Magnetic metal-organic frameworks as sensitive aptasensors for coronavirus spike protein.

Electrochemical biosensors, known for their low cost, sensitivity, selectivity, and miniaturization capabilities, are ideal for point-of-care devices. The magnetic metal-organic framework (MMOF), synthesized using the in-situ growth method, consists of ferric salt, magnetic nanoparticles, histidine, and benzene tetracarboxylic acid. MMOF was sequentially modified with aptamer-biotin and streptavidin-horseradish peroxidase, serving as a detector for spike protein and a transducer converting electrochemical signals using H2O2-hydroquinone on a screen-printed electrode. MMOF facilitates easy washing and homogeneous deposition on the working electrode with a magnet, enhancing sensitivity and reducing noise. The physical and electrochemical properties of the modified MMOFs were thoroughly characterized using various analytical techniques. The aptasensors' performance achieved a detection limit of 6 pM for voltammetry and 5.12 pM for impedance spectroscopy in human serum samples. This cost-effective, portable MMOF platform is suitable for rapid point-of-care testing for SARS-CoV-2 spike proteins.

Magnetic graphene oxide nanoflakes for dual RNA interfering delivery and gene knockdown in prostate and liver cancers.

The development of synthetic carriers for small interfering RNA (siRNA) and plasmids is crucial for effective gene therapy. In this study, we synthesized magnetic graphene oxide nanoflakes as carriers for siRNA delivery, with the goal of knockdown specific genes such as the green fluorescence protein (GFP). Our approach combined magnetically reduced graphene oxide with polyethylenimine (PEI) crosslinked to its surface using carbonyl diimidazole. To evaluate the adsorption capacity of the PEI-modified nanocomposite, we investigated its ability to bind two types of nucleic acids: short-hairpin (sh)RNA plasmids and siRNA targeting GFP. The nanocomposite exhibited significant adsorption, with maximum capacities of 426 ng/µg for shRNA and 71 ng/µg for siRNA, respectively. Simultaneous delivery of siRNA and shRNA using our designed nanocomposites was successfully achieved in human hepatoma and prostate cancer cells. Under magnetic guidance, the knockdown efficiencies reached 73.5 % in hepatoma cells for dual delivery of siRNA and shRNA. Our findings revealed that the nanocomplexes were internalized by the cells through a caveolae-dependent endocytosis mechanism. The demonstrated ability of the nanoflakes to efficiently transport siRNA and shRNA, with high loading capacity, controlled release, and magnetic targeting, resulted in effective GFP knockdown in vitro. These findings highlight the potential of magnetic graphene oxide nanoflakes as promising carriers for siRNA delivery and gene knockdown in therapeutic applications.

Synthesis and characterization of Au-decorated graphene oxide nanocomposite for magneto-electrochemical detection of SARS-CoV-2 nucleocapsid gene.

Fast and effective diagnosis is the first step in monitoring the current coronavirus 2 (CoV-2) pandemic. Herein, we establish a simple and sensitive electrochemical assay using magnetic nanocomposite and DNA sandwich probes to rapidly quantify the CoV-2 nucleocapsid (N) gene down to the 0.37 fM level. This assay uses a pair of specific DNA probes. The capture probe is covalently conjugated to Au-decorated magnetic reduced graphene oxide (AMrGO) nanocomposite for efficiently capturing target RNA. In contrast, the detection probe is linked to peroxidase for signal amplification. The probes target the COV-2 gene, allowing for specific magnetic separation, enzymatic signal amplification, and subsequent generation of voltammetric current with a total assay time of 45 min. The developed biosensor has high selectivity and can discriminate non-specific gene sequences. Synthetic COV-2 N-gene can be detected efficiently in serum and saliva, while 1-bp mismatch gene yielded a low response. The performance of the genosensor was good in an extensive linear range of 5 aM-50 pM. For synthetic N-gene, we achieved the detection limit of 0.37, 0.33, and 0.19 fM in human saliva, urine, and serum. This simple, selective, and sensitive genosensor could have various genetics-based biosensing and diagnostic applications.

Multifunctional magnetic nanocarriers for delivery of siRNA and shRNA plasmid to mammalian cells: Characterization, adsorption and release behaviors.

Nucleic acids are promising candidates for treating various diseases. Nucleic acid is negatively charged and hydrophilic; therefore, it is not efficiently taken up by cells. Successful gene therapy requires the development of carriers for efficient delivery of gene-expressing DNA plasmid and small interfering RNA (siRNA) duplex. In this study, we developed MNP-CA-PEI, a citric acid (CA)-modified magnetic nanoparticle (MNP) cross-linked with polyethyleneimine (PEI), using carbonyldiimidazole as the crosslinker. The physical properties of MNP-CA-PEI (particle size, morphologies, surface coating, surface potentials, magnetic hystereses, superparamagnetic behaviors, and infrared spectra) were systematically characterized by transmission electron microscopy imaging, dynamic light scattering, thermogravimetric analysis, superconducting quantum interference device, and Fourier transform infrared spectroscopy. The adsorption isotherm and kinetics were determined by the Langmuir model, the Freundlich model, a pseudo-first-order equation, and a pseudo-second-order equation. MNP-CA-PEI could form polyelectrolyte complexes with negatively charged nucleic acids, enabling the efficient delivery of nucleic acids into cells. Using MNP-CA-PEI nanoparticles, we magnetically triggered the intracellular delivery of green fluorescence protein (GFP)-expressing DNA plasmid, plasmid-expressing short hairpin RNA (shRNA) against GFP, or siRNA targeting GFP into different cell lines. Nucleic acid/MNP-CA-PEI displayed the enhanced cellular uptake of GFP-expressing DNA plasmid, and it improved the silencing efficiency of shRNA and siRNA, determined by fluorescence imaging. Gene knockdowns mediated by shRNA and siRNA were also confirmed by a quantitative real-time polymerase chain reaction. MNP-CA-PEI delivered nucleic acids into cytosol through caveolae-mediated endocytosis. This study introduces a new MNP functionalization that can be used for the magnetically driven intracellular delivery of nucleic acids.

Tetraethylenepentamine-Coated ß Cyclodextrin Nanoparticles for Dual DNA and siRNA Delivery.

Nucleic acid reagents, including plasmid-encoded genes and small interfering RNA (siRNA), are promising tools for validating gene function and for the development of therapeutic agents. Native ß-cyclodextrins (BCDs) have limited efficiency in gene delivery due to their instable complexes with nucleic acid. We hypothesized that cationic BCD nanoparticles could be an efficient carrier for both DNA and siRNA. Tetraethylenepentamine-coated ß-cyclodextrin (TEPA-BCD) nanoparticles were synthesized, characterized, and evaluated for targeted cell delivery of plasmid DNA and siRNA. The cationic TEPA coating provided ideal zeta potential and effective nucleic acid binding ability. When transfecting plasmid encoding green fluorescent protein (GFP) by TEPA-BCD, excellent GFP expression could be achieved in multiple cell lines. In addition, siRNA transfected by TEPA-BCD suppressed target GFP gene expression. We showed that TEPA-BCD internalization was mediated by energy-dependent endocytosis via both clathrin-dependent and caveolin-dependent endocytic pathways. TEPA-BCD nanoparticles provide an effective means of nucleic acid delivery and can act as potential carriers in future pharmaceutical application.

Voltammetric biosensor for coronavirus spike protein using magnetic bead and screen-printed electrode for point-of-care diagnostics.

The rapid spread of the novel human coronavirus 2019 (COVID-19) and its morbidity have created an urgent need for rapid and sensitive diagnostics. The real-time polymerase chain reaction is the gold standard for detecting the coronavirus in various types of biological specimens. However, this technique is time consuming, labor intensive, and expensive. Screen-printed electrodes (SPEs) can be used as point-of-care devices because of their low cost, sensitivity, selectivity, and ability to be miniaturized. The ability to detect the spike protein of COVID-19 in serum, urine, and saliva was developed using SPE aided by magnetic beads (MBs) and a portable potentiostat. The antibody-peroxidase-loaded MBs were the captured and catalytic units for the electrochemical assays. The MBs enable simple washing and homogenous deposition on the working electrode using a magnet. The assembly of the immunological MBs and the electrochemical system increases the measuring sensitivity and speed. The physical and electrochemical properties of the layer-by-layer modified MBs were systematically characterized. The performance of these immunosensors was evaluated using spike protein in the range 3.12-200 ng mL-1. We achieved a limit of detection of 0.20, 0.31, and 0.54 ng mL-1 in human saliva, urine, and serum, respectively. A facile electrochemical method to detect COVID-19 spike protein was developed for quick point-of-care testing.

MicroRNA-126 inhibits pathological retinal neovascularization via suppressing vascular endothelial growth factor expression in a rat model of retinopathy of prematurity.

Vascular endothelial growth factor (VEGF) is the principal growth factor responsible for the retinal neovascularization in the pathogenesis of retinopathy of prematurity (ROP). Current therapies for ROP include laser ablation and intravitreal anti-VEGF injection. However, these treatments either destroy the peripheral retina or associate with problems of persistent peripheral avascular retina or later recurrence of ROP. In the present study we investigated a new therapeutic approach by exploring the potential role of a specific microRNA, miR-126, in regulating VEGFA expression and retinal neovascularization in a rat oxygen-induced retinopathy (OIR) model. We demonstrated that miR-126 mimic and plasmid effectively suppresses VEGFA mRNA expression in both human and rat retinal pigment epithelium cell lines, quantified with qRT-PCR. Animal experiments on rat OIR model revealed that intravitreal injection of miR-126 plasmid efficiently downregulated VEGFA expression in the intraocular fluid and retinal tissues measured by ELISA, and significantly suppressed retinal neovascularization, which was confirmed by calculating sizes of neovascularization areas on fluorescence microscopic images of flat mounted retina stained with Alexa Fluor 594-conjugated isolectin B4 to visualize blood vessels. Together, these results showed that intravitreal injection of miR-126 plasmid could inhibit retinal neovascularization by down-regulating VEGFA expression, suggesting a potential therapeutic effect for ROP.

Electrochemical immunosensor for serum parathyroid hormone using voltammetric techniques and a portable simulator.

An electrochemical platform based on a screen-printed carbon electrode (SPCE) is developed to detect parathyroid hormone (PTH). A nanocomposite of multi-walled carbon nanotube (MWCNT) and gold nanoparticles (AuNP) was deposited on the SPCE to immobilize antibodies and horseradish peroxidase (HRP). MWCNT improved the stability and conductivity of the immunosensor because of its good electron-transfer ability and tubular structure. The AuNP not only provided a large surface area for antibody immobilization, but it also enhanced the electrochemical signal for enzyme-linked immunosensing. Cyclic voltammetry showed both electron transfer and the effective surface area were increased on the modified electrode. The characteristics of the modified SPCE were assayed by Raman spectroscopy, scanning electron microscopy, atomic force microscopy, and electrochemical techniques. The linear detection range of this PTH immunosensor was within 1-300 pg/ml, and the electrochemical performance was not affected by interference from protein components in human serum. After storage at 4 °C for 28 days, 85% PTH sensing ability of this immunosensor was maintained compared to the freshly prepared one using the SWV and DPV methods. The relative standard deviations of all measurements were within 3-8% for both voltammetric methods. These results indicated the developed immunosensor had good stability and reproducibility. This PTH immunosensor had a detection limit of 0.886 and 0.065 pg/ml for the differential pulse voltammetry and square wave voltammetry, respectively. We provided a quick analysis of serum PTH which might be used as an electrochemical immunosensing platform for point-of-care testing.

Encapsulating curcumin in ethylene diamine-ß-cyclodextrin nanoparticle improves topical cornea delivery.

Curcumin is a powerful scavenger of reactive oxygen species and could prevent the corneal cells from oxidative damage. However, the clinical efficacy of curcumin is limited by its low aqueous solubility and stability, leading to poor bioavailability. ß-cyclodextrin, with a hydrophilic surface and a hydrophobic cavity and self-assembling properties, can form inclusion complexes with lipophilic drugs such as curcumin for ocular delivery. We synthesized ethylene diamine (EDA)-modified ß-cyclodextrin and prepared the curcumin complexation using the solvent evaporation method. The EDA-ß-cyclodextrin provided a better thermodynamic stability and higher complex yield for curcumin complexes, compared to ß-cyclodextrin, which were demonstrated on the analysis of their van't Hoff plots and phase solubility diagrams. We characterized EDA-ß-cyclodextrin curcumin nanoparticles and determined that the EDA modified ß-cyclodextrin is a more suitable carrier than parental ß-cyclodextrin, using FT-IR, XRD, TEM, and analyses of solubility and storage stability. In addition, the curcumin-EDA-ß-cyclodextrin nanoparticles had better in vitro corneal penetration and 3 -h cumulative flux in a porcine cornea experiment, and displayed an improved biocompatibility, confirmed by the histological examination of porcine corneas and cell viability of bovine corneal epithelial cells. These results together revealed a role of EDA modification in the ß-cyclodextrin carrier, including the improvement of curcumin complex formation, thermodynamic properties, cytotoxicity, and the in vitro corneal penetration. The EDA-ß-cyclodextrin inclusion can provide curcumin a higher degree of aqueous solubility and corneal permeability.

Oligochitosan modified albumin as plasmid DNA delivery vector: Endocytic trafficking, polyplex fate, in vivo compatibility.

Cationic macromolecules condense DNA into small nanoparticles and form polyplex. The composition of the polyplex determines the endocytic process, the intracellular routing and the fate of the polyplex. Previously, oligochitosan-modified vectors with different protein moieties are used as gene delivery vector and the types of protein moiety can influence the endosome escape ability and transfection efficiency. Among the modified vectors, oligochitosan-modified bovine serum albumin (BSA) showed 90% transfection efficeincy compared to the modified zein and ovalbumin. These data encouraged us to investigate the mechanism of internalization involved in the superior transfection efficiency of modified BSA/ plasmid polyplex. The effect of specific endocytic inhibitors was studied in two adherent cell lines. The caveolae-mediated and lipid-mediated pathways play a significant role in the polyplex internalization. Next, a colocation of polyplex with lysosome was investigated in the presence of LysoTracker using confocal microscopy. Up to 70% of polyplex successfully escaped the lysosome without degradation. Four non-adherent cell lines showed above than 60% transfection efficiency at an optimized vector/plasmid ratio. Moreover, no significant hemolytic effect was observed up to 500 µg/mL of cationic BSA, indicating no detectable cell membrane disruption. Overall, the hybrid biomacromolecule showed good intracellular delivery and safety in a mice model.

Design and Verification of a Dry Sensor-Based Multi-Channel Digital Active Circuit for Human Brain Electroencephalography Signal Acquisition Systems.

A brain-computer interface (BCI) is a type of interface/communication system that can help users interact with their environments. Electroencephalography (EEG) has become the most common application of BCIs and provides a way for disabled individuals to communicate. While wet sensors are the most commonly used sensors for traditional EEG measurements, they require considerable preparation time, including the time needed to prepare the skin and to use the conductive gel. Additionally, the conductive gel dries over time, leading to degraded performance. Furthermore, requiring patients to wear wet sensors to record EEG signals is considered highly inconvenient. Here, we report a wireless 8-channel digital active-circuit EEG signal acquisition system that uses dry sensors. Active-circuit systems for EEG measurement allow people to engage in daily life while using these systems, and the advantages of these systems can be further improved by utilizing dry sensors. Moreover, the use of dry sensors can help both disabled and healthy people enjoy the convenience of BCIs in daily life. To verify the reliability of the proposed system, we designed three experiments in which we evaluated eye blinking and teeth gritting, measured alpha waves, and recorded event-related potentials (ERPs) to compare our developed system with a standard Neuroscan EEG system.

Multivariate analysis of metabolic parameters and optimization of antibody production using high cell density hybridoma in hollow fiber bioreactors.

OBJECTIVES: The relationships of manipulation of culture temperature and medium circulation rate on the metabolic parameters were regressed by multiple linear regression analysis in hollow fiber bioreactors (HFB). RESULTS: The high circulation rate could significantly enhance the oxygen consumption of the hybridoma cells and the medium's oxidation-reduction potential. A mildly hypothermic condition of 36 °C and a circulation rate of 182.5 mL/min could support the hybridoma had the maximal antibody titer of 60.75 µg/mL for 20 days. When the ammonium ion was 65 ppm or lactate close to 2.6 g/L, the medium was replaced to maintain the stable and healthy cells at the high cell concentration of 3.33 × 108/mL for continuous antibody production. Two serum-free media could be successfully applied to this perfusion system and maintain hybridoma growth and antibody production. CONCLUSION: The single-use HFBs could provide the advantages including high cell density, low shear stress, and continuous antibody production.

Biocompatible quantum dot-antibody conjugate for cell imaging, targeting and fluorometric immunoassay: crosslinking, characterization and applications.

Quantum dots (QDs) are important fluorescent probes that offer great promise for bio-imaging research due to their superior optical properties. However, QDs for live cell imaging and the tracking of cells need more investigation to simplify processing procedures, improving labeling efficiency, and reducing chronic toxicity. In this study, QDs were functionalized with bovine serum albumin (BSA) via a chemical linker. Anti-human immunoglobulin antibodies were oxidized by sodium periodate to create reactive aldehyde groups for a spontaneous reaction with the amine groups of BSA-modified QDs. An antibody-labeled QD bioconjugate was characterized using agarose gel electrophoresis, dynamic light scattering, and zeta potential. Using fluorescence spectroscopy, we found that the fluorescence of QDs was retained after multiple conjugation steps. The cell-labeling function of the QD bioconjugate was confirmed using an image analyzer and confocal microscopy. The QD bioconjugate specifically targeted human immunoglobulin on the membrane surface of recombinant cells. In addition, the QD bioconjugate applied in fluorometric immunoassay was effective for the quantitative analysis of human immunoglobulin in an enzyme-linked immunosorbent assay. The developed QD bioconjugate may offer a promising platform to develop biocompatible tools to label cells and quantify antibodies in the immunoassay.

Facile development of medium optimization for antibody production: implementation in spinner flask and hollow fiber reactor.

Most bio-industrial mammalian cells are cultured in serum-free media to achieve advantages, such as batch consistency, suspended growth, and simplified purification. The successful development of a serum-free medium could contribute to a reduction in the experimental variation, enhance cell productivity, and facilitate biopharmaceuticals production using the cell culture process. Commercial serum-free media are also becoming more and more popular. However, the cell line secrets its own recombinant product and has special nutritional requirements. How can the composition of the proprietary medium be adjusted to support the specific cell's metabolism and recombinant protein? This article uses statistical strategies to modify the commercial medium. A design of experiments is adopted to optimize the medium composition for the hybridoma cell in a serum-free condition. The supplements of peptone, ferric citrate, and trace elements were chosen to study their impact on hybridoma growth and antibody production using the response surface methodology. The stimulatory effect of the developed formulation on hybridoma growth was confirmed by the steepest ascent path. The optimal medium stimulated the hybridoma growth and antibody production in three diverse systems: a static plate, an agitated spinner flask, and a hollow fiber reactor. The cells in the developed serum-free medium had a better antibody production as compared to that in the commercial medium in the hollow fiber reactor. Our results demonstrated that the facile optimization for medium and antibody production was successfully accomplished in the hybridoma cells.

Protein moiety in oligochitosan modified vector regulates internalization mechanism and gene delivery: Polyplex characterization, intracellular trafficking and transfection.

Oligochitosan-modified proteins have gained attention as efficient non-viral vectors for gene delivery. However, little information exists if protein moieties can serve as an important role for internalization and endosome escape ability of the genetic material. To explore this issue, we designed two cationic oligochitosan-modified vectors that consist of different proteins, namely a hydrophobic plant protein (zein) and a hydrophilic animal protein (ovalbumin (OVA)) to deliver pDNA to epithelial cell line CHO-K1 and HEK 293 T. These cationic vectors were systematically characterized by molecular weight, infrared (IR) structural analysis, transmission electron microscopy (TEM) morphology, and surface charge. A remarkable impact of protein moieties was observed on physiochemical properties of the developed vectors. Oligochitosan-modified zein containing hydrophobic protein exhibited high buffering capacity and excellent DNA binding ability compared to the oligochitosan-modified OVA. The data on transfection in the presence of endocytic inhibitors indicated that the caveolae-mediated pathway (CvME) played a key role in the internalization of the zein-based polyplex. However, the OVA-based polyplex was internalized in CHO-K1 cells via CvME and in HEK 293 T cells via the lipid-mediated pathway. Moreover, oligochitosan-modified zein exhibited lower cytotoxicity, greater lysosomal escape ability, better plasmid stability, and better transfection efficiency than the oligochitosan-modified OVA. This study offers a facile procedure for the synthesis of cationic vectors and elucidates the relationship that exists between protein moieties and transfection activity, thus providing an alternative, non-viral platform for the gene delivery.

The Preparation of Graphene Oxide-Silver Nanocomposites: the Effect of Silver Loads on Gram-Positive and Gram-Negative Antibacterial Activities.

In this work, silver nanoparticles (Ag NPs) were decorated on thiol (-SH) grafted graphene oxide (GO) layers to investigate the antibacterial activities in Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Pseudomonas aeruginosa). The quasi-spherical, nano-sized Ag NPs were attached to the GO surface layers, as confirmed by using field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM), respectively. The average size of GO-Ag nanocomposites was significantly reduced (327 nm) from those of pristine GO (962 nm) while the average size of loaded Ag NPs was significantly smaller than the Ag NPs without GO. Various concentrations of AgNO3 solutions (0.1, 0.2, and 0.25 M) were loaded into GO nanosheets and resulted in the Ag contents of 31, 43, and 65%, respectively, with 1-2 nm sizes of Ag NPs anchored on the GO layers. These GO-Ag samples have negative surface charges but the GO-Ag 0.2 M sample (43% Ag) demonstrated the highest antibacterial efficiency. At 10 ppm load of GO-Ag suspension, only a GO-Ag 0.2 M sample yielded slight bacterial inhibition (5.79-7.82%). As the GO-Ag content was doubled to 20 ppm, the GO-Ag 0.2 M composite exhibited ~49% inhibition. When the GO-Ag 0.2 M composite level was raised to 100 ppm, almost 100% inhibition efficiencies were found on both Staphylococcus aureus (S.A.) and Pseudomonas aeruginosa (P.A.), which were significantly higher than using pristine GO (27% and 33% for S.A. and P.A.). The combined effect of GO and Ag nanoparticles demonstrate efficient antibacterial activities.

Efficient gene delivery by oligochitosan conjugated serum albumin: Facile synthesis, polyplex stability, and transfection.

Chitosan and its derivatives have shown to be potential gene carriers with biocompatiblility and safety. However, their practical delivery is far from being ideal because of the low transfection efficiency. The present work describes the potential of a natural protein, bovine serum albumin (BSA), conjugated with a natural oligosaccharide, oligochitosan (OC), as a considerable promising approach for a safe and efficient non-viral gene delivery vector. The FTIR spectra proved the effective conjugation of BSA with OC through covalent bond. The condensation ability of plasmid DNA (pDNA) with a BSA-OC biopolymer was analyzed by gel retardation assay, competition binding assay, and dynamic light scattering used to measure the nanoparticle size. In addition, the BSA-OC biopolymer showed the protection of pDNA from enzymatic degradation by DNase I and showed good stability when exposed to 50% fetal bovine serum. The transfection efficiency was evaluated in the presence of 10% serum-supplemented media or serum-free media on three kinds of mammalian cells. Our results showed that the BSA-OC biopolymer is a good non-viral vehicle for gene delivery. We investigated the parameters such as the pDNA payload, temperature, incubating duration, and biopolymer/pDNA ratio on the transfection efficiency. This hybrid vehicle had the ability to transfect 90% of cells and to maintain 80% of cell viability. The aforementioned results suggest that the facile synthesis of the BSA-OC biopolymer could overcome the cytotoxicity problem and transfection barriers during in vitro gene delivery.

Increased anti-biofilm efficacy of toluidine blue on Staphylococcus species after nano-encapsulation.

BACKGROUND: Photodynamic therapy has been studied as a method for inactivating bacterial growth. Workers have used planktonic bacterial as well as biofilm bacterial cultures to evaluate the potential of photodynamic therapy in inactivating bacteria. However, almost all the studies use a photosensitiser in aqueous solution, which could be detrimental to the efficiency of photodynamic therapy. METHODS: In this study, the photodynamic killing effect of toluidine blue O (TBO) has been investigated on Staphylococcal biofilms in-vitro. The sensitivity of the in-vitro biofilms to photodynamic killing action was compared using different formulations of TBO, different dosages of photosensitiser and different light irradiation strengths. Effect of TBO formulations on bacterial quorum sensing system was evaluated using a colorimetric assay. Finally, dual staining using hoechst and propidium iodide stains was carried out on the photodynamically treated biofilms to visualise and compare the effects of photodynamic therapy. Scanning electron microscope imagery was also carried out to evaluate the photodynamic killing effect on the in-vitro biofilms. RESULTS: The sensitivity of biofilms to the photodynamic killing effect increased proportionally with the photosensitiser dosage and the light irradiation duration. TBO encapsulated in microemulsion was more effective in killing the biofilm bacteria than only TBO in water. The combination of TBO in microemulsion with EDTA was another effective way of increasing the photodynamic killing effect on the bacterial biofilms. Effect of encapsulated TBO on the quorum sensing system of bacteria was greater than the effect of aqueous solution of TBO. The in-vitro Staphylococcal biofilms could thus be inhibited by the photodynamic effect, and TBO encapsulated in microemulsion was much more effective than only TBO in water. CONCLUSIONS: The encapsulation of a photosensitiser is an effective way of increasing the likelihood of the complete and successful inactivation of the biofilm growth. The encapsulated photosensitiser achieves higher inactivation of the bacterial biofilm than that of the aqueous solution of a photosensitiser.

Systemic effects after intravitreal injection of bevacizumab in new born rabbit eyes.

PURPOSE: To determine the systemic impact of intravitreal injection of bevacizumab (IVB), an anti-vascular endothelium growth factor antibody, in newborn rabbits. MATERIALS AND METHODS: We used four groups of rabbits. Group 1 rabbits received a single injection of IVB starting from the age of 6 weeks. Group 2 rabbits received a single injection of balanced salt solution (BSS, 0.025 ml) and served as controls for group 1. Group 3 rabbits received two consecutive injections of IVB at the ages of 6 and 10 weeks. Group 4 rabbits received two consecutive injections of BSS at the ages of 6 and 10 weeks and served as controls for group 3. During the experiment, a complete blood count (CBC), clinical biochemistry, weight gain, food intake, body temperature, blood pressure, pulse, and mortality were measured in the animals. Two months after IVB injection, the animals were sacrificed, and histology of the major organs was checked. Immunohistochemistry was assessed to explore the neurons in the central nervous system (CNS). RESULTS: We found there were no morphological or functional changes in the eyes following IVB injection. Furthermore, there were no differences in CBC, biochemistry, or other measured parameters among the four groups of animals. We checked the histology of the major organs and neurons in the CNS and they did not reveal significant differences among the four groups of animals. CONCLUSIONS: Conclusively, IVB of either one or two injections (0.625 mg) in newborn rabbit eyes is well tolerated and does not cause noticeable systemic organ pathology.

Photosensitizer in lipid nanoparticle: a nano-scaled approach to antibacterial function.

Photosensitization-based antimicrobial therapy (PAT) is an alternative therapy aimed at achieving bacterial inactivation. Researchers use various photosensitizers to achieve bacterial inactivation. However, the most widely used approach involves the use of photosensitizers dispersed in aqueous solution, which could limit the effectiveness of photodynamic inactivation. Therefore, the approaches to encapsulate the photosensitizer in appropriate vehicles can enhance the delivery of the photosensitizer. Herein, Toluidine Blue O (TBO) was the photosensitizer, and lipid nanoparticles were used for its encapsulation. The lipid nanoparticle-based delivery system has been tailor-made for decreasing the average size and viscosity and increasing the formulation stability as well as the wettability of skin. Usage of an appropriate vehicle will also increase the cellular uptake of the photosensitizer into the bacterial cells, leading to the damage on cell membrane and genomic DNA. Evidence of effectiveness of the developed PAT on planktonic bacteria and biofilms was examined by fluorescence microscopy and scanning electron microscopy. Lipid nanoparticles protected the photosensitizer from aggregation and made the application easy on the skin as indicated in data of size distribution and contact angle. The use of lipid nanoparticles for encapsulating TBO could enhance photosensitization-based antimicrobial therapy as compared to the aqueous media for delivering photosensitizers.