Tareq Youssef

Biophysical characterization of gold nanoparticles-loaded liposomes

Gold nanoparticles were prepared and loaded into the bilayer of dipalmitoylphosphatidylcholine (DPPC) liposomes, named as gold-loaded liposomes. Biophysical characterization of gold-loaded liposomes was studied by transmission electron microscopy (TEM) and Fourier transform infrared (FTIR) spectroscopy as well as turbidity and rheological measurements. FTIR measurements showed that gold nanoparticles made significant changes in the frequency of the CH(2) stretching bands, revealing that gold nanoparticles increased the number of gauche conformers and create a conformational change within the acyl chains of phospholipids. The transmission electron micrographs (TEM) revealed that gold nanoparticles were loaded in the liposomal bilayer. The zeta potential of DPPC liposomes had a more negative value after incorporating of Au NPs into liposomal membranes. Turbidity studies revealed that the loading of gold nanoparticles into DPPC liposomes results in shifting the temperature of the main phase transition to a lower value. The membrane fluidity of DPPC bilayer was increased by loading the gold nanoparticles as shown from rheological measurements. Knowledge gained in this study may open the door to pursuing liposomes as a viable strategy for Au NPs delivery in many diagnostic and therapeutic applications.

Evaluation of hypericin-loaded solid lipid nanoparticles: Physicochemical properties, photostability and phototoxicity

Hypericin (HYP), a natural photosensitizer, has powerful photo-oxidizing ability, tumor-seeking characteristics, and minimal dark toxicity; nevertheless, it has proven high lipid solubility compared to its sparingly water soluble nature. Therefore, its formulation into solid lipid nanoparticles (SLNs) has attracted increasing attention as a potential drug-delivery carrier. Two HYP-loaded SLNs formulations were prepared utilizing microemulsion-based technique. Thereafter, the physicochemical properties of the formulations were investigated and evaluated. HYP-loaded SLNs showed spherical shape with mean particle size ranging from 200–300 nm for both formulations (FA and FB). The encapsulation efficiencies reached above 80% and FA showed significant higher encapsulation than FB (P < 0.05), also, the thermal analysis using differential scanning calorimetry (DSC) indicated good compatibility between hypericin and lipids forming the cores in both formulations. Spectroscopic measurements of the photostability study showed that hypericin encapsulation into SLNs improved its photostability, compared to free HYP in 0.1% ethanolic solution. However, photocytotoxicity studies on HepG2 cells revealed an evident inhibition of the photodynamic efficacy of HYP-loaded SLNs, compared to free HYP. In conclusion, although the elevated entrapment efficiency of HYP into SLNs increased its photostability, it decreased its phototoxicity which might be due to the quenching deactivation of HYP molecules resulting from SLN compactness and thickness structure.

Photosensitized Structural Modifications of the Lens Protein α-Crystallin: Do All Modifications Impair Chaperone-like Activity?¶

Among chaperone‐like functioning proteins, the lens α‐crystallins are of particular interest because they are not renewed, and even minor alterations can hurt their function of maintaining the proper refractive index and avoiding cataract formation in the lens. Several reports have suggested the occurrence of remarkable structural modifications in lens proteins in the presence of endogenous and exogenous sensitizers upon exposure to light. In particular, it has been shown in vitro that hypericin, the active ingredient of Hypericum, can bind to and, in the presence of light, cause the photopolymerization of α‐crystallin. On the basis of these results it has also been suggested that a subsequent significant impairment of the protein function can occur. Using absorption and emission spectroscopic techniques, as well as circular dichroism, we have studied the structural modifications of α‐crystallin resulting from its interaction with hypericin after irradiation with visible light. To investigate the chaperone‐like function of α‐crystallin, the heat‐induced aggregation kinetics of another lens protein, βLow‐crystallin, was monitored by measuring the apparent absorption due to scattering at 360 nm as a function of time, and no apparent damage to its functional role was observed. Spectroscopic results, on the contrary, show a prominent reduction in both tryptophan and hypericin fluorescence emission intensity after light irradiation, suggesting an alteration in the tryptophan microenvironment and a high degree of packing of the chromophore due to photoinduced modification of the molecular framework. Control experiments on α‐crystallin structurally modified by light in the presence of hypericin indicated that the protein still retains its ability to chaperone both lens crystallins and insulin.

Application of quercetin and its bio-inspired nanoparticles as anti-adhesive agents against Bacillus subtilis attachment to surface

The aim of this study was directed to reveal the repulsive effect of coated glass slides by quercetin and its bio-inspired titanium oxide and tungsten oxide nanoparticles on physical surface attachment of Bacillus subtilis as an ab-initio step of biofilm formation. Nanoparticles were successfully synthesized using sol-gel and acid precipitation methods for titanium oxide and tungsten oxide, respectively (in the absence or presence of quercetin). The anti-adhesive impact of the coated-slides was tested through the physical attachment of B. subtilis after 24h using Confocal Laser Scanning Microscopy (CLSM). Here, quercetin was presented as a bio-route for the synthesis of tungsten mixed oxides nano-plates at room temperature. In addition, quercetin had an impact on zeta potential and adsorption capacity of both bio-inspired amorphous titanium oxide and tungsten oxide nano-plates. Interestingly, our experiments indicated a contrary effect of quercetin as an anti-adhesive agent than previously reported. However, its bio-inspired metal oxide proved their repulsive efficiency. In addition, quercetin-mediated nano-tungsten and quercetin-mediated amorphous titanium showed anti-adhesive activity against B. subtilis biofilm.

Fluorescence study on the interaction between hypericin and lens protein "α-crystallin".

Hypericin has been reported as a potent photosensitizing agent exhibiting antiviral, antibacterial, antineoplastic activities. Although its photophysics and mode of action are strongly modulated by the binding protein, detailed information about its mechanism of interaction with possible cellular targets, including proteins, is still lacking. Previous in vitro studies demonstrated that hypericin can be uptaken by intact lens and is able to bind to the major lens protein “α‐crystallin.” In this study, the mechanism of interaction of this potent drug with α‐crystallin was studied using the chemical denaturant guanidine hydrochloride (GdnHCl) and the hydrophobic surface probe, 8‐anilino‐1‐naphthalenesulfonic acid (ANS). Fluorescence measurements showed that the increased exposure of tryptophan resulting from partial unfolding of α‐crystallin incubated with 1.0 mol L−1 of GdnHCl corresponds to the maximum accessibility of hydrophobic sites to ANS at the same GdnHCl concentration. Interestingly at this additional hydrophobicity of the protein, hypericin exhibited its maximum fluorescence intensity. This in vitro study implied that hydrophobic sites of α‐crystallin play a significant role in its interaction with hypericin. The binding between α‐crystallin and hypericin was found to be enhanced by partial perturbation of the protein.

Role of ZnS shell on stability, cytotoxicity, and photocytotoxicity of water-soluble CdSe semiconductor quantum dots surface modified with glutathione

Abstract. The present study examines the stability and cytotoxicity of two quantum dots (QDs) systems in cell culture medium in the presence and absence of a thin layer of a ZnS shell. The two systems were built from a core, CdSe, and surface modified with glutathione (GSH), named CdSe∼GSH and CdSe/ZnS∼GSH. CdSe/ZnS∼GSH QDs exhibited a high photostability with a pronounced enhancement in photoluminescence in cell culture medium. Both systems showed insignificant reduction in cell viability of HFB-4 and MCF-7 cell lines in the dark. Following 60 min of low laser power exposure (irradiance of 10  mW cm−2), CdSe∼GSH QDs showed a remarkable decrease in cell viability, which may result from the detachment of GSH molecules, whereas CdSe/ZnS∼GSH QDs showed an insignificant decrease either immediately after irradiation or even 2 h post-exposure, which can be attributed to the high affinity between ZnS and GSH coatings. This study demonstrated that a thin layer of ZnS shell played a crucial role in the stability of CdSe/ZnS∼GSH QDs in cell culture medium with an improvement in luminescence efficiency, whereas surface modification with GSH molecules in the presence of ZnS showed no significant cytotoxic effects before or after photoirradiation, which makes this system attractive for several biomedical applications.

Photodynamic efficacy of hypericin targeted by two delivery techniques to hepatocellular carcinoma cells

The photocytotoxic effect of hypericin (Hyp) targeted by two different delivery techniques, namely, liposomes and anti-hepatocyte specific antigen (anti-HSA) was investigated. Optical absorption and steady-state fluorescence were used to analyze the conjugation of Hyp with anti-HSA model and to evaluate the encapsulation capacity and drug release in a liposome model. Particle size and thermal analysis of the prepared liposomes were performed using laser-light scattering and differential scanning calorimetry (DSC), respectively. Viability study of HepG2 cells exposed to Hyp in the two delivery systems, in the dark and following visible light irradiation, was performed in comparison to free Hyp. The intracellular uptake and localization of Hyp in HepG2 cells were analyzed by means of spectrofluorometry and fluorescence microscopy. Spectroscopic measurements demonstrated that Hyp binds to anti-HSA in its monomeric form. The photocytotoxic effect of Hyp depended clearly on the form of Hyp administered, either in free form, loaded into liposomes or conjugated with anti-HSA. While liposomes loaded with Hyp (Lip-Hyp) did not induce significant phototoxicity, both free Hyp or anti-HSA-Hyp inflicted substantial cell mortality, after photoirradiation. The intracellular uptake of Lip-Hyp by HepG2 cells was estimated to be 20% less compared to free Hyp or anti-HSA-Hyp. In spite of the equal uptake of both free Hyp and anti-HSA-Hyp, HepG2 cells demonstrated a relatively higher mortality with anti-HSA-Hyp compared to free Hyp.

Photosensitized Effects of Rose Bengal on Structure and Function of Lens Protein “Alpha‐Crystallin”

The conformational changes of the bovine lens protein “α‐crystallin” have been investigated in the presence of the photosensitizer Rose Bengal (RB), in the dark as well as after visible light irradiation. Absorption and fluorescence emission spectra of RB [5 × 10−6 m] and Fourier transform‐IR spectra of α‐crystallin [5 mg mL−1] were significantly altered upon RB α‐crystallin complex formation. RB was found to bind to α‐crystallin in a molecular pocket characterized by a low polarity, with Trp most likely involved in this interaction. The binding constant (Kb) has been estimated to be of the order of 2.5 (mg/mL)−1. The intrinsic fluorescence of α‐crystallin was quenched through both dynamic and static mechanisms. Light‐induced photosensitized effects showed structural modifications in α‐crystallin, including tertiary and secondary structure (an increase in unordered structure) alterations. Notwithstanding those photoinduced structural variations detected in α‐crystallin when complexed with RB, the protein still retains its ability to play the role of chaperone for β‐crystallin.

Gelatin nanoparticles enhance delivery of hepatitis C virus recombinant NS2 gene

Background Development of an effective non-viral vaccine against hepatitis C virus infection is of a great importance. Gelatin nanoparticles (Gel.NPs) have an attention and promising approach as a viable carrier for delivery of vaccine, gene, drug and other biomolecules in the body. Aim of work The present study aimed to develop stable Gel.NPs conjugated with nonstructural protein 2 (NS2) gene of Hepatitis C Virus genotype 4a (HCV4a) as a safe and an efficient vaccine delivery system. Methods and results Gel.NPs were synthesized and characterized (size: 150±2 nm and zeta potential +17.6 mv). NS2 gene was successfully cloned and expressed into E. coli M15 using pQE-30 vector. Antigenicity of the recombinant NS2 protein was confirmed by Western blotting to verify the efficiency of NS2 as a possible vaccine. Then NS2 gene was conjugated to gelatin nanoparticles and a successful conjugation was confirmed by labeling and imaging using Confocal Laser Scanning Microscope (CLSM). Interestingly, the transformation of the conjugated NS2/Gel.NPs complex into E. coli DH5-α was 50% more efficient than transformation with the gene alone. In addition, conjugated NS2/Gel.NPs with ratio 1:100 (w/w) showed higher transformation efficiency into E. coli DH5-α than the other ratios (1:50 and 2:50). Conclusion Gel.NPs effectively enhanced the gene delivery in bacterial cells without affecting the structure of NS2 gene and could be used as a safe, easy, rapid, cost-effective and non-viral vaccine delivery system for HCV.

Photo-extracellular synthesis of gold nanoparticles using Baker's yeast and their anticancer evaluation against Ehrlich ascites carcinoma cells

The chemical methods for the synthesis of gold nanoparticles lead to the formation of some toxic chemicals adsorbed on the surface that may have adverse effects on their medical applications. Hence, the need to develop environmentally benign nanoparticles has attracted growing interest. Gold nanoparticles with 13.0 ± 0.9 nm size have been biosynthesized using an aqueous extract of Bakers yeast (Saccharomyces cerevisiae) under visible light. The existing components in the aqueous yeast extract were identified for the first time as trimethylsilyl derivatives of butan-2,3-diol, glucose, indole-3-acetic acid and undecanoic acid. This extract acts as a capping and reducing agent for gold nanoparticles. The cytotoxicity of the biosynthesized gold nanoparticles, chemically synthesized gold nanoparticles of ∼5.0 ± 2.0 nm size and aqueous extract of Bakers yeast towards Ehrlich ascites carcinoma cancer cells has been studied using the Trypan blue exclusion method. The results show that the killed percentage of Ehrlich ascites carcinoma cells under dark incubation with an aqueous extract of Bakers yeast, chemically synthesized gold nanoparticles and the photo-biosynthesized gold nanoparticles are 9.7%, 12.5% and 24.6%, respectively. These percentages are increased to 10.63%, 60.6% and 86.5% under visible light incubation, respectively. The killing enhancement of the Ehrlich ascites carcinoma cells under visible light incubation is proposed based on the photothermal properties of the formed plasmonic gold nanoparticles that conjugated with anti-epidermal growth factor receptor antibodies beside the phagocytosis of the excess yeast extract that is present in the photo-extracellular synthesized gold nanoparticles sample. The combination between two different treatment ways can give a third one with high affinity for the treatment of cancer.