Liposome bilayer stability: emphasis on cholesterol and its alternatives.

Liposomes are spherical lipidic nanocarriers composed of natural or synthetic phospholipids with a hydrophobic bilayer and aqueous core, which are arranged into a polar head and a long hydrophobic tail, forming an amphipathic nano/micro-particle. Despite numerous liposomal applications, their use encounters many challenges related to the physicochemical properties strongly affected by their constituents, colloidal stability, and interactions with the biological environment. This review aims to provide a perspective and a clear idea about the main factors that regulate the liposomes' colloidal and bilayer stability, emphasising the roles of cholesterol and its possible alternatives. Moreover, this review will analyse strategies that offer possible approaches to provide more stable in vitro and in vivo liposomes with enhanced drug release and encapsulation efficiencies.

Jordanian Kaolinite with TiO2 for Improving Solar Light Harvesting Used in Dye Removal.

TiO2-Kaolinite nanocomposite photocatalysts were synthesized using the sol-gel method, with titanium isopropoxide/HCl as reactants and Jordanian kaolinite clay as a support material. The samples' TiO2 content ranged from 10% to 70% (m/m). TiO2-Kaolinite composites were characterized using FTIR, SEM, XRF, and XRD. According to XRD measurements of the nano-composite samples, the intensity of the anatase peaks increased as the TiO2 percentage of the composition increased. As the percentage of TiO2 increased, so did the peaks of Ti-O-Si in FTIR. The extent of photocatalytic degradation of Congo-red dye was used to evaluate the photocatalytic activity of the prepared nanocomposites. After four hours under the sun, the percentage of Congo-red degradation ranged from 27 to 99 percent depending on the TiO2 content of the used nanocomposite. Meanwhile, the concentration drop in the dark did not exceed 10%. Photodegradation outperforms traditional treatment methods in terms of target degradation. Using naturally abundant materials such as clay in conjunction with metal oxides is widely regarded as an effective method of modifying the photoresponse properties of TiO2 particles, thereby improving solar light harvesting for target degradation.

Liposomes: structure, composition, types, and clinical applications.

Liposomes are now considered the most commonly used nanocarriers for various potentially active hydrophobic and hydrophilic molecules due to their high biocompatibility, biodegradability, and low immunogenicity. Liposomes also proved to enhance drug solubility and controlled distribution, as well as their capacity for surface modifications for targeted, prolonged, and sustained release. Based on the composition, liposomes can be considered to have evolved from conventional, long-circulating, targeted, and immune-liposomes to stimuli-responsive and actively targeted liposomes. Many liposomal-based drug delivery systems are currently clinically approved to treat several diseases, such as cancer, fungal and viral infections; more liposomes have reached advanced phases in clinical trials. This review describes liposomes structure, composition, preparation methods, and clinical applications.

Cinnamaldehyde-cucurbituril complex: investigation of loading efficiency and its role in enhancing cinnamaldehyde in vitro anti-tumor activity.

This study aimed to clarify the physico-chemical properties of cucurbit[7]uril (CB[7]) and cinnamaldehyde (Cinn) inclusion complexes (CB[7]-Cinn) and their resulting antitumor activity. CB[7]-Cinn inclusion complexes were prepared by a simple experimental approach and fully characterized for their stoichiometry, formation constant, particle size and morphology. Quantum chemical calculations were performed to elucidate the stable molecular structures of the inclusion complexes and their precursors and to investigate the probable stoichiometry and direction of interaction using three different DFT functionals at the 6-31G(d,p) basis set. The UV-vis spectrophotometric titrations as well as the Job plot, based on 1H NMR spectroscopy, suggested 1 : 1 and 1 : 2 stoichiometries of CB[7] : Cinn. The formation constants of the complexes were calculated using Benesi-Hildebrand equations and non-linear fittings. Moreover, the theoretical calculations confirmed the potential formation of 1 : 1 and 1 : 2 stoichiometries and clarify the orientation of binding from the Cinn phenyl moiety. The nanoparticles' TEM images showed a crystal-like spherical shape, smooth surface, with a small tendency to agglomerate. CB[7]-Cinn inclusion complexes were analyzed for their antitumor activity against MDA-MB-231 breast cancer and U-87 glioblastoma cell lines. The IC50 values were calculated after 72 hours of incubation with different concentrations of CB[7]-Cinn inclusion complexes and compared to free Cinn and free CB[7]. The IC50 values for free Cinn and CB[7]-Cinn inclusion complexes were 240.17 ± 32.46 µM and 260.47 ± 20.83 µM against U-87 cells and 85.93 ± 3.35 µM and 176.3 ± 7.79 µM against MDA-MB-231 cells, respectively, despite the enhanced aqueous solubility. No significant cytotoxicity was noticed for the free CB[7].

Preparation, characterization, and biological activity study of thymoquinone-cucurbit[7]uril inclusion complex.

In this study, the formation of a host-guest inclusion complex between cucurbit[7]uril (CB[7]) and thymoquinone (TQ) was investigated in aqueous solution. The formation of a stable inclusion complex, CB[7]-TQ, was confirmed by using different techniques, such as 1H NMR and UV-visible spectroscopy. The aqueous solubility of TQ was clearly enhanced upon the addition of CB[7], which provided an initial indication for supramolecular complexation. The complexation stoichiometry and the binding constant of the inclusion complex were determined through a combination of two sets of titration methods, including UV-visible and fluorescence displacement titrations. Both methods suggested the formation of a 1 : 1 stoichiometry between CB[7] and TQ with moderate binding affinity of 3 × 103 M-1. Density functional theory (DFT) calculations were also performed to verify the structure of the resulted host-guest complex and to support the complexation stoichiometry. The theoretical calculations were in agreement with experimental results obtained by 1H NMR spectroscopy. Most importantly, the cytotoxic effect of the CB[7]-TQ complex was investigated against cancer and normal cell lines. The results showed that the anticancer activity of TQ against MDA-MB-231 cells was enhanced by the complexation with CB[7], while no significant effect was observed in MCF-7 cells. The results also confirmed the low toxicity of the CB[7] host molecule that supports the use of CB[7] as a drug carrier.

Synthesis of Mono-Amino Substituted γ-CD: Host-Guest Complexation and In Vitro Cytotoxicity Investigation.

Cyclodextrins (CDs) are cyclic oligosaccharides which can trap hydrophobic molecules and improve their chemical, physical, and biological properties. γ-CD showed the highest aqueous solubility with the largest cavity diameter among other CD types. The current study describes a direct and easy method for nucleophilic mono-aminos to be substituted with γ-CD and tested for their ability to host the guest curcumin (CUR) as a hydrophobic drug model. The mass spectrometry and NMR analyses showed the successful synthesis of three amino-modified γ-CDs: mono-6-amino-6-deoxy-cyclodextrine (γ-CD-NH2), mono-6-deoxy-6-ethanolamine-γ-cyclodextrine (γ-CD-NHCH2CH2OH), and mono-6-deoxy-6-aminoethylamino)-γ-cyclodextrin (γ-CD-NHCH2CH2NH2). These three amino-modified γ-CDs were proven to be able to host CUR as native γ-CDs with formation constants equal to 6.70 ± 1.02, 5.85 ± 0.80, and 8.98 ± 0.90 mM-1, respectively. Moreover, these amino-modified γ-CDs showed no significant toxicity against human dermal fibroblast cells. In conclusion, the current work describes a mono-substitution of amino-modified γ-CDs that can still host guests and showed low toxicity in human dermal fibroblasts cells. Therefore, the amino-modified γ-CDs can be used as a carrier host and be conjugated with a wide range of molecules for different biomedical applications, especially for active loading methods.

Green Synthesis of Silver Nanoparticles as an Effective Antibiofouling Material for Polyvinylidene Fluoride (PVDF) Ultrafiltration Membrane.

Silver nanoparticles (AgNPs) were successfully synthesized using the aqueous extract of the Paronychia argentea Lam (P. argentea) wild plant. The results showed that the conversion of Ag+ to Ag0 nanoparticles ratio reached 96.5% as determined by Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES), with a negative zeta potential (ζ) of -21.3 ± 7.68 mV of AgNPs expected to improve the stability of synthesized AgNPs. AgNP antibacterial activity has been examined against Streptococcus aureus (S. aureus) and Escherichia coli (E. coli) bacteria. The minimum inhibition concentration (MIC) was 4.9 µL/mL for both E. coli and S. aureus bacteria, while the minimum bactericidal concentrations (MBC) were 19.9 µL/mL and 4.9 µL/mL for S. aureus and E. coli, respectively. The synthesized AgNPs were incorporated in ultrafiltration polyvinylidene Fluoride (PVDF) membranes and showed remarkable antibiofouling behavior against both bacterial strains. The membranes were characterized using Fourier transform infrared spectroscopy (FTIR), scanning electron microscope (SEM), and X-ray diffraction (XRD). The contact angle and porosity of the membrane were also determined. The efficiency of the membranes regarding rejection rate was assessed using bovine serum albumin (BSA). It was found in the flux experiments that membranes BSA rejection was 99.4% and 98.7% with and without AgNPs, respectively.

Lipid nanostructures for targeting brain cancer.

Advancements in both material science and bionanotechnology are transforming the health care sector. To this end, nanoparticles are increasingly used to improve diagnosis, monitoring, and therapy. Huge research is being carried out to improve the design, efficiency, and performance of these nanoparticles. Nanoparticles are also considered as a major area of research and development to meet the essential requirements for use in nanomedicine where safety, compatibility, biodegradability, biodistribution, stability, and effectiveness are requirements towards the desired application. In this regard, lipids have been used in pharmaceuticals and medical formulations for a long time. The present work focuses on the use of lipid nanostructures to combat brain tumors. In addition, this review summarizes the literature pertaining to solid lipid nanoparticles (SLN) and nanostructured lipid carriers (LNC), methods of preparation and characterization, developments achieved to overcome blood brain barrier (BBB), and modifications used to increase their effectiveness.

Fabrication of a novel nontoxic trichlorophenol-epichlorohydrin-based compound with high antimicrobial activity and thermal stability.

The aim of this study was to modify a discontinued, toxic antiseptic agent 2,4,5-trichlorophenol (TCP) by reacting it with epichlorohydrin (ECH) to obtain a nontoxic novel compound with similar antimicrobial effectiveness. A novel compound named {[1,3-bis(2,4,5-trichlorophenoxy) propan-2-yl] oxy}-3-(2,4,5-trichlorophenoxy) hexan-2-ol (TPTH) was synthesized from this reaction. Chemical and physical structures of the product were characterized by FTIR, MS, Uv-vis, NMR, SEM and TEM. The thermal stability of TPTH was evaluated by conducting thermogravimetric analysis. Biological interactions of the compound were investigated by performing antimicrobial activity and cytotoxicity assays. The compound displayed a good antimicrobial activity where minimum inhibitor concentrations were found to be 0.02, 0.08, and 0.15 µg mL-1 against Staphylococcus aureus (S. aureus), Methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli) respectively. Additionally, well diffusion assay demonstrated that, the zone of inhibitions for S. aureus, MRSA and E. coli were 24 mm, 22 mm and 18 mm, respectively. Cytotoxicity assay results revealed that TPTH is nontoxic against cells at effective anti-microbial concentrations. TPTH shows thermal stability up to 220 °C. Results here demonstrate the successful conversion of toxic TCP to a nontoxic form; TPTH with a good anti-microbial activity and thermal stability.

Grafting of anti-nucleolin aptamer into preformed and remotely loaded liposomes through aptamer-cholesterol post-insertion.

A new combination strategy of an active loading and active targeting approach was applied in this work. The liposomes actively loaded with Curcumin (CRM) (LipCRM) were decorated with cholesterol tagged-anti-nucleolin AS1411 aptamer (NCL) via a new post-insertion approach, utilizing the cholesterol as a wedge to incorporate aptamer into the surface of the liposome bilayer. A successful NCL post-insertion was verified by agarose gel electrophoresis and dynamic light scattering (DLS). The cellular uptake of AptNCL-Lip was investigated using flow cytometry and Confocal Laser Scanning Microscopy (CLSM) on two different human breast cancer cell lines (MCF-7 and MDA-MB-231). The uptake and cytotoxicity of loaded CRM were investigated using flow cytometry and MTT assay. Our results showed successful post insertion of NCL aptamer to the surface of Lip. Also, higher cellular uptake was noted for AptNCL-Alexa-LipRhod compared to blank LipRhod in both cell lines. Moreover, CLSM showed prominent endocytosis and uptake of AptNCL-Alexa-LipRhod into the cytoplasm of breast cancer cells. Furthermore, the results showed a significant increase in the uptake and cytotoxicity of AptNCL-LipCRM compared to LipCRM in both cell lines. Overall, our results demonstrate a successful post-insertion of cholesterol-tagged aptamer into liposomes and the possible combination between active loading and active targeting.

Developing and Characterization of Chemically Modified RNA Aptamers for Targeting Wild Type and Mutated c-KIT Receptor Tyrosine Kinases.

The c-KIT receptor represents an attractive target for cancer therapy. Aptamers are emerging as a new promising class of nucleic acid therapeutics. In this study, a conventional SELEX approach was applied against the kinase domain of a group of c-KIT proteins (c-KITWT, c-KITD816V, and c-KITD816H) to select aptamers from a random RNA pool that can bind to the kinase domain of each target with high affinity and can selectively interfere with their kinase activities. Interestingly, our data indicated that one candidate aptamer, called V15, can specifically inhibit the in vitro kinase activity of mutant c-KITD816V with an IC50 value that is 9-fold more potent than the sunitinib drug tested under the same conditions. Another aptamer, named as H5/V36, showed the potential to distinguish between the c-KIT kinases by modulating the phosphorylation activity of each in a distinct mechanism of action and in a different potency.

Co-encapsulation of thymoquinone with docetaxel enhances the encapsulation efficiency into PEGylated liposomes and the chemosensitivity of MCF7 breast cancer cells to docetaxel.

Combinatorial therapeutic strategies to eradicate tumors can be superior to a single therapeutic modality. Docetaxel (DT) has been approved for the treatment of local or metastasized breast cancer alone or in combination with other chemotherapeutic agents. Thymoquinone (TQ) originated from the seeds of Nigella Sativa plant has been reported to possess in vitro and in vivo antitumor activity against variety of tumors. In the current study, we have investigated the synergistic anticancer efficacy of a novel combination of DT and TQ on MCF7 breast cancer cell line using MTT cell viability assay. Moreover, this study describes for the first time the co-encapsulation of DT and TQ into PEGylated liposomes. The results showed that the combination of DT and TQ resulted in significant synergistic cytotoxicity compared to DT and TQ alone. Moreover, DT and TQ have been successfully co-encapsulated into PEGylated liposomes with higher encapsulation efficiency compared to DT and TQ alone. In conclusion, DT and TQ combination poses a synergistic effect and may aid in decreasing the required doses of DT. Also, the co-encapsulation of DT and TQ into PEGylated liposomes can provide a promising DT and TQ delivery system into cancer cells.

Aptamers Chemistry: Chemical Modifications and Conjugation Strategies.

Soon after they were first described in 1990, aptamers were largely recognized as a new class of biological ligands that can rival antibodies in various analytical, diagnostic, and therapeutic applications. Aptamers are short single-stranded RNA or DNA oligonucleotides capable of folding into complex 3D structures, enabling them to bind to a large variety of targets ranging from small ions to an entire organism. Their high binding specificity and affinity make them comparable to antibodies, but they are superior regarding a longer shelf life, simple production and chemical modification, in addition to low toxicity and immunogenicity. In the past three decades, aptamers have been used in a plethora of therapeutics and drug delivery systems that involve innovative delivery mechanisms and carrying various types of drug cargos. However, the successful translation of aptamer research from bench to bedside has been challenged by several limitations that slow down the realization of promising aptamer applications as therapeutics at the clinical level. The main limitations include the susceptibility to degradation by nucleases, fast renal clearance, low thermal stability, and the limited functional group diversity. The solution to overcome such limitations lies in the chemistry of aptamers. The current review will focus on the recent arts of aptamer chemistry that have been evolved to refine the pharmacological properties of aptamers. Moreover, this review will analyze the advantages and disadvantages of such chemical modifications and how they impact the pharmacological properties of aptamers. Finally, this review will summarize the conjugation strategies of aptamers to nanocarriers for developing targeted drug delivery systems.

Encapsulation of echinomycin in cyclodextrin inclusion complexes into liposomes: in vitro anti-proliferative and anti-invasive activity in glioblastoma.

Echinomycin, a DNA bis-intercalator peptide, belongs to the family of quinoxaline antibiotics. Echinomycin exhibits potent antitumor and antimicrobial activity. However, it is highly water insoluble and suffers from low bioavailability and unwanted side effects. Therefore, developing new formulations and delivery systems that can enhance echinomycin solubility and therapeutic potency is needed for further clinical application. In this study, echinomycin has been complexed into the hydrophobic cavity of γ-cyclodextrin (γCD) then encapsulated into PEGylated liposomes. The anti-proliferative and anti-invasive effect has been evaluated against U-87 MG glioblastoma cells. Echinomycin-in-γCD inclusion complexes have been characterized by phase solubility assay, TLC, and 1H-NMR. The echinomycin-in-γCD inclusion complexes have been loaded into liposomes using a thin film hydration method to end up with echinomycin-in-γCD-in-liposomes. Drug-loaded liposomes were able to inhibit cell proliferation with IC50 of 1.0 nM. Moreover, echinomycin-in-γCD-in-liposomes were found to inhibit the invasion of U-87 MG cells using the spheroid gel invasion assay. In conclusion, the current work describes for the first time γCD-echinomycin complexes and their encapsulation into PEGylated liposomes.

Remote loading of curcumin-in-modified ß-cyclodextrins into liposomes using a transmembrane pH gradient.

Curcumin (CRM) is a natural polyphenol with antioxidative, anti-inflammatory, and anticancer therapeutic properties. However, CRM therapeutic potential is limited by low water solubility and bioavailability. Intraliposomal remote loading describes the retention of drugs in liposome cores in response to transmembrane pH gradient. The current study describes for the first time the remote loading of CRM into liposomes using secondary (E-ßCD) and tertiary (D-ßCD) amino-modified ß-cyclodextrins (ßCDs) as carriers and solubilizers. ßCDs were chemically modified to prepare the ionizable weak base functional group followed by forming a guest-host complex of CRM in the modified ßCDs hydrophobic cavities via a solvent evaporation encapsulation technique. These complexes were then actively loaded into preformed liposomes, composed of DPPC/cholesterol (65/35 molar ratio) via pH gradient. The formation of CRM-ßCDs inclusion complexes was characterized using UV-Vis spectroscopy, thermal analysis, and NMR spectroscopy. The complex stoichiometric ratio was determined to be 1 : 1 of CRM-ßCDs based on Job's plot which was also confirmed by the modified Benesi-Hildebrand equation with increasing probability of forming the 1 : 2 ratio of CRM-ßCDs. The apparent formation constants (K f) of 51.6, 100.9 and 55.4 mM-2 were determined for CRM-ßCD, CRM-E-ßCD, and CRM-D-ßCD complexes, respectively. Liposome size, charge and polydispersity index indicate the presence of a homogeneous population before and after active loading. The encapsulation efficiencies of CRM-ßCD complexes into pH gradient preformed liposomes were 16.5, 51.1, and 41.7 for CRM-ßCD, CRM-E-ßCD, and CRM-D-ßCD, respectively, showing more than 5 fold increase compared to normal liposomes. The current study provides a novel remote loading approach utilizing chemically modified cyclodextrins to incorporate hydrophobic drugs into liposomes.

Correction: Selection and targeting of EpCAM protein by ssDNA aptamer.

[This corrects the article DOI: 10.1371/journal.pone.0189558.].

Selection and targeting of EpCAM protein by ssDNA aptamer.

Aptamers are molecules that reveal highly complex and refined molecular recognition properties. These molecules are capable of binding with high affinity and selectivity to targets, ranging from small molecules to whole living cells. Several aptamers have been selected for targeting cellular proteins and they have also used in developing therapeutics and diagnostic strategies. Epithelial cell adhesion molecule (EpCAM) is considered as a cancer stem cell (CSC) biomarker and one of the most promising targets for aptamer selection against CSCs. In this study, we have developed a ssDNA aptamer with high affinity and selectivity of targeting the EpCAM protein extracellular domain. The SELEX technique was applied and the resulted sequences were tested on EpCAM-positive human gastric cancer cell line, KATO III, and the EpCAM-negative mouse embryonic fibroblast, NIH/3T3 cells. Ep1 aptamer was successfully isolated and showed selective binding on EpCAM-positive KATO III cells when compared to EpCAM-negative NIH/3T3 cells, as observed by the flow cytometry and the confocal imaging results. Additionally, the binding of Ep1 to EpCAM protein was assessed using mobility shifting assay and aptamers-protein docking. Furthermore, the binding affinity of Ep1 was measured against EpCAM protein using EpCAM-immobilized on magnetic beads and showed apparent affinity of 118 nM. The results of this study could suggest that Ep1 aptamer can bind specifically to the cellular EpCAM protein, making it an attractive ligand for targeted drug delivery and as an imaging agent for the identification of cancer cells.

Thymoquinone efficiently inhibits the survival of EBV-infected B cells and alters EBV gene expression.

Epstein--Barr virus (EBV) is a human virus with oncogenic potentials that is implicated in various human diseases and malignancies. In this study, the modulator activity of the potent herbal extract drug thymoquinone on EBV was assessed in vitro. Thymoquinone was tested for cytotoxicity on human cells of lymphoblastoid cells, Raji Burkitt's lymphoma, DG-75 Burkitt's lymphoma, peripheral blood mononuclear cells, and periodontal ligament fibroblast. Apoptosis induction was analyzed via TUNEL assay and activity studies of caspase-3. The effect of thymoquinone on EBV gene expression was determined using real-time polymerase chain reaction. We report here, for the first time, a promising selective inhibitory affect of thymoquinone on EBV-infected B cell lines in vitro, compared with lower activity on EBV negative B cell line and very low toxicity on human peripheral blood mononuclear cells and periodontal ligament fibroblasts. Moreover, the drug was found to efficiently suppress the RNA expression of EBNA2, LMP1, and EBNA1 genes. Specifically, EBNA2 expression levels were the most affected indicating that this gene might have a major contribution to thymoquinone potency against EBV infected cells. Overall, our results suggest that thymoquinone has the potential to suppress the growth of EBV-infected B cells efficiently.

Thymoquinone in liposomes: a study of loading efficiency and biological activity towards breast cancer.

Thymoquinone (2-isopropyl-5-methyl-1,4-benzoquinone) is a herbal-derived drug with potential chemopreventive and chemotherapeutic activity. However, thymoquinone suffers from high hydrophobicity causing poor solubility which limits its bioavailability and high lipophilicity causing poor formulation characteristics. Liposomes are versatile drug carriers that can be used to solve problems of drug solubility, instability, and bio-distribution. In this study, we were able to prepare thymoquinone-loaded liposomes (TQ-LP) and thymoquinone loaded in liposomes modified with Triton X-100 (XLP) with diameters of about 100 nm, and entrapment efficiency of more than 90% for TQ-LP and of 49.6% for XLP. The TQ-LP liposomes were effective in suppressing the proliferation of breast cancer cell lines MCF-7 and T47D, and at the same time exerting very low toxicity on normal periodontal ligament fibroblast. Altogether, this report describes the first successful encapsulation of thymoquinone into liposome; which maintains stability, improves bioavailability and maintains its anticancer activity.