Entrapment of Ovalbumin into Liposomes—Factors Affecting Entrapment Efficiency,Liposome Size,and Zeta Potential

Various amounts of Ovalbumin (OVA) were encapsulated into positively and negatively charged multilamellar liposomes, with the aim to investigate the entrapment efficiency in different buffers and to study their effects on the liposome size and zeta potential. Results showed that the entrapment efficiency of OVA in anionic liposomes was the same in 10 mM Phosphate Buffer (PB) as in Phosphate-Buffered Saline (PBS; PB?+?0.15 M NaCl). Also, liposome size was approximately 1200 nm for all anionic liposomes incorporating OVA. The entrapment efficiency of OVA in cationic liposomes was highly dependent on ionic strength. The size of cationic liposomes was approximately 1200 nm in PBS, regardless of protein content, but increased with the amount of the incorporated protein in PB. Aggregation of cationic liposomes in PB was observed when the mass of the protein was 2.5 mg or greater. The zeta potential of anionic liposomes was negative and of cationic liposomes positive in the whole range of protein mass tested. These results show how different compositions of lipid and aqueous phases can be used to vary the entrapment efficiency, liposome size, and zeta potential—the factors that are of great importance for the use of liposomes as drug carriers.     

Evaluation of the high-pressure extrusion technique as a method for sizing plasmid DNA-containing cationic liposomes

A promising strategy to improve the immunogenic potential of DNA vaccines is the formulation of plasmid DNA (pDNA) with cationic liposomes. In this respect, particle size may be of crucial importance. This study aimed at the evaluation of high-pressure extrusion as a method for sizing cationic liposomes after entrapment of pDNA. This is a well-known sizing method for liposomes, but so far, it has not been applied for liposomes that are already loaded with pDNA. Liposomes composed of egg PC, DOTAP, and DOPE with entrapped pDNA were prepared by the dehydration-rehydration method and subjected to various extrusion cycles, comparing different membrane pore sizes and extrusion frequencies. At optimized extrusion conditions, liposome diameter (Zave) and polydispersity index (PDI) were reduced from 560 nm and 0.56-150 nm and 0.14 respectively, and 35% of the pDNA was retained. Importantly, gel electrophoresis and transfection experiments with pDNA extracted from these extruded liposomes demonstrated the preservation of the structural and functional integrity of the pDNA. The reduction in size resulted in enhanced transfection of HeLa cells, as detected by functional expression of the fluorescent protein, eGFP. In addition, these liposomes were able to stimulate Toll-like receptor 9, indicating efficient endosomal uptake and release of the included pDNA. In conclusion, high-pressure extrusion is a suitable technique to size cationic liposomes with entrapped pDNA and allows preparation of well-defined nanosized pDNA-liposomes, with preserved pDNA integrity. Their improved transfection efficiency and ability to activate an important pattern-recognition receptor are favorable properties for DNA vaccine delivery vehicles.     

Effect of diglucosamine on the entrapment of protein into liposomes

Liposomes, which had entrapped bovine serum albumin (BSA), were modified with diglucosamine by two methods. The liposome was prepared by a freeze-thawing method in the presence of the disaccharide, or the disaccharide was added to the liposome prepared in advance without it. To examine the effects of diglucosamine, the morphology, mean particle size, and zeta potential of both liposomes were compared with those of BSA-entrapping liposome prepared without the disaccharide. Diglucosamine caused no remarkable change in shape and no aggregation of the liposome. The presence of the disaccharide was confirmed on the surfaces of modified liposomes, and the entrapment of BSA into the liposomes was increased by the disaccharide. The entrapment behavior was affected by the way the disaccharide was added, and the difference in the way the BSA was entrapped was also indicated.     

Effect of Diglucosamine on the Entrapment of Protein into Liposomes

Liposomes, which had entrapped bovine serum albumin (BSA), were modified with diglucosamine by two methods. The liposome was prepared by a freeze-thawing method in the presence of the disaccharide, or the disaccharide was added to the liposome prepared in advance without it. To examine the effects of diglucosamine, the morphology, mean particle size, and zeta potential of both liposomes were compared with those of BSA-entrapping liposome prepared without the disaccharide. Diglucosamine caused no remarkable change in shape and no aggregation of the liposome. The presence of the disaccharide was confirmed on the surfaces of modified liposomes, and the entrapment of BSA into the liposomes was increased by the disaccharide. The entrapment behavior was affected by the way the disaccharide was added, and the difference in the way the BSA was entrapped was also indicated.     

Oral delivery of antigens in liposomes with some lipid compositions modulates oral tolerance to the antigens

Several liposomes containing ovalbumin (OVA), a model antigen, with different lipid compositions were prepared in order to evaluate their ability to induce oral tolerance. Oral administration of these liposomal OVAs induced suppression of the proliferative responses of popliteal lymph node cells from the treated mice to OVA, suggesting that these treated mice were tolerized. The efficiency of the induction of oral tolerance was affected by the liposome composition. OVA entrapment in these liposomes could modulate the tolerizing dose of OVA itself. These results suggest that some liposomes can be suitable antigen-delivery systems for modulated and/or effective induction of oral tolerance.     

Electrostatics of carboxylated anionic vesicles for improving entrapment capacity

Electrostatic interaction is an important secondary force affecting the structure, stability, and function of lipid vesicles (liposomes). For this study, a negatively charged lipid with carboxylic acid was mixed with phospholipid to produce anionic vesicles. The electrostatics of the carboxylated anionic vesicle (ca. 200 nm diameter) was determined and correlated with entrapment capacity of the vesicles. Correlative analysis revealed the zeta potential of the vesicles as a factor quantitatively affecting the entrapment capacity for a water-soluble marker, in which the entrapment capacity reached its maximum level in less than −30 mV of zeta potential. Transmission electron microscopy (TEM) revealed that the vesicles with high entrapment capacity are composed of a unilamellar membrane. This finding is expected to be useful for efficient encapsulation of water-soluble pharmaceuticals within vesicles.     

Temperature-controlled interaction of thermosensitive polymer-modified cationic liposomes with negatively charged phospholipid membranes

To obtain cationic liposomes of which affinity to negatively charged membranes can be controlled by temperature, cationic liposomes consisting of 3beta-[N-(N', N'-dimethylaminoethane)carbamoyl]cholesterol and dioleoylphosphatidylethanolamine were modified with poly(N-acryloylpyrrolidine), which is a thermosensitive polymer exhibiting a lower critical solution temperature (LCST) at ca. 52 degrees C. The unmodified cationic liposomes did not change its zeta potential between 20-60 degrees C. The polymer-modified cationic liposomes revealed much lower zeta potential values below the LCST of the polymer than the unmodified cationic liposomes. However, their zeta potential increased significantly above this temperature. The unmodified cationic liposomes formed aggregates and fused intensively with anionic liposomes consisting of egg yolk phosphatidylcholine and phosphatidic acid in the region of 20-60 degrees C, due to the electrostatic interaction. In contrast, aggregation and fusion of the polymer-modified cationic liposomes with the anionic liposomes were strongly suppressed below the LCST. However, these interactions were enhanced remarkably above the LCST. In addition, the polymer-modified cationic liposomes did not cause leakage of calcein from the anionic liposomes below the LCST, but promoted the leakage above this temperature as the unmodified cationic liposomes did. Temperature-induced conformational change of the polymer chains from a hydrated coil to a dehydrated globule might affect the affinity of the polymer-modified cationic liposomes to the anionic liposomes.     

The transfection of Jurkat T-leukemic cells by use of pH sensitive immunoliposomes

A gene transfer vector has been developed utilising anionic liposomes as a carrier of plasmid DNA (pEGlacZ, 7.6 kb) to transfect CD3+ T lymphocytes (Jurkat cells). The plasmid DNA that contained the Escherichia coli beta-galactosidase reporter gene was condensed using poly-l-lysine of molecular mass 20,700 (PLK99) to form a polyplex which was interacted with several anionic liposome formulations to form lipopolyplexes. The liposome formulations where based on dioleoylphosphatidylethanolamine (DOPE) in combination with cholesterol and dioleoylphosphatidylcholine (DOPC) and oleic acid, or dimyristoylphosphatidylethanolamine (DMPE). For targeting to the Jurkat cells distearoylphosphatidylethanolamine (DSPE) linked to poly (ethylene glycol) molecular mass 2,000 and coupled to anti-CD3 antibody was incorporated. The polyplexes and lipopolyplexes were characterised in terms of size, zeta potential, agarose gel electrophoresis and electron microscopy and the permeability of the lipopolyplexes to liposome-encapsulated glucose was determined. The polyplexes consisted of a mixed population of rod-like structures (53-160 nm long and 23-31 nm diameter) and spheres (18-30 nm diameter). The lipopolyplexes retained a permeability barrier although were more permeable to glucose than their component liposomes. The poly-l-lysine condensing agent was still susceptible to pronase digestion suggesting that the polyplex was associated with the outer surface of the liposome. The lipopolyplexes with lipid composition DOPE/cholesterol/OA/DSPE-PEG2000 anti-CD3+ PLK99-plasmid DNA had significant gene transfer activity, as monitored by beta-galactosidase expression, that depended on the charge ratio of the component polyplex and the lipid/DNA weight ratio. The anti-CD3 antibody, the liposomal lipid and pH sensitivity were essential for transfection activity.     

Effect of DNA complexation and freeze-drying on the physicochemical characteristics of cationic liposomes

We describe the use of saccharides, such as sorbitol, mannitol, sucrose, maltodextrin, and dextran, as cyoprotectants for freeze-drying cationic liposomes. Saccharides can protect liposomes either by interacting with phospholipid headgroups or by forming an amorphous glass surrounding the vesicles, thus preventing aggregation, mechanical rupture of membrane, fusion of liposomes, and drug leakage. We have particularly considered liposome characteristics, such as size, zeta potential, and ability in complexing DNA, before and after freeze-drying. Our study indicates that cationic liposomes are able to maintain liposome characteristics after lyophilization and rehydration and maintain the ability to complex DNA even if the strength of the interaction forces was of lower intensity with respect to liposomes before lyophilization.     

Investigations of the effect of the lipid matrix on drug entrapment, in vitro release, and physical stability of olanzapine-loaded solid lipid nanoparticles

The purpose of this research was to study the effect of the lipid matrix on the entrapment of olanzapine (OL). OL-loaded solid lipid nanoparticles (SLNs) were prepared using lipids like glyceryl monostearate (GMS), Precirol ATO 5 (PRE), glyceryl tristearate (GTS), and Witepsol E85 (WE 85)--and poloxamer 407 and hydrogenated soya phosphatidylcholine as stabilizers--using a hot melt emulsification high-pressure homogenization technique, and then characterized by particle size analysis, zeta potential, differential scanning calorimetry (DSC), and powder X-ray diffraction (pXRD). Homogenization at 10,000 psi for 3 cycles resulted in the formation of SLNs with a mean particle size of approximately 190 nm for the 4 lipids investigated. The highest partition coefficient for OL between the melted lipid and pH 7.4 phosphate buffer (pH 7.4 PB) was obtained with GTS. The entrapment efficiency was in the following order: GTS SLNs > PRE SLNs > WE 85 SLNs > GMS SLNs. DSC and pXRD showed that much of the incorporated fraction of OL existed in the amorphous state after incorporation into SLNs. A sharp increase in the flocculation of the SLN dispersions was observed upon addition of 0.6 M aqueous sodium sulfate solution. Nanoparticle surface hydrophobicity was in the following order: GTS SLNs > PRE SLNs > WE 85 SLNs > GMS SLNs. A significant increase in size and zeta potential was observed for GTS SLN and WE 85 SLN dispersions stored at 40 degrees C. Release of OL from the SLNs was sustained up to 48 hours in pH 7.4 PB and obeyed Higuchi's release kinetics.     

Lipid composition of cationic nanoliposomes implicate on transfection efficiency

Abstract

Cationic liposome (CL)-DNA complexes (lipoplexes) have appeared as leading nonviral gene carriers in worldwide gene therapy clinical trials. Arriving at therapeutic dosages requires the full understanding of the mechanism of transfection. However, using CLs to deliver therapeutic nucleic acids and drugs to target organs have some problems, including low transfection efficiency. The aim of this study was developing novel CLs containing four neutral lipids; cholesterol, 1,2-dioleoyl phosphatidylethanolamine, distearoylphosphatidylcholine and dipalmitoylphosphatidylcholine as a helper lipid and dimethyl dioctadecyl ammonium bromide as a cationic lipid to increase transfection efficiency. We have investigated the correlation between number of lipid composition and transfection efficiency. The morphology, size and zeta potential of liposomes and lipoplexes were measured and lipoplexes formation was monitored by gel retardation assay. Transfection efficiency was assessed using firefly luciferase reporter assay. It was found that transfection efficiency markedly depended on liposome to plasmid DNA (pDNA) weight ratio, lipid composition and efficiency of pDNA entrapment. High transfection efficiency of plasmid by four component lipoplexes was achieved. Moreover, lipoplexes showed lower transfection efficiency and less cytotoxicity compared to Lipofectamine?. These results suggest that lipid composition of nanoliposomes is an important factor in control of their physical properties and also yield of transfection.     

Entrapment of soy bean trypsin inhibitor and alpha1-antitrypsin by multilamellar liposomes.

The broad-spectrum protease inhibitors, soy bean trypsin inhibitor and α₁-antitrypsin, were entrapped within anionic multilamellar liposomes; the efficiency of entrapment was 12% for α₁-antitrypsin and 14% for soy bean trypsin inhibitor. Entrapment of ³H-labeled d-glucose, a marker for the aqueous compartment, was dependent upon the nature and concentration of coentrapped antiprotease. Rechromatography of the pooled liposome fractions following solubilization with the detergent Triton X-100 demonstrated latency of the entrapped materials. In addition, entrapment of soy bean trypsin inhibitor, as well as ³H-labeled d-glucose, was shown to be proportional to the net anionie surface charge in the lipid bilayers, suggesting sequestration within the aqueous compartments of the liposomes. This localization was also indicated by the minimal adsorption of antiproteases to anionic liposomes, contrasted with the extensive electrostatic binding of the antiproteases to cationic liposomes.     

Nanostructure of cationic lipid-oligonucleotide complexes

Complexes (lipoplexes) between cationic liposomes and single-strand oligodeoxynucleotides (ODN) are potential delivery systems for antisense therapy. The nanometer-scale morphology of these assemblies is relevant to their transfection efficiency. In this work the monocationic lipid dioleoyloxytrimethylammoniumpropane, the neutral "helper" lipid cholesterol, and an 18-mer anti-bcl2 ODN were combined at different ratios. The lipoplexes formed were characterized for the quantity of ODN bound, for the degree of lipid mixing, and for their size. The nanostructure of the system was examined by cryogenic-temperature transmission electron microscopy, augmented by small-angle x-ray scattering. Addition of ODN to cationic liposomes induced both liposome aggregation and the formation of a novel condensed lamellar phase. This phase is proposed to be stabilized by anionic single-strand ODN molecules intercalated between cationic bilayers. The proportion of cholesterol present apparently did not affect the nature of lipoplex microstructure, but changed the interlamellar spacing.     

Preparation and characterization of galactose-modified liposomes by a nonaqueous enzymatic reaction

In this study, NOH (NOH?=?N-octadecyl-4-[(D-galactopyranosyl)oxy]-2,3,5,6-tetrahydroxy hexanamide) was enzymatically synthesized as a targeting molecule and incorporated into liposomes to prepare a liposome surface modified with galactose. Glycyrrhetinic-acid-loaded liposome (GA-LP) and glycyrrhetinic-acid-loaded liposome surface modified with galactose (NOH-GA-LP) were prepared by the ethanol-injection method. NOH-GA-LP was characterized by morphology, particle size, zeta potential, encapsulation efficiency, release in vitro, and stability. The size of spherical particles was in the range of 179-211?nm. Spherical particles exhibit a positive electrical charge (38.7 mV) and possess high encapsulation efficiency (91.3%) and show sustained release (72% over 48 hours) in vitro. This novel approach for the liposome surface modified with galactose by enzymatic synthesis is expected to provide potential application as a drug carrier for active targeted delivery to hepatocytes.     

Ultradeformable liposomes with terpenes for delivery of hydrophilic compound

Ultradeformable liposomes containing penetration enhancers were created to deliver NaFl. Vesicles were investigated for their particle size, zeta potential, NaFl entrapment efficiency (%EE), loading efficiency, and in vitro skin penetration. The vesicles obtained were spherical in shape, with a particle size of less than 100 nm and a negative surface charge (-6 to -11 mV). The %EE of NaFl loaded in vesicles ranged from 37 to 48%. Ultradeformable liposomes with monoterpenes (d-limonene, 1,8-cineole and geraniol) significantly improved NaFl penetration through the skin. Confocal laser scanning microscopy analysis confirmed skin-penetration results and was used to evaluate the behavior of hydrophilic compounds penetrating through the skin.     

The transfection of Jurkat T-leukemic cells by use of pH-sensitive immunoliposomes

An effective pH-sensitive gene transfer vector has been developed utilising anionic liposomes with various formulations as a carrier of plasmid DNA (pEGlacZ, 7.6 kb) to transfect CD3 T+ lymphocytes (Jurkat cells). The plasmid DNA was condensed using poly-l-lysines with a range of molecular masses to form polyplexes that were interacted with several anionic liposome formulations to form lipopolyplexes. For targeting to the Jurkat cells, distearoylphosphatidylethanolamine (DSPE) linked to poly (ethylene glycol) molecular mass 2000 and coupled to anti-CD3 antibody was incorporated in the liposomes. The polyplexes and lipopolyplexes were characterised in terms of size, zeta potential, gel electrophoresis and electron microscopy. The gene transfer activity of the lipopolyplexes, assessed from beta-galactosidase expression, depended on the charge ratio (NH(3)+/PO(4)-) of the component polyplex and the lipid/DNA weight ratio of the lipopolyplex.     

6-mercaptopurine and daunorubicin double drug liposomes-preparation, drug-drug interaction and characterization

This article addresses and investigates the dual incorporation of daunorubicin (DR) and 6-mercaptopurine (6-MP) in liposomes for better chemotherapy. These drugs are potential candidates for interaction due to the quinone (H acceptor) and hydroxyl (H donor) groups on DR and 6-MP, respectively. Interactions between the two drugs in solution were monitored by UV/Vis and fluorescence spectroscopy. Interaction between the two drugs inside the liposomes was evaluated by HPLC (for 6-MP) and by fluorescence spectroscopy (for daunorubicin) after phospholipase-mediated liposome lysis. Our results provide evidence for the lack of interaction between the two drugs in solution and in liposomes. The entrapment efficiencies of 6-MP in the neutral Phosphatidyl choline (PC):Cholesterol (Chol):: 2:1 and anionic PC:Chol:Cardiolipin (CL) :: 4:5:1 single and double drug liposomes were found to be 0.4% and 1.5% (on average), respectively. The entrapment efficiencies of DR in the neutral and anionic double drug liposomes were found to be 55% and 31%, respectively. The corresponding entrapment of daunorubicin in the single drug liposomes was found to be 62% on average. Our thin layer chromatography (TLC) and transmission electron microscopy (TEM) results suggest stability of lipid and liposomes, thus pointing plausible existence of double drug liposomes. Cytotoxicity experiments were performed by using both single drug and double drug liposomes. By comparing the results of phase contrast and fluorescence microscopy, it was observed that the double drug liposomes were internalized in the jurkat and Hut78 (highly resistant cell line) leukemia cells as viewed by the fluorescence of daunorubicin. The cytotoxicity was dose dependent and had shown a synergistic effect when double drug liposome was used.     

Ultradeformable liposomes with terpenes for delivery of hydrophilic compound

Ultradeformable liposomes containing penetration enhancers were created to deliver NaFl. Vesicles were investigated for their particle size, zeta potential, NaFl entrapment efficiency (%EE), loading efficiency, and in vitro skin penetration. The vesicles obtained were spherical in shape, with a particle size of less than 100 nm and a negative surface charge (–6 to –11 mV). The %EE of NaFl loaded in vesicles ranged from 37 to 48%. Ultradeformable liposomes with monoterpenes (d-limonene, 1,8-cineole and geraniol) significantly improved NaFl penetration through the skin. Confocal laser scanning microscopy analysis confirmed skin-penetration results and was used to evaluate the behavior of hydrophilic compounds penetrating through the skin.     

Physicochemical Characterization and Study of in vitro Interactions of pH-sensitive Liposomes with the Complement System

Complement activation is an important step in the acceleration of liposome clearance. The anaphylatoxins released following complement activation may motivate a wide variety of physiologic changes. We performed physicochemical characterization and in vitro studies of the interaction of complement system with both noncirculating and long-circulating pH-sensitive and nonpH-sensitive liposomes. The liposomes were characterized by diameter, zeta potential, and atomic force microscopy (AFM). The study of liposome interactions with complement system was conducted using hemolytic assay in rat serum. All liposomes presented a similar mean diameter (between 99.8 and 124.3 nm). The zeta potential was negative in all liposome preparations, except in liposomes modified with aminopoly (ethyleneglycol) 2000-distearoylphosphatidylethanolamine (aPEG₂₀₀₀-DSPE), which presented positive zeta potential. Atomic force microscopy images showed that non–long-circulating pH-sensitive liposomes are prone to vesicles aggregation. Non–pH-sensitive liposomes complement system activates, while pH-sensitive liposomes showed to be poor complement activators in rat serum.     

Investigation of parameters influencing incorporation,retention and cellular cytotoxicity in liposomal formulations of poorly soluble camptothecin

Abstract

Improving tumor delivery of lipophilic drugs through identifying advanced drug carrier systems with efficient carrier potency is of high importance. We have performed an investigative approach to identify parameters that affect liposomes’ ability to effectively deliver lipophilic camptothecin (CPT) to target cells. CPT is a potent anticancer drug, but its undesired physiological properties are impairing its therapeutic use. In this study, we have identified parameters influencing incorporation and retention of lipophilic CPT in liposomes, evaluating the effect of lipid composition, lipid chemical structure (head and tail group variations, polymer inclusion), zeta potential and anisotropy. Polyethyleneglycol (PEG) surface decoration was included to avoid liposome fusing and increase the potential for prolonged in vivo circulation time. The in vitro effect of the different carrier formulations on cell cytotoxicity was compared and the effect of active targeting of one of the formulations was evaluated. We found that a combination of liposome surface charge, lipid headgroup and carbon chain unsaturation affect CPT incorporation. Retention in liposomes was highly dependent on the liposomal surroundings and liposome zeta potential. Inclusion of lipid tethered PEG provided stability and prevented liposome fusing. PEGylation negatively affected CPT incorporation while improving retention. In vitro cell culture testing demonstrated that all formulations increased CPT potency compared to free CPT, while cationic formulations proved significantly more toxic to cancer cells that healthy cells. Finally, antibody mediated targeting of one liposome formulation further enhanced the selectivity towards targeted cancer cells, rendering normal cells fully viable after 1 hour exposure to targeted liposomes.