Recent Biophysical Issues About the Preparation of Solute-Filled Lipid Vesicles

Here we report some recent biophysical issues on the preparation of solute-filled lipid vesicles and their relevance to the construction of “synthetic cells.” First, we introduce the “semi-synthetic minimal cells” as the liposome-based cell-like systems, which contain a minimal number of biomolecules required to display simple and complex biological functions. Next, we focus on recent aspects related to the construction of synthetic cells. Emphasis is given to the interplay between the methods of synthetic cell preparation and the physics of solute encapsulation. We briefly introduce the notion of structural and compositional “diversity” in synthetic cell populations.     

Novel strategy for encapsulation and targeted delivery of poorly water-soluble active substances

Carriers for targeted delivery and controlled release of poorly water-soluble active substances (PWSAS) are facing three challenges: (a) the encapsulation issues, (b) limitations of PWSAS water solubility, and (c) burst drug release which can be pharmacologically dangerous and economically inefficient. The present study brings a novel strategy for encapsulation and controlled release of PWSAS—caffeine in concentrations which are higher than its maximal water solubility without the possibility of burst effect. The modification of hydrophilic carrier based on poly(methacylic acid) was done using casein and liposomes. To further increase the maximal caffeine loading inside the carrier nicotinamide was used. The release study of the encapsulated PWSAS was elaborated with respect to morphology of the carriers and interactions that could be established between its structural components. The carriers swelling and the release of caffeine and nicotinamide were also investigated depending on caffeine concentration, the presence of different liposomal formulations and the volume ratio of liposomal formulation, in three media with different pH simulating the path of the carrier through the human gastrointestinal tract. The synthesized carriers are promising candidates for encapsulation of PWSAS in concentrations which are higher than its maximal water solubility and for the targeted delivery of those dosages.     

Solubilization of unilamellar liposomes by betaine-type zwitterionic/anionic surfactant systems

The mechanisms governing the solubilizing interactions between zwitterionic/anionic mixed surfactant systems at different molar fractions of the zwitterionic surfactant (X_zwitter) and neutral or electrically charged unilamellar liposomes were investigated. The mixed systems were formed by N-dodecyl-N,N-dimethylbetaine and sodium dodecyl sulfate in the presence of piperazine-1,4-bis-(2-ethanesulfonic acid) buffer at pH 7.20. Unilamellar liposomes formed by egg phosphatidylcholine, in some cases together with stearylamine or phosphatidic acid, were used. Solubilization was detected as a decrease in static light-scattering of liposomes. Two parameters were regarded as corresponding to the effective surfactant/lipid molar ratios (Re) at which the surfactant system (i) saturated the liposomes, Re_sat, and (ii) led to a total solubilization of liposomes, Re_sol. From these parameters the bilayer/aqueous medium surfactant partition coefficients for the saturation (K_sat) and complete bilayer solubilization (K_sol) were determined. When X_zwitter was 0.40, The Re and K parameters showed a maximum, whereas the critical micellar concentration (CMC) of these systems exhibited a minimum, regardless of the electrical charge of bilayers. Given that the ability of the surfactant systems to saturate or solubilize liposomes is inversely related to the Re_sat and Re_sol parameters, these capacities appear to be directly correlated with the CMC of the mixed systems. The similarity of both K_sat and K_sol (particularly for X_zwitter=0.2–0.8) suggests that a similar partition equilibrium governs both the saturation and the complete solubilization of bilayers, the free surfactant concentration (S_a,S_b), remaining almost constant with similar values to the CMC for each mixed system studied.     

Amino acid profiles and liposomes: Their role as chemosensory information carriers in the marine environment

Yellowfin tuna enhance their hunting success in the vast pelagic environment by using their sense of smell to detect intact (uninjured) prey that are beyond visual range. However, the olfactory cues that tuna use would normally face huge and rapid dilution in the open ocean. We demonstrate that these prey odors are complexed within biologically derived lipid structures that probably delay the dilution of the amino acids to subthreshold concentrations and provide persistent arousal and search cues for the tuna. This may be the first demonstration of an extracorporeal biological function for liposomes. Tuna may also form chemical search images to maximize feeding efficiency. We demonstrate that the amino acid profiles of various prey species are consistent over time and between schools, which makes the formation of search images feasible.     

Insights into the Action Mechanism of the Antimicrobial Peptide Lasioglossin III

Lasioglossin III (LL-III) is a cationic antimicrobial peptide derived from the venom of the eusocial bee Lasioglossum laticeps. LL-III is extremely toxic to both Gram-positive and Gram-negative bacteria, and it exhibits antifungal as well as antitumor activity. Moreover, it shows low hemolytic activity, and it has almost no toxic effects on eukaryotic cells. However, the molecular basis of the LL-III mechanism of action is still unclear. In this study, we characterized by means of calorimetric (DSC) and spectroscopic (CD, fluorescence) techniques its interaction with liposomes composed of a mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-rac-phosphoglycerol (POPG) lipids as a model of the negatively charged membrane of pathogens. For comparison, the interaction of LL-III with the uncharged POPC liposomes was also studied. Our data showed that LL-III preferentially interacted with anionic lipids in the POPC/POPG liposomes and induces the formation of lipid domains. Furthermore, the leakage experiments showed that the peptide could permeabilize the membrane. Interestingly, our DSC results showed that the peptide-membrane interaction occurs in a non-disruptive manner, indicating an intracellular targeting mode of action for this peptide. Consistent with this hypothesis, our gel-retardation assay experiments showed that LL-III could interact with plasmid DNA, suggesting a possible intracellular target.     

Feeding rats with liposomes or fish oil differently affects their lipid metabolism

Two n‐3 polyunsaturated fatty acid (PUFA)‐rich diets differing in their chemical and physical forms were given to rats during 2 wk. Liposomes [phospholipids (PL) organized in bilayer structures] made from a natural marine lipid extract or a mixture of fats containing fish oil [similar fatty acids esterified in triacylglycerols (TAG)] were used. The influence of n‐3 PUFA dietary sources on plasma parameters, i.e. TAG, cholesterol and PL concentrations, was investigated. A similar hypotriglyceridemic effect of n‐3 PUFA from liposomes or fish oil was observed. In contrast, feeding rats with liposomes led to different PL and cholesterol patterns. In the plasma of rats fed liposomes, total cholesterol amounts were positively correlated with PL levels. Liposome and fish oil feedings caused a marked increase in the amounts of n‐3 PUFA, which occurred mainly at the expense of n‐6 PUFA. However, the enrichment in n‐3 PUFA in the different plasma lipid classes differed substantially when ingested in the form of fish oil or liposomes. These results were interpreted in terms of different lipid bioavailability and metabolic fate during the digestive steps and in the liver, with the dietary source.     

Glycyrrhetinic Acid Liposomes Encapsulated Microcapsules from Microfluidic Electrospray for Inflammatory Wound Healing

In diabetic wound healing, M1 macrophage accumulation and elevated inflammation are prevalent issues. Intelligent delivery systems that can sustainably release antioxidizing and anti-inflammatory ingredients are expected for effective wound healing. Herein, a novel glycyrrhetinic acid (GA) liposomes encapsulated microcapsules delivery system that has desired features for inflammatory wound repair is presented. As the bacteria could break down the alginate shells, the GA liposomes could be controllably released from the microcapsules, which promotes M2 macrophage polarization and regulate their responses in the inflammatory wound microenvironment. Based on these, it is demonstrated that the GA liposomes encapsulated microcapsules delivery system exhibits an anti-inflammatory and immunomodulatory effect for diabetic wound healing in a full-thickness defect model in diabetic rats. These results indicate that the immunomodulatory capabilities of the microcapsules can be unitized for efficient wound repair, and such a delivery system is valuable for clinical wound healing applications.     

Artificial neural networks as alternative tool for minimizing error predictions in manufacturing ultradeformable nanoliposome formulations

This work was aimed at determining the feasibility of artificial neural networks (ANN) by implementing backpropagation algorithms with default settings to generate better predictive models than multiple linear regression (MLR) analysis. The study was hypothesized on timolol-loaded liposomes. As tutorial data for ANN, causal factors were used, which were fed into the computer program. The number of training cycles has been identified in order to optimize the performance of the ANN. The optimization was performed by minimizing the error between the predicted and real response values in the training step. The results showed that training was stopped at 10?000 training cycles with 80% of the pattern values, because at this point the ANN generalizes better. Minimum validation error was achieved at 12 hidden neurons in a single layer. MLR has great prediction ability, with errors between predicted and real values lower than 1% in some of the parameters evaluated. Thus, the performance of this model was compared to that of the MLR using a factorial design. Optimal formulations were identified by minimizing the distance among measured and theoretical parameters, by estimating the prediction errors. Results indicate that the ANN shows much better predictive ability than the MLR model. These findings demonstrate the increased efficiency of the combination of ANN and design of experiments, compared to the conventional MLR modeling techniques.     

Mucoadhesive liposomes as ocular delivery system: physical,microbiological, and in vivo assessment

Background: Mucoadhesive drug delivery is a promising strategy to overcome ocular biopharmaceutical constraints. Objective and methods: Ciprofloxacin HCl-loaded reverse phase evaporation liposomes were coated with different concentrations and molecular weights of mucoadhesive biocompatible chitosan polymer to form chitosomes. This colloidal mucoadhesive system was evaluated in vitro and in vivo with respect to deliver the antibiotic to ocular surface. Results and conclusion: The results obtained pointed out that liposome coating process resulted in entrapment efficiency reduction and higher chitosan concentration, and molecular weight showed a more pronounced effect. No morphological differences between coated and uncoated liposomes were observed. Diffusion was the drug release mechanism from chitosomes. Concerning rheological behavior, pseudoplastic flow was characteristic to the prepared chitosomal dispersions. In addition, chitosan coating improved the ocular permeation of ciprofloxacin HCl. Microbiologically; this formulated system enhanced antimicrobial activity of ciprofloxacin HCl against both Gram-positive and Gram-negative bacteria. Moreover, this mucoadhesive system was able to inhibit the growth of Pseudomonas aeruginosa in rabbits' eyes for 24 hours when compared to the marketed preparation. In vivo bacterial conjunctivitis model elucidated that symptoms were controlled by the prolonged release formulation such as that done by the marketed product.     

Effect of surface-mannose modification on aerosolized liposomal delivery to alveolar macrophages

Purpose: The effect of surface-mannose modification on aerosolized liposomal delivery to alveolar macrophages (AMs) was evaluated in vitro and in vivo. Method: 4-Aminophenyl-α-D-mannopyranoside (Man) was used for surface-mannose modification, and mannosylated liposomes with various mannosylation rates (particle size: 1000 nm) were prepared. Results: In the in vitro uptake experiments, the uptake of mannosylated liposomes by AMs was increased with the increase in the mannosylation rate over the range 2.4–9.1 mol% Man and became constant at over 9.1%. Thus, the most efficient mannosylation rate was 9.1 mol% Man. Furthermore, free mannose inhibited the uptake of mannosylated liposomes by AMs. This indicates that the uptake mechanism of mannosylated liposomes by AMs is mannose receptor-mediated endocytosis. In the in vivo animal experiments, the mannosylated liposomes (mannosylation rate, 9.1 mol% Man) were more efficiently delivered to AMs after pulmonary aerosolization to rats than nonmodified liposomes and did not harm lung tissues. Conclusion: These results indicate that surface-mannose modification is useful for efficient aerosolized liposomal delivery to AMs.