Uniform Liposomes Research Articles

Colorimetric sensor based on release control from liposomes by the formation of 2,2-bipyridine-Fe2+ complex

In colorimetric detection, an analyte may be determined both qualitatively and quantitatively by keeping track of color changes that seem to be perceptible to the unaided eye. The uniform liposomes with diameters of 100 nm were synthesized from sphingomyelin and cholesterol. We comprehensively assessed 2,2-bipyridine's capacity to release from liposomes as well as the color shift to dark red caused by the formation of complexes between Fe2+ and 2,2-bipyridine for colorimetric detection. In addition, we extensively evaluated and examined factors influencing liposomal membrane leakage, such as pH value change, bulkiness of the molecular structure (2,2-bipyridine and doxorubicin), and the membrane rupture mechanism of triton X100. To provide the best circumstances for the color sensor from these liposomes, pH conditions and concentrations of triton X100 and Fe2+ were optimized. In addition, it has been confirmed and thoroughly described how pH breaks the liposome membrane and how Fe2+-Bipy changes color. The capacity to release from liposomes throughout time was studied via analysis of kinetic release models, including Fick's law, the Higuchi model, and the Korsmeyer-Peppas model. Our approach develops a method to is straightforward, quick to detect, and easily applied. This research holds promise as color color-sensing method for detecting viruses and pathogens in the future.

Development of Robust Cationic Light-Activated Thermosensitive Liposomes: Choosing the Right Lipids.

Extensive research has been conducted on cationic light-activated thermosensitive liposomes (CLTSLs) as a means for site-specific and controlled drug release; however, less attention has been given to the stability of these nanoparticles. Selecting the appropriate lipids is crucial for the development of a stable and responsive system. In this study, we investigated the impact of various lipids on the physical properties of cationic light-activated liposomes. Incorporating poly(ethylene glycol) PEG molecules resulted in uniform liposomes with low polydispersity index, while the addition of unsaturated lipid (DOTAP) resulted in extremely leaky liposomes, with almost 80% release in just 10 min of incubation at body temperature. Conversely, the inclusion of cholesterol in the formulation increased liposome stability too much and decreased their sensitivity to stimuli-responsive release, with only 14% release after 2 min of light exposure. To achieve stable and functional CLTSL, we substituted an equivalent amount of unsaturated lipid with a saturated lipid (DPTAP), resulting in stable liposomes at body temperature that were highly responsive to light, releasing 90% of their content in 10 s of light exposure. We also conducted two atomistic molecular dynamics simulations using lipid compositions with saturated and unsaturated lipids to investigate the effect of lipid composition on the dynamical properties of the liposomal lipid bilayer. Our findings suggest that the nature of lipids used to prepare liposomes significantly affects their properties, especially when the drug loading needs to be stable but triggered drug release properties are required at the same time. Selecting the appropriate lipids in the right amount is therefore essential for the preparation of liposomes with desirable properties.

DNA Soccer-Ball Framework Templated Liposome Formation with Precisely Regulated Nucleation Seeds.

A soccer-ball-shaped three-dimensional DNA origami framework was assembled to serve as an exoskeleton and to direct liposome growth inside. With up to 90 available inner modification sites, cholesterol moieties were introduced as nucleation seeds, and the vesicle templating efficiency was systematically investigated with precisely regulated seed numbers and arrangements. We confirmed that a nonsaturated optimum number (n = 30) of nucleation seeds with relatively even spatial distribution was essential for achieving well-templated and highly uniform liposomes. The seed arrangement principles and effects and the liposome formation mechanisms are thoroughly discussed. The revealed key factors in the design and optimization of 3D DNA nanoframes for functional liposome production could benefit the fields of nanotechnology and molecular medicine.

Strategic application of liposomal system to R-α-lipoic acid for the improvement of nutraceutical properties

R-α-lipoic acid (RLA) and dihydrolipoic acid (DHLA), a reduced form of RLA, are potent endogenous antioxidants that can reduce oxidative damage. Despite their numerous nutraceutical potentials, clinical applications of RLA are still limited due to its poor solubility and stability problems. This study aimed to develop an RLA-loaded liposome (LIP/RLA) for the improvement of nutraceutical properties. LIP/RLA was developed by a typical solvent injection method. Uniform liposomes of LIP/RLA were observed by transmission electron microscopy, and the mean particle size was calculated to be ∼150 nm from the data of dynamic light scattering. LIP/RLA could prevent the degradation of RLA even under acidic conditions (pH 1.2) possibly due to the encapsulation of RLA into the liposomal structure. In the release test under pH6.8 with lipase, LIP/RLA showed relatively rapid release of RLA, possibly due to the lipolysis of phospholipids by lipase. After the oral administration of LIP/RLA (10 mg-RLA/kg, p.o.) in rats, the systemic exposures of RLA and DHLA increased by 2.8- and 5.8-fold, respectively. In a rat model of acute hepatic injury induced by carbon tetrachloride (CCl4) (0.7 mL-CCl4/kg, p.o.), orally dosed LIP/RLA (3 mg-RLA/kg, p.o.) resulted in 78.7% and 86.4% reductions of plasma alanine aminotransferase, and aspartate aminotransferase, respectively; however, RLA was found to be less effective possibly due to the poor oral absorption. The RLA-loaded liposomal system might be a promising carrier for poorly water-soluble materials with poor stability under acidic conditions, as well as RLA, to improve their oral absorption and nutraceutical properties.

Modified Spraying Technique and Response Surface Methodology for the Preparation and Optimization of Propolis Liposomes of Enhanced Anti-Proliferative Activity against Human Melanoma Cell Line A375.

Propolis is a honeybee product that contains a mixture of natural substances with a broad spectrum of biological activities. However, the clinical application of propolis is limited due to the presence of a myriad of constituents with different physicochemical properties, low bioavailability and lack of appropriate formulations. In this study, a modified injection technique (spraying technique) has been developed for the encapsulation of the Egyptian propolis within liposomal formulation. The effects of three variables (lipid molar concentration, drug loading and cholesterol percentage) on the particle size and poly dispersity index (PDI) were studied using response surface methodology and the Box–Behnken design. Response surface diagrams were used to develop an optimized liposomal formulation of the Egyptian propolis. A comparative study between the optimized liposomal formulation prepared either by the typical ethanol injection method (TEIM) or the spraying method in terms of particle size, PDI and the in-vitro anti-proliferative effect against human melanoma cell line A375 was carried out. The spraying method resulted in the formation of smaller propolis-loaded liposomes compared to TEIM (particle sizes of 90 ± 6.2 nm, and 170 ± 14.7 nm, respectively). Furthermore, the IC50 values against A375 cells were found to be 3.04 ± 0.14, 4.5 ± 0.09, and 18.06 ± 0.75 for spray-prepared propolis liposomes (PP-Lip), TEIM PP-Lip, and propolis extract (PE), respectively. The encapsulation of PE into liposomes is expected to improve its cellular uptake by endocytosis. Moreover, smaller and more uniform liposomes obtained by spraying can be expected to achieve higher cellular uptake, as the ratio of liposomes or liposomal aggregates that fall above the capacity of cell membrane to “wrap” them will be minimized.

Berberine-loaded solid proliposomes prepared using solution enhanced dispersion by supercritical CO2: Sustained release and bioavailability enhancement

In present study, berberine proliposomes (ber-PL) of powder state that can form liposomal structure via self-assemble in water was prepared using solution enhanced dispersion by supercritical CO2 (SEDS) to improve the oral bioavailability of berberine. Several important parameters were optimized using Box-Behnken design to get ber-PL with high entrapment efficacy (EE) of 90.3% ± 4.9%. Physicochemical characterization showed the optimum ber-PL was amorphous spherical nanoparticles and could form uniform liposomes around 80 nm easily via hydration. EE was closely related to the particle morphology of ber-PL. In vitro drug release study displayed a sustained release profile, and the drug released faster in neutral condition than acid medium. The oral bioavailability for ber-PL was 22.47 times greater than raw berberine, and the mean residence time of berberine in plasma was extended from 6.82 ± 2.99 to 10.83 ± 2.01 h as well. The proliposome formulated using SEDS is a favorable strategy to improve the bioavailability of berberine.

Abstract 4416: Reversal of chemoresistance in ovarian cancer cells by the liposomal co-delivery of MDR inhibitors and paclitaxel

Abstract Ovarian cancer is one of the most lethal gynaecologic malignancies. This can be attributed to the emergence of multidrug resistance in the clinic due to the over-expression of drug-efflux pumps such as P-gp. To overcome this, we have developed a liposomal platform for the the co-delivery of potent P-gp inhibitors with chemotherapeutic drugs. Purpose: To allow for the effective reversal of chemo-resistance in ovarian cancer cells using liposomes co-loaded with tariquidar and paclitaxel. Methods: PEGylated liposomes composed of eggphosphatidylcholine, cholesterol and DOTAP loaded with equimolar amounts of tariquidar (XR) and paclitaxel (PCT) were prepared by the thin film hydration method followed by extrusion. Evaluation of drug resistance of ovarian cancer lines SKOV3, HeyA8 and Tynku was carried out by quantifying MDR1 and MRP1 receptor expression followed by a rhodamine123 exclusion assay. The cytotoxicity of the formulations on the above cancer cell lines was evaluated using the Promega cell viability assay. The effects of the formulations were evaluated in fluorescently stained cells using the iCyte® imaging cytometer (Thorlabs, USA). Long-term kinetic analysis of unstained live cultures was perfomed by holographic imaging cytometer Holomonitor ® M4 (Phase Holographic Imaging, Sweden) and fluorescence microscopy. Results: The liposomes had a particle size distribution of about 200 nm with a zeta potential of 30 mV. As compared to MRP1, a 2.4 fold, 1.9-fold and 1.6-fold higher expression of MDR1 was seen in the HeyA8-MDR, SKOV3-TR and Tynku-R cells respectively over their drug-sensitive counterparts. Using the rhodamine exclusion assay, 14 nM of liposomal XR showed better rhodamine inclusion than 140 nM free XR. In vitro cytotoxicity experiments showed that the drug co-loaded formulation was able to effectively overcome resistance to PCT even at low PCT doses. Using live-cell imaging, paclitaxel-induced arrest of cell cycle progression and onset of apoptosis following this mitotic catastrophe was visible from 1.5 μM to 50 nM only with the co-loaded liposomes. The cell cycle arrest was further visualized using time-lapse Holomonitor M4 which showed the increasing incidence of cells arrested in mitosis over time. Using fluorescence microscopy, we were able to clearly visualize the presence of polymerized microtubules around the cell nucleus as a result of the paclitaxel-mediated microtubule dysfunction. Conclusions: Uniform liposomes co-loaded with tariquidar and paclitaxel were prepared and characterized. The co-loaded liposomes were able to effectively reverse chemoresistance and induce cytotoxicity in a variety of drug-resistant ovarian cancers. The formulations were shown to arrest cell cycle, preventing its progression to the G1 phase. This arrest of cell cycle was shown to be due to the paclitaxel-mediated micro-tubule dysfunction. Note: This abstract was not presented at the meeting. Citation Format: Shravan K. Sriraman, Yilin Zhang, Ed Luther, Ernst Lengyel, Vladimir Torchilin, Vladimir Torchilin. Reversal of chemoresistance in ovarian cancer cells by the liposomal co-delivery of MDR inhibitors and paclitaxel. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4416. doi:10.1158/1538-7445.AM2015-4416

A facile route to the synthesis of monodisperse nanoscale liposomes using 3D microfluidic hydrodynamic focusing in a concentric capillary array.

A novel microscale device has been developed to enable the one-step continuous flow assembly of monodisperse nanoscale liposomes using three-dimensional microfluidic hydrodynamic focusing (3D-MHF) in a concentric capillary array. The 3D-MHF flow technique displays patent advantages over conventional methods for nanoscale liposome manufacture (i.e., bulk-scale alcohol injection and/or sonication) through the on-demand synthesis of consistently uniform liposomes without the need for post-processing strategies. Liposomes produced by the 3D-MHF device are of tunable size, have a factor of two improvement in polydispersity, and a production rate that is four orders of magnitude higher than previous MHF methods, which can be attributed to entirely radially symmetric diffusion of alcohol-solvated lipid into an aqueous flow stream. Moreover, the 3D-MHF platform is simple to construct from low-cost, commercially-available components, which obviates the need for advanced microfabrication strategies necessitated by previous MHF nanoparticle synthesis platforms.

Liposomal Drug Products and Recent Advances in The Synthesis of Supercritical Fluid-Mediated Liposomes

Since the pioneering research of Bangham et al. in 1965, liposomes have attracted a large amount of interest as potential carriers of various bioactive molecules for clinical applications. However, scaling-up conventional methods of liposome preparation has been proven to be challenging. Compared with conventional methods, processes that use supercritical fluid (SCF)-CO2 require a reduced amount of organic solvent, are relatively fast and simple to perform, and yield stable and more uniform liposomes. A number of studies have demonstrated that SCF-CO2 methods might be suitable for industrial-scale manufacturing of liposomes. In this review there are two topics being discussed. We provide an overview of liposomal drug products and aim to describe the physicochemical properties of liposomes prepared using various SCF methods. We review all of the available literature on SCF-CO2-based liposomes and focus on the future applications of these innovative technologies in industrial-scale liposome preparation.

Preparation of giant unilamellar vesicles from damp lipid film for better lipid compositional uniformity

Giant unilamellar vesicles (GUVs) containing cholesterol often have a wide distribution in lipid composition. In this study, GUVs of 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC)/1,2-distearoyl-sn-glycero-3-phosphocholine(DSPC)/cholesterol and 1,2-diphytanoyl-sn-glycero-3-phosphocholine(diPhyPC)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine(DPPC)/cholesterol were prepared from dry lipid films using the standard electroformation method as well as a modified method from damp lipid films, which are made from compositional uniform liposomes prepared using the Rapid Solvent Exchange (RSE) method. We quantified the lipid compositional distributions of GUV by measuring the miscibility transition temperature of GUVs using fluorescence microscopy, since a narrower distribution in the transition temperature should correspond to a more uniform distribution in GUV lipid composition. Cholesterol molecules can demix from other lipids in dry state and form cholesterol crystals. Using optical microscopy, micron-sized crystals were observed in some dry lipid films. Thus, a major cause of GUV lipid compositional heterogeneity is the demixing of lipids in the dry film state. By avoiding the dry film state, GUVs prepared from damp lipid films have a better uniformity in lipid composition, and the standard deviations of miscibility transition temperature are about 2.5 times smaller than that of GUVs prepared from dry lipid films. Comparing the two ternary systems, diPhyPC/DPPC/cholesterol GUVs has a larger cholesterol compositional heterogeneity, which directly correlates with the low maximum solubility of cholesterol in diPhyPC lipid bilayers (40.2±0.5mol%) measured by light scattering. Our data indicate that cholesterol interacts far less favorably with diPhyPC than it does with other PCs. The damp lipid film method also has a potential of preparing GUVs from cell membranes containing native proteins without going through a dry state.

Conventional and Dense Gas Techniques for the Production of Liposomes: A Review

The aim of this review paper is to compare the potential of various techniques developed for production of homogenous, stable liposomes. Traditional techniques, such as Bangham, detergent depletion, ether/ethanol injection, reverse-phase evaporation and emulsion methods, were compared with the recent advanced techniques developed for liposome formation. The major hurdles for scaling up the traditional methods are the consumption of large quantities of volatile organic solvent, the stability and homogeneity of the liposomal product, as well as the lengthy multiple steps involved. The new methods have been designed to alleviate the current issues for liposome formulation. Dense gas liposome techniques are still in their infancy, however they have remarkable advantages in reducing the use of organic solvents, providing fast, single-stage production and producing stable, uniform liposomes. Techniques such as the membrane contactor and heating methods are also promising as they eliminate the use of organic solvent, however high temperature is still required for processing.

Phospholipid Nanosomes

Phospholipid nanosomes are small, uniform liposomes manufactured utilizing supercritical fluid technologies. Supercritical fluids are first used to solvate phospholipid raw materials, and then decompressed to form phospholipid nanosomes that can encapsulate hydrophilic molecules such as proteins and nucleic acids. Hydrophobic therapeutics are co-solvated with phospholipid raw materials in supercritical fluids that, when decompressed, form phospholipid nanosomes encapsulating these drugs in their lipid bilayers. Mathematical modeling and semi-empirical experiments indicate that the size and character of phospholipid nanosomes depend on the several process parameters and material properties including the size and design of decompression nozzle, bubble size, pressure and the rate of decompression, interfacial forces, charge distribution and the nature of compound being encapsulated. Examples are presented for the encapsulation of a protein and hydrophobic drugs. In vitro and in vivo data on breast cancer cells and xenografts in nude mice indicate that paclitaxel nanosomes are less toxic and much more effective than paclitaxel in Cremophor EL (Taxol). Camptothecin nanosomes demonstrate that the normally very water-insoluble camptothecin can be formulated in a biocompatible aqueous medium while retaining in vivo efficacy against lymphoma xenografts in nude mice. In vitro data for betulinic acid nanosomes demonstrate enhanced efficacy against HIV-1 (EC50 of 1.01 microg/ml versus 6.72 microg/ml for neat betulinic acid). Phospholipid nanosomes may find utility in the enhanced delivery of hydrophilic drugs such as recombinant proteins and nucleic acid as well as hydrophobic anticancer and anti-HIV drugs.

Chemotherapy with hybrid liposomes without any drug in vivo

Prolonged survival was seen in a carcinoma model in mice intraperitoneally inoculated with B-16 melanoma cells after the intraperitoneal treatment with hybrid liposomes composed of L-alpha-dimyristoylphosphatidylcholine (DMPC) and polyoxyethylenedodecyl ether (C12(EO)n, n = 10 and 23 respectivery) which had a uniform and stable structure. No drug was administered. The therapeutic effects of the single-component liposomes composed of lipids with a variety of hydrophilic head groups and different hydrophobic alkyl chains were investigated. Markedly prolonged survival (248%) of mice was achieved after treatment with DMPC liposomes. However, DMPC liposomes have the disadvantage of an unstable structure, requiring daily sonication. On the other hand, no life-prolonging effects or toxicity occurred with the administration of the other single-component liposome employed in this study. Next, we successfully prepared stable, uniform liposomes composed of 90 mol% DMPC and 10 mol% C12(EO)n (n = 10 and 23, respectively), which have diameters of 70 nm and 100 nm, respectively. Interestingly, prolonged survival (173-186%) of mice was achieved after treatment with hybrid liposomes of 90 mol% DMPC/10 mol% C12(EO)n (n = 10 and 23). Finally, we conducted toxicity tests using normal rats to determine hybrid liposome stability. There were no abnormal findings in blood chemistry or relative organ weights at autopsy of normal rats after hybrid liposome administration. In addition, hybrid liposomes were metabolized in the liver after intravenous administration to normal mice. These results suggest that hybrid liposomes could be used as a new single chemotherapeutic agent in the treatment of carcinoma with no side effects.

The Reliability of Activated Partial Thromboplastin Time Methods and the Relationship to Lipid Composition and Ultrastructure

Wide variations in procoagulant properties, lipid composition and ultrastructure of five commonly used APTT methods have been demonstrated. Performance of the methods with a range of coagulation abnormalities has been ranked. Most of the reagents obtained a high score with one or more defects, but a low score with others. A consistent good ranking throughout was only observed with one reagent. The number of significant correlations between the reagents' procoagulant activities and lipid content confirms the view that the performance of an APTT method is largely dependent upon its lipid composition. Marked differences in concentration and distribution of phospholipids, fatty acids and neutral lipids were evident. The importance of the concentration of phosphatidyl serine in regulating the procoagulant activity of an APTT method has been demonstrated. Electron microscopy provides evidence of the contrasting composition of the reagents from the more discrete uniform liposomes present in the more reliable reagents, to more ill-defined components present in those reagents which performed less well. The study highlights the need for standardisation of the APTT.