA was encapsulated in chitosan nanoparticles (CNPs), a drug carrier with high particle stability, low cellular toxicity, and robust preparation methods. Physiochemical and morphological characterization analysis through light scattering analysis showed miRNA-CNP sizes below 200 nm, with a low polydispersity index and exhibition of irregular spherical shape of the nanoparticles synthesised through FESEM analysis. Additionally, in vitro nanoparticle efficacy evaluated through scratch assay suggests a decrease in invasion ability of cancer cells exhibited by miRNA-CNP

Synthesis of a nanoparticle system for the enhanced accumulation of fluorescently-labelled amino acids encapsulated in monodispersed chitosan nanoparticle system

Poor in vivo bioavailability of nutrient is a major challenge in efficient delivery of nutraceutics. The increased bioavailability of nutraceuticals is prerequisite for efficient absorption by the gastrointestinal system. Nanotechnology-based approaches for nutraceutical applications could potentially increase absorption of nutrients and enhance its cellular accumulation due to its nanosize and promote better in vivo energy biodistribution. However, the dynamics of intracellular cell trafficking of nanoparticles and nutraceutical release has remain scarcely studied. This study describes a non-efficacious nanoparticle-mediated system for the encapsulation and delivery of fluorescently-labelled amino acids using tripolyphosphate as a crosslinker. Light scattering data showed successful formation of particle size as small as 65.69 nm with low polydispersity index (PDI) value of 0.178 at specific volume ratios of chitosan to tripolyphosphate. Following encapsulation, nanoparticle size and PDI value increased to 182.73 nm and 0.257 respectively discern successful accommodation of the fluorescently-labelled amino acid within its core. In vitro visualization of amino acids release and accumulation via fluorescence microscopy suggested that encapsulated amino acids were efficiently accumulated into Vero3 cell cytoplasm at 24 hours post treatment with localization in proximity to the cell nucleus. These results therefore suggested that the chitosan nanoparticle system developed was able to enhance the intracellular accumulation of glutamic acids and may serve as a suitable carrier for nutraceuticals delivery

HIF-1 ACTIVATION AND INFLAMMATORY RESPONSES TO HYPOXIA

Acute hypoxia is a significant physiological danger during high-altitude flying and military aircraft missions. The human brain requires a constant supply of oxygen to function properly, and is susceptible to settings with low availability of air oxygen. Hypoxia can influence inflammatory signalling, and both central and systemic responses can activate HIF pathway genes. HIFs are critical molecules that regulate inflammation andhypoxia, ensuring appropriate cell function and survival. Hypoxia is the condition in which insufficient oxygen reaches the body\u27s tissues. It can be caused by a decrease in partial oxygen pressure (PO2) in the environment, problems with breathing and/or oxygen transport, or the inability of tissues to utilise oxygen. Different organs are hypoxic due to differences in tissue oxygen tensions, which are determined by differences in aerobic metabolism. Extremely hypoxic individuals have the most dramatic systemic and neurological adaptations to persistent hypoxia. In this review, we provide an overview of central and systemic responses to hypoxia and discuss the activation of HIF-1 pathway

Superparamagnetic iron oxide and quantum dots for biomedical applications

Nanomaterials has become insanely popular in biomedical field ranging from contrast agent applications in medical imaging to carriers in gene and drug delivery. Nanomaterials have unique properties that distinguish them from bulk materials in term of size, chemical reactivity, energy absorption and biological mobility. Magnetic nanoparticles (Fe3O4) posses superparamagnetic properties, which is widely known for its application as a contrast agent in MRI and as nanocarrier in targeted drug delivery. Semiconductor quantum dots (CdSe-ZnS) have strong fluorescent properties and the wavelength emitted depend sensitively on particle size. It have extensive applications in organic light emitting diode (OLED), sensor, and fluorescent biological labels. Current work are focusing on synthesis and characterization of biocompatible magnetic nanoparticles and fluorescence quantum dots, as well as toxicity of the nanomaterials for the biomedical application and gene therapy specifically. Magnetic nanoparticles (Fe3O4) and semiconductor quantum dots (CdSe-ZnS) are both synthesized using co-precipitation method and microwave irradiation technique respectively. The synthesized nanoparticles were characterized with X-Ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR), Transmission Electron Microscopy (TEM), and Vibrating Sample Magnetometry (VSM). The toxicity of both type of nanoparticles were tested on 2 different cell line; A549 human lung carcinoma and MRC5 human lung fibroblast using MTT assay. This research explores the potentials of these two types of nanomaterials in biomedical applications

Interaction between Pasteurella multocida B: 2 and its derivatives with bovine aortic endothelial cell (BAEC)

Background: Pasteurella multocida B:2 causes bovine haemorrhagic septicaemia (HS), leading to rapid fatalities in cattle and buffaloes. An attenuated derivative of P. multocida B:2 GDH7, was previously constructed through mutation of the gdhA gene and proved to be an effective live attenuated vaccine for HS. Currently, only two potential live attenuated vaccine candidates for HS are being reported; P. multocida B:2 GDH7 and P. multocida B:2 JRMT12. This study primarily aims to investigate the potential of P. multocida B:2 GDH7 strain as a delivery vehicle for DNA vaccine for future multivalent applications. Results: An investigation on the adherence, invasion and intracellular survival of bacterial strains within the bovine aortic endothelial cell line (BAEC) were carried out. The potential vaccine strain, P. multocida B:2 GDH7, was significantly better (p ≤ 0.05) at adhering to and invading BAEC compared to its parent strain and to P. multocida B:2 JRMT12 and survived intracellularly 7 h post treatment, with a steady decline over time. A dual reporter plasmid, pSRGM, which enabled tracking of bacterial movement from the extracellular environment into the intracellular compartment of the mammalian cells, was subsequently transformed into P. multocida B:2 GDH7. Intracellular trafficking of the vaccine strain, P. multocida B:2 GDH7 was subsequently visualized by tracking the reporter proteins via confocal laser scanning microscopy (CLSM). Conclusions: The ability of P. multocida B:2 GDH7 to model bactofection represents a possibility for this vaccine strain to be used as a delivery vehicle for DNA vaccine for future multivalent protection in cattle and buffaloes

Successful transfer of plasmid DNA into in vitro cells transfected with an inorganic plasmidMg/Al-LDH nanobiocomposite material as a vector for gene expression

The delivery of a full plasmid, encoding the green fluorescent protein gene into African monkey kidney (Vero3) cells, was successfully achieved using nanobiocomposites based on layered double hydroxides. This demonstrated the potential of using the system as an alternative DNA delivery vector. Intercalation of the circular plasmid DNA, pEGFP-N2, into Mg/Al-NO−3 layered double hydroxides (LDH) was accomplished through anion exchange routes to form the nanobiocomposite material. The host was previously synthesized at the Mg2+ to Al3+ molar ratio Ri = 2 and subsequently intercalated with plasmid DNA. Size expansion of the interlamellae host from 8.8 ° A in LDH to 42 °A was observed in the resulting nanobiocomposite,indicating stable hybridization of the plasmid DNA. The powder x-ray diffraction (PXRD)results, supplemented with Fourier-transform infrared (FTIR) spectroscopy, compositional and electrophoresis studies confirmed the encapsulation episode of the biomaterial. In order to elucidate the use of this resulting nanobiocomposite as a delivery vector, an MTT assay was performed to determine any cytotoxic effects of the host towards cells. The intercalated pEGFP-N2 anion was later successfully recovered through acidification with HNO3 after treatment with DNA-degrading enzymes, thus also showing the ability of the LDH host to protect the intercalated biomaterial from degradation. Cell transfection studies on Vero3 cells were then performed, where cells transfected with the nanobiocomposite exhibited fluorescence as early as 12 h post-treatment compared to naked delivery of the plasmid itself

Optimization of process parameters influencing the sustainable construction of iron oxide nanoparticles by a novel tropical wetlands Streptomyces spp.

A Streptomyces strain isolated from the soil sediments of tropical freshwater wetlands in Malaysia demonstrated promising attributes to be developed into a versatile microbial nanofactory for the sustainable synthesis of ferric oxide nanoparticles (IONP). Process parameters such as temperature, ferric salt precursor concentration, cell free extract (CFE) concentration and biomass harvesting times are serious players in the extracellular generation of metallic nanoparticles. A statistical approach using One-Variable-At-A-Time (OVAT) Analysis followed by Response Surface Methodology (RSM) was employed towards the optimization of the microbial bioprocess and modulation of nanoparticle size dimensions. OVAT revealed that IONP production increased with increasing temperature, precursor concentration, harvesting time and CFE concentration with highest yield at 65 °C, 2 mM precursor concentration, 68 h harvesting time and 100% CFE concentration. A detailed statistical analysis using RSM (RSM) showed significantly strong negative interactive effects between temperature and CFE concentration (p = 0.0037), temperature and precursor concentration (p = 0.0093) and mild interactive effects between CFE and precursor concentration (p = 0.0301). Taking into account the interactive influence of these variables, numerical analysis using RSM proposed that for optimal generation of microbial mediated IONP, a CFE concentration of 55.58%, temperature of 55.75 °C and precursor concentration of 2.46 mM FeCl3.6H2O would be required