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.

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.

Antioxidant-antibacterial containing bi-layer scaffolds as potential candidates for management of oxidative stress and infections in wound healing.

Tissue engineering techniques are continuously evolving towards providing better microenvironment along with therapeutic potential to address the skin tissue defects. Factors such as microbial infections, presence of excessive free radicals and depletion in antioxidant based scavenging systems pose serious challenges by prolonging inflammation and delaying the repair process. Incorporation of bioactive molecules in polymer based biomimetic scaffolds may present new vistas for handling chronic wounds. In this study, chitosan/collagen scaffolds incorporating 0.5, 1 and 2% (w/w) silymarin (CS-CO-SM) were synthesized and studied for their biocompatibility, in vitro release kinetics and anti-oxidant activity. The release kinetics of silymarin from the CS-CO-SM scaffold showed an initial burst followed by sustained release. The scaffolds were biocompatible and supported the recovery of COS-7 cells from UV induced oxidative stress. Further the CS-CO-SM(2) scaffolds were used to fabricate a bi-layer scaffold by layer upon layer arrangement with CS-Ag3 (3% Ag, w/w). The Ag was incorporated to impart antimicrobial property to the scaffold. The in vivo studies on bi-layer scaffolds were carried out in Wistar rat models at 3, 7 and 10 days post injury and the skin excisions were studied for wound contraction, histology (H&E staining), and lipid peroxidation. The bi-layer scaffold accelerated the process of wound healing with no inflammatory cells, proliferation of fibroblast, neovascularization and collagen deposition. By day 10 post transplantation of the scaffold, the skin had a structure similar to normal skin with complete re-epithelization. This bi-layer scaffold with antioxidant and antimicrobial properties promotes wound healing and is proposed as a potential tissue engineering material for managing chronic wounds.

Native hypersaline sulphate reducing bacteria contributes to iron nanoparticle formation in saltpan sediment: A concern for aquaculture.

A hypersaline dissimilatory sulphate reducing bacterium, strain LS4, isolated from the sediments of Ribander saltpan, Goa, India was found to produce (Fe2O3) maghemite nanoparticles. The presence of maghemite nanoparticles was also detected in the same sediment. Strain LS4 was isolated anaerobically on modified Hatchikian's media at 300 psu, growing optimally at 30 °C, 150 psu salinity and pH 7.8. Based on biochemical characteristics and 16S rRNA sequence analysis, the strain LS4 belongs to genus Desulfovibrio. This isolate synthesized iron oxide nanoparticles in vitro when challenged with FeCl3 & FeSO4 in the growth medium. The biological nanoparticles were characterized to be Fe2O3 nanoparticle of 19 nm size by X-ray diffraction, transmission electron microscopy, fourier transform infrared spectroscopy, scanning electron microscopy and energy-dispersive x-ray spectroscopy. Maghemite nanoparticles (5.63 mg g-1) were isolated from the saltpan sediment by magnetic separation which showed similar characteristic features to the Fe2O3 nanoparticle produced by strain LS4 with an average size of 18 nm. Traditionally Goan saltpans were used for aquaculture during the non-salt making season, thus effects of these nanoparticles on Zebra fish embryo development were checked, which resulted in developmental abnormalities and DNA damage in a dose dependent manner. With the increasing nanoparticle concentration (0.1 mg.L-1 to100 mg.L-1), the mortality rate increased with a decrease in the hatching rate (93.05 ± 2.4 to 25 ± 4.16%) and heart rate (150-120 beats per minute). The nanoparticle exposed embryos developed malformed larvae with a characteristic of pericardial edema, curved body, curved notochord, curved tail and curved tail tip. These results suggest that strain LS4 might be playing a role as a contributor in the formation of iron oxide nanoparticle in the Ribander saltpan sediment, however; its high concentration will have a negative impact on aquaculture in these saltpans.

Fluorescent Lead(IV) Sulfide Nanoparticles Synthesized by Idiomarina sp. Strain PR58-8 for Bioimaging Applications.

The fabrication of nanoparticles by microorganisms presents a "green" method for generating biocompatible nanomaterials. We discovered the intracellular biosynthesis of fluorescent lead(IV) sulfide nanoparticles by the moderate halophile, Idiomarina sp. strain PR58-8. The bacterium tolerated up to 8 mM Pb(NO3)2 during growth. Non-protein thiols dose-dependently increased in response to metal exposure, which suggests they are involved in the growth of PbS2 crystals and lead detoxification. Using X-ray diffraction, transmission electron microscopy (TEM), high-resolution TEM, and energy dispersive analysis of X-rays, the nanoparticles were characterized as spherical ß-PbS2 nanoparticles (PbS2NPs) with a tetragonal crystal lattice, a crystallite domain size of 2.38 nm, and an interplanar distance of 0.318 nm. A narrow symmetric emission spectrum with a Gaussian distribution and an emission maximum at 386 nm was obtained when the particles were excited at 570 nm. The PbS2NPs exhibited a large Stokes' shift (8,362 cm-1) and a relatively high quantum yield (67%). These properties, along with fluorescence that was maintained in various microenvironments and their biocompatibility, make these nanoparticles excellent candidates for bioimaging. The particles were internalized by HeLa cells and evenly distributed within the cytoplasm, exhibiting their potential for in situ bioimaging applications. The "as-synthesized" lead(IV) sulfide nanoparticles may provide expanded opportunities for targeted bioimaging via modifying the surface of the particles.IMPORTANCE This article reports the intracellular synthesis of fluorescent lead(IV) sulfide nanoparticles (PbS2NPs) by a microorganism. All previous reports on the microbial synthesis of lead-based nanoparticles are on lead(II) sulfide that exhibits near-infrared fluorescence, requiring expensive instrumentation for bioimaging. Bioimaging using PbS2NPs can be achieved using routine epifluorescence microscopes, as it fluoresces in the visible range. The research on PbS2 nanoparticles to date is on their chemical synthesis employing toxic precursors, extreme pH, pressure, and temperature, resulting in cytotoxic products. In this context, the synthesis of PbS2 nanoparticles by Idiomarina sp. strain PR58-8, described in this work, occurs at ambient temperature and pressure and results in the generation of biocompatible nanoparticles with no hazardous by-products. The excellent fluorescence properties that these particles exhibit, as well as their abilities to easily penetrate the cells and evenly distribute within the cytoplasm, make them exceptional candidates for bioimaging applications. This study demonstrated the synthesis and fluorescence bioimaging application of microbially synthesized PbS2 nanoparticles.

Passive permeability and effective pore size of HeLa cell nuclear membranes.

Nuclear pore complexes in the nuclear membrane act as the sole gateway of transport of molecules from the cytoplasm to the nucleus and vice versa. Studies on biomolecular transport through nuclear membranes provide vital data on the nuclear pore complexes. In this work, we use fluorescein isothiocyanate-labeled dextran molecules as a model system and study the passive nuclear import of biomolecules through nuclear pore complexes in digitonin-permeabilized HeLa cells. Experiments are carried out under transient conditions in the time lapse imaging scheme using an in-house constructed confocal laser scanning microscope. Transport rates of dextran molecules having molecular weights of 4-70 kDa corresponding to Stokes radius of 1.4-6 nm are determined. Analyzing the permeability of the nuclear membrane for different sizes the effective pore radius of HeLa cell nuclear membrane is determined to be 5.3 nm, much larger than the value reported earlier using proteins as probe molecules. The range of values reported for the nuclear pore radius suggest that they may not be rigid structures and it is quite probable that the effective pore size of nuclear pore complexes is critically dependent on the probe molecules and on the environmental factors.

Anti-bacterial TeNPs biosynthesized by haloarcheaon Halococcus salifodinae BK3.

Microbial synthesis of highly structured metal sulfide and metallic nanoparticles is a benign approach of nanomaterial synthesis. Various microbes have been exploited for nanoparticle synthesis, but nanofabrication using haloarchaea is still in nascent stages. Here, we report the intracellular synthesis of hexagonal needle-shaped tellurium nanoparticles with an aspect ratio of 1:4.4, by the haloarcheon Halococcus salifodinae BK3. The isolate was able to tolerate up to 5.5 mM K2TeO3. The yield of tellurium nanoparticles was highest when the culture was exposed to 3 mM K2TeO3, even though the isolate exhibited slightly decreased growth rate as compared to the culture growing in the absence of K2TeO3. The enzyme tellurite reductase was responsible for tellurite resistance and nanoparticle synthesis in H. salifodinae BK3. These tellurium nanoparticles exhibited anti-bacterial activities against both Gram-positive and Gram-negative bacteria, with higher antibacterial activity towards Gram-negative bacteria. This is the first report on the synthesis of tellurium nanoparticles by Halophilic archaea.

Nanomaterial-Based Approaches for Prevention of Biofilm-Associated Infections on Medical Devices and Implants.

Biofilm formation is a major problem in medical device-related infections leading to failure of implant-based therapies. Though various conventional approaches to counter biofilm formation like physical and/or mechanical removal, chemical removal, and the use of antimicrobials exist, they fail due to increased resistance of biofilms. This review discusses various nanomaterial-based approaches such as the use of metallic and metal oxide nanoparticles- and polymer-based nanocomposites, which are currently being developed for prevention and treatment of biofilms. Nanoparticles of transition metals and their oxides are toxic to microorganisms and exhibit their toxicity through the generation of reactive oxygen species at concentrations that are non-toxic to eukaryotic cells. Other approaches include the entrapment of bioactive agents in polymer/ceramic nanoparticles, for enhanced anti-biofilm activity due to the synergistic effect between them. These nanomaterial-based approaches could play an important role in control and eradication of biofilm related infections and complications associated with medical devices and implants.

Laser scanning photothermal microscopy: fast detection and imaging of gold nanoparticles.

We report on the design and construction of a laser scanning photothermal microscope and present images of gold nanoparticles of size as small as 5 nm. Laser scanning method allows fast image acquisition at 80 µs pixel dwell time so that a 500 × 500 pixel image is acquired in 20 s. Photothermal imaging at fast time scales can have potential applications in variety of fields including tracking of biomolecular transport processes.

Mechanisms of metal resistance and homeostasis in haloarchaea.

Haloarchaea are the predominant microflora of hypersaline econiches such as solar salterns, soda lakes, and estuaries where the salinity ranges from 35 to 400 ppt. Econiches like estuaries and solar crystallizer ponds may contain high concentrations of metals since they serve as ecological sinks for metal pollution and also as effective traps for river borne metals. The availability of metals in these econiches is determined by the type of metal complexes formed and the solubility of the metal species at such high salinity. Haloarchaea have developed specialized mechanisms for the uptake of metals required for various key physiological processes and are not readily available at high salinity, beside evolving resistance mechanisms for metals with high solubility. The present paper seeks to give an overview of the main molecular mechanisms involved in metal tolerance in haloarchaea and focuses on factors such as salinity and metal speciation that affect the bioavailability of metals to haloarchaea. Global transcriptomic analysis during metal stress in these organisms will help in determining the various factors differentially regulated and essential for metal physiology.

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.

Synthesis and characterization of fumaric acid functionalized AgCl/titania nanocomposite with enhanced antibacterial activity.

Silver based antimicrobial agents with TiO2 as a host support (AgCl/TiO2) are increasingly being evaluated for disinfection and therapeutic applications. In this work TiO2 supported silver chloride nanocomposites were synthesized and functionalized with fumaric acid. X-ray diffraction results showed that the TiO2 was in anatase phase and AgCI was in the cubic chalcoargyrite phase. The functionalization was confirmed by FTIR analysis. The fumaric acid functionalized AgCl/TiO2 (Fu-AgCl/TiO2) showed uniform particle size distribution in the range of 4-5 nm. The intensity size distribution of Fu-AgCl/TiO2 nanocomposite by DLS showed an average particle diameter of -290 nm with a polydispersity index (P. I.) of 0.47. Further, a high BET surface area of -320.7 m2/g was observed for Fu-AgCl/TiO2 with an average pore size distribution of 3.8 nm. The antimicrobial activity of Fu-AgCl/TiO2 and AgCl/TiO2 was evaluated by determining the MIC and MBC values, and it was observed that Fu-AgCl/TiO2 exhibited better antimicrobial activity as compared to the unfunctionalized nanocomposite. The antimicrobial activity of Fu-AgCl/TiO2 is mainly attributed to its superior physicochemical properties coupled with the synergistic action of both fumaric acid and silver ions. The changes in the cytoplasmic membrane of the cells due to the formation of intracellular ROS were observed by SEM imaging.

Altered growth and enzyme expression profile of ZnO nanoparticles exposed non-target environmentally beneficial bacteria.

The extensive production and usage of nanoparticles with ultimate disposal in the environment leads to unintentional exposure of non-target environmentally beneficial bacteria thereby posing a serious threat to the native soil inhabitants. Soil microflora is an important link in the biogeochemical cycling of nutrients, affecting ecosystem functioning and productivity. This study evaluates the effect of one of the widely used nanoparticles, zinc oxide on two predominant soil bacteria, Gram-positive Bacillus subtilis and Gram-negative Pseudomonas aeruginosa with respect to their biocatalytic activities. Growth profiles of these bacteria in the presence of zinc oxide nanoparticles (ZnONPs) at a concentration of 20 ppm exhibited a prolonged lag phase in B. subtilis, whereas no significant effect was observed in the case of P. aeruginosa even at 200 ppm. Interestingly, the enzymatic profile of both the organisms was affected at non-lethal ZnONPs concentrations. The most pronounced effect was on the enzymes associated with amylolytic activity, denitrification and urea degradation wherein total inhibition of activity was noted in B. subtilis. The enzyme activities were lowered in the case of P. aeruginosa. The results presented here reiterate a critical need for exposure assessment and risk characterization of nanomaterial disposal on soil microflora while formalizing waste management strategies.

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.

Aggregation-Induced Emission-Based Material for Selective and Sensitive Recognition of Cyanide Anions in Solution and Biological Assays.

Cyanide is one of the highly poisonous pollutants to our environment and toxic to human health. It is important to develop the widely applicable methods for their recognition to secure safe uses for people coming into contact and handling cyanide and their derivatives. In this regard, the aggregation-induced emission materials possess high potential for the development of simple, fast, and convenient methods for cyanide detection through either "turn-off" or "turn-on". Among the AIE-based materials, tetraphenylethylene is a promising sensor for various sensing applications. In this paper, we have designed and synthesized a TPE-based chemosensor, which shows high sensitivity and displays good selectivity for cyanide (CN-) over others in the presence of interfering Cl-, I-, F-, Br-, HSO4 -, H2PO4 -, NO3 -, HCO3 -, and ClO4 - anions employed. The naked-eye, UV-vis, and fluorescence methods are employed to evaluate the performance of probe 1 toward CN- detection. From these experiments, CN- ions can be detected with a limit of detection as low as 67 nM, which is comparatively lower than that of the World Health Organization (WHO) permissible limit of the cyanide anion, that is, 1.9 µM. From the Job's plot, the 1:1 stoichiometric complexation reaction between probe 1 and CN- was found. The probe was efficiently applied for the detection of CN- ions using a paper strip method. The probe 1 also showed the potential of detecting CN- ions in various food items and in the cell line.

Nanocarrier Mediated siRNA Delivery Targeting Stem Cell Differentiation.

Stem cell-based regenerative medicine holds exceptional therapeutic potential and hence the development of efficient techniques to enhance control over the rate of differentiation has been the focus of active research. One of the strategies to achieve this involves delivering siRNA into stem cells and exploiting the RNA interference (RNAi) mechanism. Transport of siRNA across the cell membrane is a challenge due to its anionic property, especially in primary human cells and stem cells. Moreover, naked siRNA incites immune responses, may cause off-target effects, exhibits low stability and is easily degraded by endonucleases in the bloodstream. Although siRNA delivery using viral vectors and electroporation has been used in stem cells, these methods demonstrate low transfection efficiency, cytotoxicity, immunogenicity, events of integration and may involve laborious customization. With the advent of nanotechnology, nanocarriers which act as novel gene delivery vehicles designed to overcome the problems associated with safety and practicality are being developed. The various nanomaterials that are currently being explored and discussed in this review include liposomes, carbon nanotubes, quantum dots, protein and peptide nanocarriers, magnetic nanoparticles, polymeric nanoparticles, etc. These nanodelivery agents exhibit advantages such as low immunogenic response, biocompatibility, design flexibility allowing for surface modification and functionalization, and control over the surface topography for achieving the desired rate of siRNA delivery and improved gene knockdown efficiency. This review also includes discussion on siRNA co-delivery with imaging agents, plasmid DNA, drugs etc. to achieve combined diagnostic and enhanced therapeutic functionality, both for in vitro and in vivo applications.

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.

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.