M. R. Mozafari

Liposomes: A Review of Manufacturing Techniques and Targeting Strategies

Today, liposomes are among the most applied technologies for the encapsulation and delivery of bioactive agents and many different compounds in biological, pharmaceutical, medical and nutritional research. In this review, classification of liposomal vesicles, methods of their preparation and encapsulation, as well as their applications in food, cosmetics and pharmaceutical industries are re- viewed. In addition, the main analytical approaches used to study liposome characteristics such as size, transition temperature, surface charge, fluidity, lamellarity, stability and encapsulation efficiency are presented. In the final part of the article, mechanisms of liposome targeting are discussed.

Nanoliposomes: preparation and analysis.

Nanoliposome, or submicron bilayer lipid vesicle, is a new technology for the encapsulation and delivery of bioactive agents. The list of bioactive material that can be incorporated to nanoliposomes is immense, ranging from pharmaceuticals to cosmetics and nutraceuticals. Because of their biocompatibility and biodegradability, along with their nanosize, nanoliposomes have potential applications in a vast range of fields, including nanotherapy (e.g. diagnosis, cancer therapy, gene delivery), cosmetics, food technology and agriculture. Nanoliposomes are able to enhance the performance of bioactive agents by improving their solubility and bioavailability, in vitro and in vivo stability, as well as preventing their unwanted interactions with other molecules. Another advantage of nanoliposomes is cell-specific targeting, which is a prerequisite to attain drug concentrations required for optimum therapeutic efficacy in the target site while minimising adverse effects on healthy cells and tissues. This chapter covers nanoliposomes, particularly with respect to their properties, preparation methods and analysis.

Nutritional and medical applications of spirulina microalgae.

Spirulina spp. and its processing products are employed in agriculture, food industry, pharmaceutics, perfumery and medicine. Spirulina has several pharmacological activities such as antimicrobial (including antiviral and antibacterial), anticancer, metalloprotective (prevention of heavy-metal poisoning against Cd, Pb, Fe, Hg), as well as immunostimulant and antioxidant effects due to its rich content of protein, polysaccharide, lipid, essential amino and fatty acids, dietary minerals and vitamins. This article serves as an overview, introducing the basic biochemical composition of this algae and moves to its medical applications. For each application the basic description of disease, mechanism of damage, particular content of Spirulina spp. for treatment, in vivo and/or in vitro usage, factors associated with therapeutic role, problems encountered and advantages are given.

Role of nanocarrier systems in cancer nanotherapy

Cancer continues to be a major cause of morbidity and mortality worldwide. While discovery of new drugs and cancer chemotherapy opened a new era for the treatment of tumors, optimized concentration of drug at the target site is only possible at the expense of severe side effects. Nanoscale carrier systems have the potential to limit drug toxicity and achieve tumor localization. When linked with tumor-targeting moieties, such as tumor-specific ligands or monoclonal antibodies, the nanocarriers can be used to target cancer-specific receptors, tumor antigens, and tumor vasculatures with high affinity and precision. This article is an overview of advances and prospects in the applications of nanocarrier technology in cancer therapy. Applications of nanoliposomes, dendrimers, and nanoparticles in cancer therapy are explained, along with their preparation methods and targeting strategies.

Comparative study of the oxidative and physical stability of liposomal and nanoliposomal polyunsaturated fatty acids prepared with conventional and Mozafari methods

The relative oxidative stability of freshly prepared and stored liposomal and nanoliposomal systems of docosahexaenoic acid (DHA, 22:6 n-3) and eicosapentaenoic acid (EPA, 20:5 n-3) were investigated. The effects of organic solvents on the oxidative stability of liposomal polyunsaturated fatty acids (PUFAs) produced by two methods, the Bangham thin-film hydration (conventional rotary evaporation method and using organic solvents) and Mozafari (direct hydration and without using organic solvents) methods, were compared. The highest physicochemical stability was observed in PUFA liposomes prepared by the Mozafari method, followed by conventional liposomes and bulk PUFAs. There was no significant change in physicochemical stability during 10 months of cold storage (4°C) in the dark. Moreover, the comparison between liposomes (>200 nm) and nanoliposomes (50-200 nm) revealed that the surface charge, physical stability and oxidative stability of liposomal PUFAs increased as the size of the liposomes decreased. The differences in the oxidative stability of PUFAs may be due to the protective effects of aqueous systems, which indicate the advantage of using non-organic solvent (water and CO(2)) techniques in liposome manufacturing.

A review of scanning probe microscopy investigations of liposome-DNA complexes.

Liposome-DNA complexes are one of the most promising systems for the protection and delivery of nucleic acids to combat neoplastic, viral, and genetic diseases. In addition, they are being used as models in the elucidation of many biological phenomena such as viral infection and transduction. In order to understand these phenomena and to realize the mechanism of nucleic acid transfer by liposome-DNA complexes, studies at the molecular level are required. To this end, scanning probe microscopy (SPM) is increasingly being used in the characterization of lipid layers, lipid aggregates, liposomes, and their complexes with nucleic acid molecules. The most attractive attributes of SPM are the potential to image samples with subnanometer spatial resolution under physiological conditions and provide information on their physical and mechanical properties. This review describes the application of scanning tunneling microscopy and atomic force microscopy, the two most commonly applied SPM techniques, in the characterisation of liposome-DNA complexes.

Mechanism of calcium ion induced multilamellar vesicle-DNA interaction

The effect of Ca2+ on the DNA interaction with anionic and neutral multilamellar vesicles (MLV) has been investigated. DNA from wheat (Triticum aestivum L. Gerek) was introduced to a suspension of MLV, composed of phosphatidylcholine (PC):dicetylphosphate (DCP):cholesterol (CHOL) at different molar ratios, to which Ca2+ (5-75 mM) was subsequently added. Indication of aggregation and/or fusion was obtained via light-scattering examination following the addition of Ca2+ and DNA to the MLV medium. Using a UV spectrophotometric assay, it was observed that although DNA alone has no effect on negatively charged MLV, it enhances liposomal interaction in the presence of calcium ions. The minimal Ca2+ concentration required to promote the interaction was detected to be 10 mM, and the highest level of interaction was observed at 75 mM. The aggregation/fusion of vesicles was detected for uncharged MLV (with no DCP in their structure), as well as for the anionic ones containing c. 10% CHOL, but not for anionic MLV containing 40% CHOL. This is explained in terms of cholesterol decreasing the membrane fluidity (above the Tc of components) as a result of which more rigid vesicles become less prone to aggregation/fusion interactions.

Formation of supramolecular structures by negatively charged liposomes in the presence of nucleic acids and divalent cations

Cationic liposomes are being increasingly studied as delivery vehicles for bioactive agents such as DNA and other polynucleotides. The mechanism of interaction of DNA with liposomes and the organization of these interacting structures during and after the interaction are still poorly understood. Nucleic acids are known to induce aggregation and size enlargement of liposomes. In the case of phosphatidylcholine (PC) vesicles, these processes depend on the presence and concentration of divalent metal cations and the amount of cholesterol in the liposomes. In this study, anionic small unilamellar vesicles (SUV) and multilamellar vesicles (MLV) composed of dicetylphosphate (DCP):PC:cholesterol at 2:7:1 molar ratios were prepared and incubated with the DNA (from wheat) and Ca(2+) (50 mM) at 25 degrees C with the aim of transferring the genetic material into the liposomes by inducing fusion of liposome-liposome aggregates created in the presence, and with the help, of DNA. The organization and the nature of the resultant liposome-DNA-Ca(2+) complexes were investigated by scanning tunneling microscopy (STM) and fluorescence microscopy. Observations of complexes with similar appearances with both SUV and MLV, as shown by two quite different microscopic approaches, prove that the resultant forms are real and not artifacts of the methodology used. At this stage it is not clear whether the detected complexes represent an intermediate state before fusion of liposomes which will lead to engulfing of the genomic material by the fused liposomes, or the final form. In either case the structures consisting of some adhered or semifused liposomes bearing the nucleic acid seem to be candidates as vehicles for in-vitro and in-vivo transfection.

Formulation and characterization of nanoliposomal 5-fluorouracil for cancer nanotherapy

Abstract A scalable and safe method was developed to prepare nanoliposome carriers for the entrapment and delivery of 5-fluorouracil (5-FU). The carrier systems were composed of endogenously occurring dipalmitoylphosphatidylcholine (DPPC), negatively charged dicetylphosphate (DCP), cholesterol (CHOL) and glycerol (3%, v/v). Nanoliposomes were prepared by the heating method in which no harmful chemical or procedure is involved. Results indicated fast and reproducible formation of non-toxic liposomes that possess high entrapment efficiency (up to 96.9%) and vesicle size range of ca. 530–620 nm. Transmission electron and optical micrographs of the 5-FU liposomes revealed that they were spherical and some were multilayered. There was an increase in the release rate of 5-FU from the liposomes prepared with a high ratio of drug:lipid. The release data showed that the highest release rates were obtained for nanoliposomes containing 5-FU with the drug concentration of 500 mM and that it followed the diffusion model. Nanoliposome preparation method introduced here has the potential of large-scale manufacture of safe and efficient carriers of 5-FU.

Applications of nanoliposomes in cheese technology

Among microencapsulation techniques used in food production, nanoliposomes are known to be one of the most interesting methods for the encapsulation of flavours, essential oils, amino acids, vitamins, minerals, enzymes, micro-organisms, redox agents, colourants, antioxidants and antimicrobials. Research has also been conducted on possible applications of nanoliposomes in cheese production by the encapsulation of ferrous glycinate, ferrous sulphate, antioxidants, nisin, β-galactosidase and cheese-ripening enzymes. In this article, nanoliposome application in cheese production has been reviewed under three main themes, namely (i) acceleration of cheese ripening, (ii) fortification of cheese with vitamins and minerals and (iii) increasing shelf life of cheese products. Various aspects of nanoliposome application in cheese technology including currently available preparation methods, efficiency of nanoliposomal enzymes, their effects on the acceleration of cheese ripening as well as rheological and chemical properties of the cheese curd are discussed.