Biocompatible hydroxyapatite-based nano vehicle bypasses viral transduction and enables sustained silencing of a pluripotency marker gene, demonstrating desired differentiation in mouse embryonic stem cells.

BACKGROUND: Differentiation of pluripotent stem cells into desired lineages is the key aspect of regenerative medicine and cell-based therapy. Although RNA interference (RNAi) technology is exploited extensively for this, methods for long term silencing of the target genes leading to differentiation remain a challenge. Sustained knockdown of the target gene by RNAi is often inefficient as a result of low delivery efficiencies, protocol induced toxicity and safety concerns related to viral vectors. Earlier, we established octa-arginine functionalized hydroxyapatite nano vehicles (R8HNPs) for delivery of small interfering RNA (siRNA) against a pluripotency marker gene in mouse embryonic stem cells. Although we demonstrated excellent knockdown efficiency of the target gene, sustained gene silencing leading to differentiation was yet to be achieved. METHODS: To establish a sustained non-viral gene silencing protocol using R8HNP, we investigated various methods of siRNA delivery: double delivery of adherent cells (Adh-D), suspension delivery followed by adherent delivery (Susp + Adh), single delivery in suspension (Susp-S) and multiple deliveries in suspension (Susp-R). Sustained knockdown of a pluripotent marker gene followed by differentiation was analysed by reverse transcriptase-PCR, fluoresence-activated cell sorting and immunofluorescence techniques. Impact on cell viability as a result of repeated exposure of the R8HNP was also tested. RESULTS: Amongst the protocols tested, the most efficient knockdown of the target gene for a prolonged period of time was obtained by repeated suspension delivery of the R8HNP-siRNA conjugate. The long-term silencing of a pluripotency marker gene resulted in differentiation of R1 ESCs predominantly towards the extra embryonic and ectodermal lineages. Cells displayed excellent tolerance to repeated exposures of R8HNPs. CONCLUSIONS: The results demonstrate that R8HNPs are promising, biocompatible, non-viral alternatives for prolonged gene silencing and obtaining differentiated cells for therapeutics.

Superwettable surfaces and factors impacting microbial adherence in microbiologically-influenced corrosion: a review.

Microbiologically-influenced corrosion (MIC) is a common operational hazard to many industrial processes. The focus of this review lies on microbial corrosion in the maritime industry. Microbial metal attachment and colonization are the critical steps in MIC initiation. We have outlined the crucial factors influencing corrosion caused by microorganism sulfate-reducing bacteria (SRB), where its adherence on the metal surface leads to Direct Electron Transfer (DET)-MIC. This review thus aims to summarize the recent progress and the lacunae in mitigation of MIC. We further highlight the susceptibility of stainless steel grades to SRB pitting corrosion and have included recent developments in understanding the quorum sensing mechanisms in SRB, which governs the proliferation process of the microbial community. There is a paucity of literature on the utilization of anti-quorum sensing molecules against SRB, indicating that the area of study is in its nascent stage of development. Furthermore, microbial adherence to metal is significantly impacted by surface chemistry and topography. Thus, we have reviewed the application of super wettable surfaces such as superhydrophobic, superhydrophilic, and slippery liquid-infused porous surfaces as "anti-corrosion coatings" in preventing adhesion of SRB, providing a potential avenue for the development of practical and feasible solutions in the prevention of MIC. The emerging field of super wettable surfaces holds significant potential for advancing efficient and practical MIC prevention techniques.

Tailoring of hydroxyapatite nanoparticle surfaces of varying morphologies to facilitate counterion diffusion and subsequent protein denaturation.

Rapid advances in nanotechnology have led to the synthesis and development of various nanomaterials with complex structures and appropriate surface functionalization in recent years. Specifically designed and functionalized nanoparticles (NPs) are increasingly researched and hold great potential in biomedical applications (for example, imaging, diagnostics and therapeutics). Yet, the surface functionalization and biodegradability of NPs play a significant role in their application. Understanding the interactions occurring at the interface between the NPs and the biological components is thus crucial for predicting the fate of the NPs. In this work we study the effect of trilithium citrate functionalization of the hydroxyapatite NPs (HAp NPs) with and without cysteamine modification and their subsequent interaction with hen egg white lysozyme and corroborate the conformational changes of the protein with effective diffusion of the lithium (Li+) counter ion.

Self-fluorescence property of octa-arginine functionalized hydroxyapatite nanoparticles aids in studying their intracellular fate in R1 ESCs.

Deciphering the endocytosis mechanisms of nanoparticle entry in cells is crucial to understand the fate of nanoparticles and the biological activity of the transported cargo. Such studies require the use of reporter agents such as fluorescent markers. Previously, we have reported the synthesis of self-fluorescent HAp nanoparticles as efficient nucleic acid delivery agents in prokaryotic and eukaryotic cells. Here, we show the application of biocompatible self-fluorescent nano delivery vehicle based on HAp and CPP- octa-arginine as an efficient system to study the mechanisms of intracellular fate of a gene delivery agent. The pathway of octa-arginine functionalized HAp NP (R8HNP) and HAp NP uptake in R1 ESCs was elucidated using confocal microscopy with the help of endocytic inhibitors. The NPs mainly enter R1 ESCs by clathrin mediated and macropinocytosis pathways. It was established that the NPs escape endosomal degradation by proton sponge effect owing to their ability to buffer the pH and trigger osmotic rupture. The functionalization of CPP, effectively enhanced the internalization and endosomal escape in R1 ESCs. The detailed results of these studies are outlined in this manuscript.

Design of a Biocompatible Hydroxyapatite-Based Nanovehicle for Efficient Delivery of Small Interference Ribonucleic Acid into Mouse Embryonic Stem Cells.

The small interference RNA (siRNA)-assisted RNA interference approach in stem cells for differentiating into cell-specific lineages is gaining importance for its therapeutic potential. An effective gene delivery platform is crucial to achieve this goal. In this context, self-fluorescent, cell-penetrating peptide (CPP)-functionalized hydroxyapatite nanoparticles (R8HNPs) were synthesized by a modified sol gel technique. R8HNPs were crystalline, displayed characteristic bands, and exhibited broad emission spectra from 350 to 750 nm corresponding to green and red fluorescence. The biocompatible R8HNPs displayed robust binding with siRNA and excellent uptake in R1 ESCs. This was attributed to functionalization with CPP. Moreover, the R8HNP-complexed siRNA exhibited excellent serum and room temperature stability. The NPs protected the siRNA from sonication, pH, and temperature-induced stress and efficiently delivered siRNA to trigger 80% silencing of a pluripotency marker gene, Oct4, in R1 ESCs at 48 h. The transient downregulation was also observed at the protein level. Our findings demonstrate R8HNPs as a promising delivery agent for siRNA therapeutics with the potential for lineage-specific differentiation and future applications in regenerative medicine.

Solid-Supported Amplification of Aggregation Emission: A Tetraphenylethylene-Cucurbit[6]uril@Hydroxyapatite-Based Supramolecular Sensing Assembly for the Detection of Spermine and Spermidine in Human Urine and Blood.

The development of sensitive and selective tools for the detection and quantification of biomarkers is important in the diagnosis and treatment of clinical diseases. Spermine (SP) and spermidine (SPD) act as biomarkers for early-stage diagnosis of cancer in humans as their increased levels in urine are indicative of abnormal biological processes associated with this fatal disease. In this study, we introduced a strategy for solid-supported amplification of the effective aggregation-induced-emission (AIE) effect of a water-soluble tetraphenylethylene (TPE)-based probe in developing a supramolecular sensing platform for the rapid, sensitive, and selective detection of SP and SPD in water. The nonemissive TPE derivative (TPEHP) forms a less emissive conjugate with hydroxyl cucurbit[6]uril (CB[6]OH) in water, which undergoes several-fold enhancement of effective emission upon electrostatic interaction with the solid surface of hydroxyapatite nanoparticles (HAp NPs), dispersed in the aqueous media. The corresponding three-component supramolecular assembly disrupts by the intrusion of SP and SPD in the CB[6] portal because of the stronger binding ability with CB[6], resulting in a turn-off fluorescence sensor for SP and SPD with enhanced sensitivity. The assembly-disassembly-based sensing mechanism was thoroughly demonstrated by carrying out isothermal titration calorimetry (ITC), spectroscopic, and microscopic experiments. The sensing system showed low limits of detection (LODs) of 1.4 × 10-8 and 3.6 × 10-8 M for SP and SPD, respectively, which are well below the required range for the early diagnosis of cancer. Besides, a good linear relationship was obtained for both SP and SPD. Nominal interference from various metal ions, anions, common chemicals, amino acids, and other biogenic amines makes this sensing platform suitable for the real-time, low-level measurement of spermine (and spermidine) in human urinary and blood samples.

Novel one step transformation method for Escherichia coli and Staphylococcus aureus using arginine-glucose functionalized hydroxyapatite nanoparticles.

Bacterial gene transformation is one of the important techniques in molecular biology which has significant applications in gene cloning technology. In this study, we have developed arginine-glucose functionalized hydroxyapatite nanoparticle (R-G-HAp NPs) mediated novel one step transformation method, effective for both Gram-positive and Gram-negative bacteria. R-G-HAp NPs served as carriers to deliver pDNA into Escherichia coli and Staphylococcus aureus at room temperature, without the need for preparation of competent cells. High transformation efficiency was achieved in Gram-positive, S. aureus (107 cfu/µg of pDNA) as well as Gram-negative, E. coli (109 cfu/µg of pDNA). This demonstrates the efficacy of R-G-HAp NPs as a nano-vehicle to achieve high plasmid transformation efficiency, even in Gram-positive bacteria which is usually a challenge, exhibiting their potential as promising synthetic non-viral vectors for efficient bacterial gene transformation.

A novel method for genetic transformation of C. albicans using modified-hydroxyapatite nanoparticles as a plasmid DNA vehicle.

In modern biological research, genetic transformation is an important molecular biology technique with extensive applications. In this work, we describe a new method for the delivery of plasmid DNA (pDNA) into a yeast species, Candida albicans. This method is based on the use of novel arginine-glucose-PEG functionalized hydroxyapatite nanoparticles (M-HAp NPs) as a vehicle which delivers pDNA into Candida albicans with a high transformation efficiency of 106 cfu µg-1 of pDNA, without the need for preparation of competent cells. A four-fold higher transformation efficiency as compared to that of the electroporation method was obtained. This new method could provide exciting opportunities for the advancement of the applications of yeasts in the field of biotechnology.

Self-Activated Fluorescent Hydroxyapatite Nanoparticles: A Promising Agent for Bioimaging and Biolabeling.

Bioimaging has drastically transformed the field of medicine, and made the process of diagnosis easy and fast. Visualization of complete organ to complex biological processes has now become possible. Among the various imaging processes, fluorescence imaging using nontoxic fluorescent nanomaterials is advantageous for several beneficial features including high sensitivity, minimal invasiveness, and safe detection limit. In this study, we have synthesized and characterized a new class of nontoxic, self-activated fluorescent hydroxyapatite nanoparticles (fHAps) with different aspect ratios (thin-rods, short-rods, rods) by changing the stabilizing agents (triethyl amine and acetyl acetone) and solvents (water and dimethyl sulfoxide). fHAps showed excellent fluorescence with a broad emission spectrum ranging from 350 to 750 nm and maximum at 502 nm. The presence of fluorescence was attributed to the electronic transition in the asymmetric structure of fHAps as confirmed by ESR spectroscopy and the absence of fluorescence in symmetric HAp NPs. In addition to exceptional fluorescence behavior, these NPs were found to be nontoxic in nature and could be easily internalized in both prokaryotic and eukaryotic systems. We propose that the fHAps provide a safe and a potential alternative to the current fluorescent materials in use for biolabeling and bioimaging applications.

Synthesis of silver nanoparticles using haloarchaeal isolate Halococcus salifodinae BK3.

Numerous bacteria, fungi, yeasts and viruses have been exploited for biosynthesis of highly structured metal sulfide and metallic nanoparticles. Haloarchaea (salt-loving archaea) of the third domain of life Archaea, on the other hand have not yet been explored for nanoparticle synthesis. In this study, we report the intracellular synthesis of stable, mostly spherical silver nanoparticles (AgNPs) by the haloarchaeal isolate Halococcus salifodinae BK3. The culture on adaptation to silver nitrate exhibited growth kinetics similar to that of the control. NADH-dependent nitrate reductase was involved in silver tolerance, reduction, synthesis of AgNPs, and exhibited metal-dependent increase in enzyme activity. The AgNPs preparation was characterized using UV-visible spectroscopy, XRD, TEM and EDAX. The XRD analysis of the nanoparticles showed the characteristic Bragg peaks of face-centered cubic silver with crystallite domain size of 22 and 12 nm for AgNPs synthesized in NTYE and halophilic nitrate broth (HNB), respectively. The average particle size obtained from TEM analysis was 50.3 and 12 nm for AgNPs synthesized in NTYE and HNB, respectively. This is the first report on the synthesis of silver nanoparticles by haloarchaea.