Efficiency Of Liposomes Research Articles

Liposomes-Based Drug Delivery Systems of Anti-Biofilm Agents to Combat Bacterial Biofilm Formation.

All currently approved antibiotics are being met by some degree of resistance by the bacteria they target. Biofilm formation is one of the crucial enablers of bacterial resistance, making it an important bacterial process to target for overcoming antibiotic resistance. Accordingly, several drug delivery systems that target biofilm formation have been developed. One of these systems is based on lipid-based nanocarriers (liposomes), which have shown strong efficacy against biofilms of bacterial pathogens. Liposomes come in various types, namely conventional (charged or neutral), stimuli-responsive, deformable, targeted, and stealth. This paper reviews studies employing liposomal formulations against biofilms of medically salient gram-negative and gram-positive bacterial species reported recently. When it comes to gram-negative species, liposomal formulations of various types were reported to be efficacious against Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, and members of the genera Klebsiella, Salmonella, Aeromonas, Serratia, Porphyromonas, and Prevotella. A range of liposomal formulations were also effective against gram-positive biofilms, including mostly biofilms of Staphylococcal strains, namely Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus subspecies bovis, followed by Streptococcal strains (pneumonia, oralis, and mutans), Cutibacterium acnes, Bacillus subtilis, Mycobacterium avium, Mycobacterium avium subsp. hominissuis, Mycobacterium abscessus, and Listeria monocytogenes biofilms. This review outlines the benefits and limitations of using liposomal formulations as means to combat different multidrug-resistant bacteria, urging the investigation of the effects of bacterial gram-stain on liposomal efficiency and the inclusion of pathogenic bacterial strains previously unstudied.

Preparation and Physical Characterization of DNA Binding Cationic Liposomes.

Cationic liposomes are routinely employed as one of the major nonviral transfecting agents for intracellular delivery of hydrophilic molecules such as nucleic acids, peptides, and proteins. Cationic liposomes when complexed with DNA form a strong positively charged cationic liposome-DNA complex or lipoplex. The chapter discusses, primarily, the major preparation technique for cationic liposomes and its physical characterization, with a focus on SYBR Green I dye exclusion assay and DNA encapsulation enhancement by freeze-thaw technique. SYBR Green I dye exclusion assay is a technique to determine the total amount of liposomal lipids required to bind a unit weight of DNA, which is critical for transfection experiments. Freeze-thaw technique on the other hand is one of the major techniques to improve DNA encapsulation efficiency in liposomes.

Preparation, Characterization and Preliminary <i>in vivo</i> Testing of Liposomated Antiidiotypic Antibodies Against Morphine Derivatives

Artificial membrane structures containing medicinal substances are highly promising for the development of new drugs. Liposomal preparations are actively used in medical practice due to their high efficacy and relatively low toxicity. Our aim was to encapsulate anti-idiotypic antibodies into a liposomal composition with the purpose of improving their immunogenic properties. Following the preparation of a liposomal composition by the dehydration/rehydration method using ultrasonic treatment, the size, zeta potential, and loading efficiency of liposomes were investigated. Preliminary in vivo studies were conducted to evaluate the adjuvant properties of liposomes of varying size. Loaded liposomes of the smallest diameter (about 110 nm) showed the potential of enhancing the immune response similar to that obtained using Freund’s adjuvant. These results justify further research into the properties of liposomes loaded with antibodies.

A study on chitosan-coated liposomes as a carrier of bovine serum albumin as oral protein drug

The development of protein drug liposomes was limited due to the instability of the structure of liposomes, such as easy aggregation, easy fusion, and drug leakage. The object of this work was to prepare bovine serum albumin (BSA) loaded liposomes (BSA-Lip) using BSA as a protein model drug and then BSA-Lip was further coated with chitosan (CH-BSA-Lip) in order to improve the stability of phospholipid bilayer membrane. It was found that the particle size of BSA-Lip increased after being coated with chitosan and the surface potential changed from negative charge to positive charge due to the electrostatic interaction between liposomes and chitosan. After chitosan coating, the encapsulation efficiency of liposomes also increased due to the interaction between positively charged chitosan and negatively charged BSA. Fourier transform infrared spectroscopy analysis confirmed that BSA was encapsulated into liposomes and chitosan was successfully coated on the surface of liposomes. Transmission electron microscopy showed that the particles were nanosized and spherical. The physical stability and in vitro digestion stability of liposomes were examined by observing the changes of particle size and potential. It was found that chitosan coating enhanced the stability of liposomes significantly. The in vitro release profile of CH-BSA-Lip possessed a slow release behavior, and BSA release was governed by both diffusion and dissolution from phospholipid bilayer.

Cationic amphiphiles based on malonic acid amides as transfection mediators

Objectives. The aim of this work is to synthesize cationic amphiphiles based on malonic acid amides. The target compounds should contain saturated and unsaturated alkyl chains in the hydrophobic portion, and one or two positive charges in the polar head as created by ethylenediamine and amino acid L-ornithine. For such cationic amphiphiles, we determined physicochemical properties and transfection efficiency of liposomes based on them.Methods. The initial compound in the synthesis is diethylmalonate. We used C-alkylation to add the first hydrophobic chain (with octylbromide, dodecylbromide, or octadecylbromide). N-oleylamine was used as the second hydrophobic chain, which was attached at the carboxyl group of the malonic acid via amide bond formation. The polar head was represented by ethylenediamine, which was then attached at the second carboxyl group of the malonic acid. Further, L-ornithine was attached to ethylenediamine to produce cationic lipids with two positive charges in the head group. The structures of the compounds were characterized by infrared spectroscopy, nuclear magnetic resonance spectroscopy, and elemental analysis. Particle size distribution was evaluated by photon correlation spectroscopy. The luciferase test was used to determine transfection efficiency using HeLa cells.Results. We have developed a synthesis scheme to produce new cationic amphiphiles with an asymmetric hydrophobic part. The obtained liposomal particles are approximately 120 nm in size and have a relatively high zeta potential of 29–30 mV.Conclusions. The size of these liposomes allows them to penetrate into cells, which makes it possible to use these compositions for transfection. The high zeta potential shows that the particles are stable. Our results demonstrate that the transfection efficiency of our liposomes (mixed with cholesterol) is comparable to a commercial formulation. Cationic amphiphiles based on malonic acid amides have great potential for liposome development for transfection.

Liposomal Nanovesicles for Efficient Encapsulation of Staphylococcal Antibiotics.

Liposomes are attractive vehicles for localized delivery of antibiotics. There exists, however, a gap in knowledge when it comes to achieving high liposomal loading efficiencies for antibiotics. To address this issue, we investigated three antibiotics of clinical relevance against staphylococcal infections with different hydrophilicity and chemical structure, namely, vancomycin hydrochloride, teicoplanin, and rifampin. We categorized the suitability of different encapsulation techniques on the basis of encapsulation efficiency, lipid requirement (important for avoiding lipid toxicity), and mass yield (percentage of mass retained during the preparation process). The moderately hydrophobic (teicoplanin) and highly hydrophobic (rifampin) antibiotics varied significantly in their encapsulation load (max 23.4 and 15.5%, respectively) and mass yield (max 74.1 and 71.8%, respectively), favoring techniques that maximized partition between the aqueous core and the lipid bilayer or those that produce oligolamellar vesicles, whereas vancomycin hydrochloride, a highly hydrophilic molecule, showed little preference to any of the protocols. In addition, we report significant bias introduced by the choice of analytical method adopted to quantify the encapsulation efficiency (underestimation of up to 24% or overestimation by up to 57.9% for vancomycin and underestimation of up to 61.1% for rifampin) and further propose ultrafiltration and bursting by methanol as the method with minimal bias for quantification of encapsulation efficiency in liposomes. The knowledge generated in this work provides critical insight into the more practical, albeit less investigated, aspects of designing vesicles for localized antibiotic delivery and can be extended to other nanovehicles that may suffer from the same biases in analytical protocols.

The effect of superparamagnetic iron oxide nanoparticles surface engineering on relaxivity of magnetoliposome.

The purpose of this work is evaluating the effect of ultra small superparamagnetic iron oxide nanoparticles (USPIONs) coatings on encapsulation efficiency in liposomes and cellular cytotoxicity assay. Moreover, we assessed the effects of surface engineering on the relaxivity of magnetoliposome nanoparticles in order to create a targeted reagent for the intelligent diagnosis of cancers by MRI. For estimating the effect of nanoparticle coatings on encapsulation, several kinds of USPIONs coated by dextran, PEG5000 and citrate were used. All kinds of samples are monodispersed and below 100 ± 10 nm and the coatings of USPIONs have no significant effect on magnetoliposome diameter. The coating of USPIONs could have effect on percentage of encapsulation. The dextran coated USPIONs have more stability and quality accordingly the encapsulation increased up to 92%, then the magnetoliposome nano particles have been targeted by Herceptin and anti-HER2 VHH, separately. Over storage period of four weeks the resulting particles were stable and physico-chemical properties such as size and zetapotential did not show any significant changes. The relaxivity of contrast agents was measured using a 1.5 T MRI. The r2/r1 ratio was more than two for all samples which demonstrate the negative contrast enhancing of all SPION embedded specimens. The high ratio of r2/r1 as well as high r2 is the best combination of a negative contrast agent as it is obtained for pure magnetite. The value of r2/r1 for all other samples including Herceptin targeted magnetoliposome, anti-HER2 VHH targeted magnetoliposome and non-targeted magnetoliposome were between ~21 to ~28, which show the magnetite embedded samples have enough negative contrast to be detectable by MRI. Therefore the HER2 targeted magnetoliposomes are a good and stable candidate as contrast agents in clinical radiology and biomedical research with minimal cytotoxicity and biocompatibility effects. Copyright © 2016 John Wiley & Sons, Ltd.

Liposomal Toxicity – a‘SEEM TO BE’ Concern for Formulation Technologists

Liposomal drug delivery (LDD) has become a promising strategy & is continuing to be a big success for the Pharmaceutical Industry. Toxicity associated with LDD did not pin point intricately making it impossible to construe. Although the induction of Liposomal toxicity is mysterious & there has been a limited exploration, there was some evidence from animal studies which include haemolysis platelet aggregation, emboli, seizures & ‘proton sponge effect’. The mechanism responsible for induction of immune responses is revealed by protein kinase-C mediated down regulation of TNF-α, NO, PGE-2. Reports at the molecular level has been proved that Liposomal efficiency/ toxicity profile was influenced by factors like molecular weight, ionic strength particle size & the amount of drug load into the liposome i.e., anticancer drug loaded excessively into liposo me has lead to cardio toxicity, skin toxicity. Increased concentration of drug hampered the efficiency due to crystalline formation & altered rate kinetics of drug release. The present objective of the study is to signify the causes of toxicity & attempts to wipe out the harmful effects associated with LDD. The task of minimizing the toxic effects of LDD is challenging aspect for the pharmaceutical technologist & makes this novel drug therapy safe, effective & unblemished.

Effect of Different Pretreatment Methods on the Measurement of the Entrapment Efficiency of Liposomes and Countermeasures in Free Drug Analysis

Further investigation into the methods for the analysis of the entrapment efficiency of liposomes has been prompted by the urgent need to improve traditional methods which offer more risk to liposome leakage. The aim of this study was to investigate the suitability of hollow fiber centrifugal ultrafiltration (HF-CF-UF) coupled with HPLC as an alternative method for the routine determination of the entrapment efficiency of liposomes and to compare it with previously developed nonequilibrium procedures based on size-exclusion chromatography (SEC). By comparison, evaluation of the entrapped fraction with SEC resulted in 3 or 4 % lower entrapment values. More importantly, the free drug concentrations were three- to sixfold higher than with HF-CF-UF. In addition, we investigated the reasons for the different values obtained with SEC and a dynamic equilibrium theory was put forward based on this. In addition, we have developed and characterized an equilibrium method of separating free and liposomal drugs for liposomal formulations based on HF-CF-UF. The method was validated over the concentration range of 7.9–235 μg mL−1 for indomethacin and 0.258–8.24 μg mL−1 for vitamin A. Inter- and intra-day precision (RSD %) were ≤1.2 % for indomethacin and ≤1.8 % for vitamin A, respectively. The recoveries of both free drug and total drug were higher than 96.0 % with RSD ≤1.3 % (n = 5). Taken together, our improved method differs from those previously reported with regard to higher recovery, small sample volume, less laborious, and most importantly without damaging or disturbing liposomes. Validation results suggested that our method was sufficiently accurate and sensitive to be used to evaluate the entrapment efficiency of a liposome formulation without complicated pretreatment.

AFM in Advanced Pharmaceutical Technology

The necessity for development of new drug delivery systems for poorly soluble drugs and therapeutics which targets directly the pathogenic area requires the design of complex systems in nanoscale. Atomic Force Microscopy (AFM) since its discovery offers a unique tool to develop novel drug delivery systems. Its capability to investigate, characterise surfaces and measure forces with spatial resolution at nano-scale respectively contributes to develop and analyse pharmaceutical systems and biomedical devices with complex structures and chemistries. Specifically, polymeric nanoparticles and liposomal drug formulation have been studied extensively by using AFM where their size and morphology where revealed [1]. However, apart from topographical information, AFM can provide details on the local compositions of the sample. There are many modes that can be used to achieve this but the most common is to monitor the phase shift of the oscillating cantilever in tapping mode Such ‘Phase imaging’ can be used to detect nanoscale variation in composition, adhesion, friction, viscoelasticity, and other properties of the materials. Changes in the phase lag often indicate changes in the properties of the sample surface. Phase imaging has proved an extremely useful tool for pharmaceutical characterization. Phase imaging has been used to reveal polymeric forms from single crystal measurements [2] to confirm phase separation of two copolymers for drug delivery [3], to establish the stability of the formulation on different environments [4] and to identify formation of amorphous domains during milling of crystalline salbutamol [5]. Moreover, the ability of AFM to operate on different conditions of temperature humidity and in liquid allows studying the kinetics of range of phenomena. An interesting work was published from Miyazaki et al. where the crystal growth rate of Nifedipine, dispersed in PEG polymer matrix, was quantified [6]. Also, the dissolution process of aspirin on different facets of the crystal was studied by Danesh et al . showing their differences in dissolution rate [7]. The ability of AFM to measure force measurements have been used to investigate interactions among the different compounds in the formulation. To achieve this, the AFM cantilever is modified by attaching a particle directly onto the probe tip and is brought in contact with the substrate of interest, the force required to separate them is the measure of their adhesion. This application of AFM has been used extensively to aerosol drug formulation where the API is associated with a carrier which is released in the site of actions. For instance, AFM has been used to investigate the adhesion force of salbutamol with different excipients [8]. Moreover, localised surface energy and young’s modulus of various compounds have been measured. For example, Young’s modulus were derived from both PLA and PLA/everolimus directly from stent surface on different temperatures. The results showed that everolimus does not affect the mechanical properties of PLA up to 1/1 (w/w) drug loading of the mixture. Also, a significant drop of the Young’s modulus in solution was observed at 36°C, suggests that in vivo the Tg of the polymer is below body temperature [9]. Furthermore, force measurements have been used to identify the encapsulation the efficiency of liposomes as the rigidity of encapsulating liposomes is higher [10].

Influence of some physical properties of 5-fluorouracil on encapsulation efficiency in liposomes

The aim of this study is to encapsulate two drugs: 5-fluorouracil (5-FU) with the hydrophobic properties and 1-β-D-arabinofuranosylcytosine (Ara-C) with the amphiphilic properties into liposomes prepared by the modified reverse-phase evaporation method (mREV) from L-α-phosphatidylcholine dipalmitoyl (DPPC). We studied the thermotropic phase behavior of liposome entrapped 5-FU and Ara-C. It is known that the stability of liposomes depends not only on the method of chemical gradient loading, the use of membrane stabilizer such as sterols, but also on the phase transition temperature (Tc) of phospholipids, which undergoes an alteration after encapsulation of drugs to liposomes. The competition of these two drugs entrapped in liposomes was analyzed by the use of two spectroscopies: 1H NMR and UV on the basis of the analysis of the signals of each drug in the liposome—drug system. The percent of encapsulation in DPPC/Ara-C/5-FU liposome obtained by the use of UV spectroscopy amounted 93.84 and 96.05% for 5-FU and Ara-C, respectively. Phase transition temperature Tc of liposomes containing Ara-C did not significantly change while for the liposomes containing 5-FU it increased in comparison with Tc of the reference liposomes formed from DPPC.

Structural and biophysical properties of a synthetic channel-forming peptide: Designing a clinically relevant anion selective pore

The design, synthesis, modeling and in vitro testing of channel-forming peptides derived from the cys-loop superfamily of ligand-gated ion channels are part of an ongoing research focus. Over 300 different sequences have been prepared based on the M2 transmembrane segment of the spinal cord glycine receptor α-subunit. A number of these sequences are water-soluble monomers that readily insert into biological membranes where they undergo supramolecular assembly, yielding channels with a range of selectivities and conductances. Selection of a sequence for further modifications to yield an optimal lead compound came down to a few key biophysical properties: low solution concentrations that yield channel activity, greater ensemble conductance, and enhanced ion selectivity. The sequence NK4-M2GlyR T19R, S22W (KKKKPARVGLGITTVLTMRTQW) addressed these criteria. The structure of this peptide has been analyzed by solution NMR as a monomer in detergent micelles, simulated as five-helix bundles in a membrane environment, modified by cysteine-scanning and studied for insertion efficiency in liposomes of selected lipid compositions. Taken together, these results define the structural and key biophysical properties of this sequence in a membrane. This model provides an initial scaffold from which rational substitutions can be proposed and tested to modulate anion selectivity. This article is part of a Special Issue entitled: Protein Folding in Membranes.

A Comparative Study of Transfection Efficiency between Liposomes, Immunoliposomes and Brain-specific Immunoliposomes

This study investigated the transfection ability and efficiency of liposomes and immunoliposomes for exogenous gene delivery into the brain via the venous system. Four groups of rats underwent tail vein injection with one of the following: liposomes encapsulating pCMV (human cytomegalovirus promoter)-LacZ plasmid 80 microg (low dose) or 300 microg (high dose); general immunoliposomes encapsulating 80 microg transferrin receptor antibodies (OX26)-pCMV-LacZ plasmid; or brain-specific immunoliposomes encapsulating 80 microg OX26-pGFAP (glial fibrillary acidic protein promoter)-LacZ plasmid. A control group received no injected agent. The LacZ mRNA levels (1 h post-injection) and beta-galactosidase activity (48 h post-injection) in the brain and peripheral organs were assayed using real-time reverse transcription-polymerase chain reaction and histochemical staining, respectively. Both immunoliposomes delivered exogenous DNA containing the LacZ gene into the brain after venous injection, resulting in extensive LacZ expression in the brain. Furthermore, the brain-specific OX26-pGFAP-LacZ immunoliposome decreased the non-specific expression of LacZ in peripheral organs without affecting transfection efficiency in the brain. Thus, brain-specific immunoliposomes are an efficient and brain-specific targeting vector.

Preparation of liposomes containing zedoary turmeric oil using freeze-drying of liposomes via TBA/water cosolvent systems and evaluation of the bioavailability of the oil

The purpose of this study was to enhance the absorption of zedoary turmeric oil (ZTO) in vivo and develop new formulations of a water-insoluble oily drug. This study described a method for preparing ZTO liposomes, which involved freeze-drying (FD) of liposomes with TBA/water cosolvent systems. The TBA/water cosolvent systems were used to investigate a feasible method of liposomes manufacture; the two factors, sugar/lipid mass ratio and TBA content (concentration), of the preparation process were evaluated in this study. The results showed that the addition of TBA content could significantly enhance the sublimation of ice resulting in short FD cycles time, and reduce the entrapment efficiency of liposomes. In addition, the residual TBA solvents levels were determined to be less than 0.37% under all optimum formulations and processing conditions. Several physical properties of liposomes were examined by H-600 transmission electron microscope (TEM) and zetamaster analyser system. The results revealed that the liposomes were smooth and spherical with an average particle size of 457 ± 7.8 nm and the zeta potential was more than 3.65 Mv. The bioavailability of the liposomes was evaluated in rabbits, compared with the conventional self-emulsifying formulation for oral administration. Compared with the conventional self-emulsifying formulation, the plasma concentration–time profiles with improved sustained-release characteristics were achieved after oral administration of the liposomes with a bioavailability of 257.7% (a good strategy for improving the bioavailability of an oily drug). In conclusion, the present experimental findings clearly demonstrated the usefulness of ZTO liposome vesicles in improving therapeutic efficacy by enhancing oral bioavailability. Our study offered an alternative method for designing sustained-release preparations of oily drugs.

Enhanced stability and in vitro bioactivity of surfactant-loaded liposomes containing Asiatic Pennywort extract

The objective of this work has been the microencapsulation of Asiatic Pennywort (AP) extract with lecithin from soybean. The effect of various quantities of non-ionic surfactant (Montanov82®) on liposomes upon physicochemical characteristics as well as their in vitro bio-activities was investigated. An addition of surfactant resulted in a decrease in particle size and an increase in percentage AP entrapment efficiency of liposomes. The surfactant-loaded liposomes demonstrated higher stability than surfactant-free liposomes where higher percentage AP remaining of liposomes can be achieved depending on surfactant concentration. No significant difference was found on AP release profiles among varied surfactant concentrations, although a presence of surfactant resulted in prolonged AP release rate. Liposomal AP with 20% w/w surfactant or higher demonstrated low cytotoxicity, a stronger anti-oxidation effect and collagen production on dermal fibroblast cells when compared with free AP and surfactant-free liposomes, possibly due to better cell internalization and less AP degradation in cells.

Efficiency of Liposomes in the Delivery of a Photosensitizer Controlled by the Stereochemistry of a Gemini Surfactant Component

Liposomes formulated with dimyristoyl-sn-glycero-phosphatidylcholine, DMPC, and either one of the cationic gemini surfactants (S,S)-2,3-dimethoxy-1,4-bis(N-hexadecyl-N,N-dimethylammonio)butane bromide, 1a, and (S,R)-2,3-dimethoxy-1,4-bis(N-hexadecyl-N,N-dimethylammonio)butane bromide, 1b, were investigated as vehicles of the photosensitizer m-tetrahydroxyphenylchlorin, m-THPC, to cell models of malignant glioma. The delivery efficiency of DMPC/1a and DMPC/1b liposome formulations were evaluated on the murine glioblastoma cell line C6 and on the human glioblastoma cell line LN229 by flow cytometry and laser scanning confocal microscopy. The stereochemistry of the spacer of the gemini was found to strongly influence the delivery efficiency of m-THPC to cells, the mode of interaction with the cell membrane, and the intracellular distribution of m-THPC. The physicochemical features of liposomes were investigated with the aim of explaining the parameters that control their biological features. Differences that could account for the different biological activity of the formulations concern the values of surface potential and the environment of m-THPC at the water/liposome interface.

Formulation and Comparative Characterization of Chitosan, Gelatin, and Chitosan–Gelatin-Coated Liposomes of CPT-11–HCl

Liposomes containing phosphatidylcholine and cholesterol (uncoated) and coated by chitosan, gelatin, and combination of chitosan and gelatin were prepared by the modified ethanol injection method. The aim of this work was to formulate and characterize liposomes of camptothecin (CPT)-11–HCl (Irinotecan HCl) containing chitosan, gelatin, and both polymers as coating materials; and also to increase its circulation longevity when compared with the free drug while maintaining the agent in its active lactone form. Size, shape, zeta potential, encapsulation efficiency, stability study, in vitro, and in vivo release study were used for characterization of liposomes. The size of liposomes was in the order of uncoated < chitosan coated < gelatin coated < combination of chitosan and gelatin coated. The zeta potential of liposomes was in the order of combination of chitosan and gelatin coated > chitosan coated > gelatin coated > uncoated. The formulations showed the long-term stability. The encapsulation efficiency of liposomes was in order of combination of chitosan and gelatin coated > gelatin coated > chitosan coated > uncoated. The in vitro and in vivo release of drug was observed in the order of combination chitosan and gelatin coated > gelatin coated > chitosan coated > uncoated.

Daidzein as an Antioxidant of Lipid: Effects of the Microenvironment in Relation to Chemical Structure

Isoflavone daidzein (D, pK a1 = 7.47 +/- 0.02 and pK a2 = 9.65 +/- 0.07) was, through a study of the parent compound and its three methyl anisol derivatives 7-methyldaidzein (7-Me-D, pK a = 9.89 +/- 0.05), 4'-methyldaidzein (4'-Me-D, pK a = 7.43 +/- 0.03), and 7,4'-dimethyldaidzein (7,4'-diMe-D), found to retard lipid oxidation in liposomal membranes through two mechanisms: (i) radical scavenging for which the 4'-OH was more effective than the 7-OH group in agreement with the oxidation potentials: 0.69 V for 4'-OH and 0.92 V for 7-OH versus Ag/AgCl in acidic solution and 0.44 V for 4'-O(-) and 0.49 V for 7-O(-) in alkaline solution and (ii) change in membrane fluidity through incorporation of the isoflavones, in effect hampering radical mobility. The radical scavenging efficiency measured by the rate of the reaction with the ABTS(*)(+) in aqueous solution followed the order D > 7-Me-D > 4'-Me-D > 7,4'-diMe-D, as also found for antioxidant efficiency in liposomes when oxidation was initiated with the water-soluble AAPH radical and monitored as the formation of conjugate dienes. For oxidation initiated by the lipid-soluble AMVN radical, the antioxidant efficiency was ranked as 4'-Me-D > D > 7,4'-diMe-D > 7-Me-D, and change in fluorescence anisotropy of fluorescent probes bound to the membrane surface or inside the lipid bilayer confirmed the effects of isoflavones on the membrane fluidity, especially for 7,4'-diMe-D.

Anti-PEG IgM elicited by injection of liposomes is involved in the enhanced blood clearance of a subsequent dose of PEGylated liposomes

Earlier we reported that PEGylated liposomes lose their long-circulating characteristic when they are administrated twice in the same animal with certain intervals (referred to as the accelerated blood clearance (ABC) phenomenon). We proposed that anti-PEG IgM, induced by the PEGylated liposomes, is responsible for the phenomenon, based on the observation that IgM thus produced selectively binds to the surface of PEGylated liposomes, subsequently leading to substantial complement activation. Interestingly, we found that under certain circumstances administration of conventional liposomes without PEG-coating also caused a strong ABC response upon injection of a second dose of PEGylated liposomes, but not of conventional liposomes. This suggests that also conventional liposomes not modified with PEG can promote an IgM response against PEG. We report here that, irrespective of the presence or absence PEG-coating, a single first dose of liposomes is capable of inducing a strong anti-PEG IgM response and, under certain circumstances, also weak responses against other lipid components. A good correspondence was observed between the amount of IgM associating with both PEGylated and conventional liposomes, concomitant complement activation triggered by those liposomes and the magnitude of the ABC phenomenon against those liposomes. Taken together, our results demonstrate that the ABC phenomenon is fully attributable to production of anti-PEG IgM by the first dose of liposomes and the subsequent complement activation upon a second dose of PEGylated but not conventional liposomes. Although the responsible immunogenic epitopes of the liposomes remain to be determined, the immunogenicity of ‘empty’ liposomes presents a serious concern in the development of liposomal formulations and their use in the clinic. Furthermore, our findings as described here raise important concerns with regard to the safety and efficiency of liposomes currently under development for clinical use.

Preparation and evaluation of liposomes encapsulating synthetic MMP inhibitor (Ro 28-2653)—cyclodextrin complexes

In this study, preparation and evaluation of liposomes, intended for intravenous administration, encapsulating synthetic MMP inhibitor (Ro 28-2653) – cyclodextrin complexes were realized. An increase in Ro solubility, via formation of binary (Ro/HPβCD) or ternary (Ro/HPβCD/L-lysine) complexes, permitted a similar increase in encapsulation efficiency of liposomes (Table 1). Moreover, Ro release kinetics depend on the encapsulation efficiency.