Nebulization of Liposomal rh-Cu/Zn–SOD with a Novel Vibrating Membrane Nebulizer

Liposomes are potential drug carriers for pulmonary drug delivery: They can be prepared from phospholipids, which are endogenous to the respiratory tract as a component of pulmonary surfactant, and at an appropriate dose liposomes do not pose a toxicological risk to this organ. Among the various categories of drug that benefit from liposomal entrapment is the anti-inflammatory enzyme superoxide dismutase, thus prolonging its biological half-life. The delivery of liposomes by nebulization is hampered by stability problems, like physical and chemical changes that may lead to chemical degradation and leakage of the encapsulated drug. Here we present data of liposomes aerosolized with a novel electronic nebulizer based on a vibrating membrane technology (PARI eFlow?), which amends drawbacks like liposomes degradation and product release. The data acquisition included aerosol properties such as aerodynamic particle size, nebulization efficiency, and liposome leakage upon nebulization. In conclusion, this study shows the ability of the PARI eFlow? to nebulize high amounts of liposomal recombinant human superoxide dismutase with reduced vesicle disruption tested in an enclosing experimental protocol.     

Delivery of fluorescent-labeled cyclodextrin by liposomes: role of transferrin modification and phosphatidylcholine composition

Drug-in-CD-in-liposome (DCL) systems which encapsulate the drug/CD inclusion complexes into inner aqueous phase of liposomes have been applied as a novel strategy to improve efficacy of lipophilic antitumor drugs. The aim of this work was to assess the role of transferrin (Tf) modification and phosphatidylcholine (PC) composition on the properties of liposomes containing hydroxypropyl-β-cyclodextrin (HP-β-CD). Fluorescence dye, FITC, was conjugated with HP-β-CD to facilitate the analysis. The resulting FITC-HP-β-CD was further encapsulated into liposomes and then the liposomes were modified with Tf. The FITC-HP-β-CD-loaded liposomes with different PC compositions were compared in terms of particle size, zeta potential, FITC content, FITC-HP-β-CD leakage, phase transition temperature (T_m) and cellular uptake. The apparent partition coefficient values of different PCs were also determined. Compared to PEGylated liposomes, FITC-HP-β-CD-loaded liposomes modified with Tf had been proved to significantly increase vesicle stability and specific cellular uptake. Moreover, PC composition affected the properties of liposomes. Soybean phosphatidylcholine (SPC) liposomes modified with Tf were found to be more easily internalized into tumor cells than 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and hydrogenated soybean phosphatidylcholine (HSPC) while Tf density on the liposomal surface was similar. And the lipophilicity of SPC was found to be much higher than DPPC and HSPC. Collectively, by the optimization of PC composition, the development of DCL modified with Tf might represent a potential strategy for the antitumor application of lipophilic drugs.     

Generation of fatty acids from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/cardiolipin liposomes that stabilize recombinant human serum albumin

Abstract

Context: At elevated temperatures, studies have shown that serum albumin undergoes irreversible changes to its secondary structure. Anionic fatty acids and/or anionic surfactants have been shown to stabilize human serum albumin (HSA) against thermal denaturation through bridging hydrophobic domains and cationic amino acids residues of the protein.

Objective: As albumin can readily interact with a variety of liposomes, this study proposes that cardiolipin delivered via 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes can improve the thermal stability of recombinant HSA produced in Saccharomyces cerevisiae (ScrHSA) in a similar manner to anionic fatty acids.

Materials and methods: Thermal stability and structure of ScrHSA in the absence and presence of DPPC/cardiolipin liposomes was assessed with U/V circular dichroism spectropolarimetry and protein thermal stability was confirmed with differential scanning calorimetry.

Results: Although freshly prepared DPPC/cardiolipin liposomes did not improve the stability of ScrHSA, DPPC/cardiolipin liposomes incubated at room temperature for 7?d (7dRT) dramatically improved the thermal stability of the protein. Mass spectrometry analysis identified the presence of fatty acids in the 7dRT liposomes, not identified in freshly prepared liposomes, to which the improved stability was attributed.

Discussion and conclusion: The generation of fatty acids is attributed to either the chemical hydrolysis or oxidative cleavage of the unsaturated acyl chains of cardiolipin. By modulating the lipid composition through the introduction of lipids with higher acyl chain unsaturation, it may be possible to generate the stabilizing fatty acids in a more rapid manner.     

In vitro evaluation of archaeosome vehicles for transdermal vaccine delivery

Abstract

Archaeosomes composed of archaeal total polar lipids (TPL) or semi-synthetic analog vesicles have been used as vaccine adjuvants and delivery systems in animal models for many years. Typically administered by intramuscular or subcutaneous injections, archaeosomes can induce robust, long-lasting humoral and cell-mediated immune responses against entrapped antigens and provide protection in murine models of infectious disease and cancer. Herein, we evaluated various archaeosomes for transdermal delivery, since this route may help eliminate needle-stick injuries and needle re-use, and therefore increase patient compliance. Archaeosomes composed of TPL from different archaea (Halobacterium salinarum, Methanobrevibacter smithii, Haloferax volcanii) and various semi-synthetic glycolipid combinations were evaluated for their ability to diffuse across the skin barrier using an ex vivo pig skin model and the results were compared to conventional synthetic ester liposomes. Physicochemical characteristics were determined for selected formulations including vesicle size, size distribution, zeta potential, fluidity, antigen (ovalbumin) incorporation efficiency and release. Archaeosomes, in particular those composed of M. smithii TPL or the synthetic glycolipid sulfated S-lactosylarchaeol (SLA) mixed with uncharged glycolipid lactosyl archaeol (LA), appeared to be effective carriers for ovalbumin, achieving much better antigen distribution and vesicle accumulation in the skin epidermis than conventional liposomes. The enhanced skin permeation of archaeosomes may be attributed to their chemical structure and physicochemical properties such as particle size, surface charge, stability, and fluidity of their lipid bilayer.     

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.     

Physicochemical characterization of stealth liposomes encapsulating an organophosphate hydrolyzing enzyme

The present studies were focused on the preparation and characterization of stericaly stabilized liposomes (SLs) encapsulating a recombinant organophosphorus hydrolyzing phosphotriesterase (OPH) enzyme for the antagonism of organophosphorus intoxication. Earlier results indicate that the liposomal carrier system provides an enhanced protective effect against the organophosphorus molecule paraoxon, presenting a more effective therapy with less toxicity than the most commonly used antidotes. Physicochemical characterization of the liposomal OPH delivery system is essential in order to get information on its in vitro stability and in vivo fate. Osmolarity, pH, viscosity, and encapsulation efficiency of the SL preparation and the surface potential of the vesicles were determined. The membrane rigidity and the impact of OPH enzyme on it was studied by electron-paramagnetic resonance spectroscopy, using spin probes. The in vitro stability of the liposomal preparations, the vesicle size distribution, and its alteration during a 3-week storage were followed by dynamic light-scattering measurements. Further, the stability of encapsulated and nonencapsulated OPH was compared in puffer and plasma.     

LIPOSOME-INCORPORATED SANTOLINA INSULARIS ESSENTIAL OIL: PREPARATION,CHARACTERIZATION AND IN VITRO ANTIVIRAL ACTIVITY

The effect of liposomal inclusion on the stability and in vitro antiherpetic activity of Santolina insularis essential oil was investigated. In order to study the influence of vesicle structure on the liposome properties, multilamellar and unilamellar vesicles were prepared by the film method and sonication, respectively. Vesicles were obtained from hydrogenated soya phosphatydilcholine and cholesterol. Formulations were examined for their stability for over one year monitoring the drug leakage from vesicles and the average size distribution. The stability of the incorporated oil was verified by studying its quali-quantitative composition. The antiviral activity was studied against Herpes simplex virus type 1 (HSV-1) by plaque reduction and yield reduction assays. Results showed that Santolina insularis essential oil can be incorporated in high amounts in the prepared liposomes, which successfully prevented its degradation. Moreover, stability studies pointed out that vesicle dispersions were stable for at least one year and neither oil leakage nor vesicle size alteration occurred during this period. Antiviral activity assays demonstrated that Santolina insularis essential oil is effective in inactivating HSV-1 and that the activity is principally due to direct virucidal effects. Free essential oil proved to be more effective than liposomal oil and a different activity was discovered which related to the vesicular structure. The ED₅₀ values, significantly lower when cells were pre-incubated with the essential oil before the virus adsorption, indicate an intracellular mechanism in the antiviral activity of Santolina insularis. Moreover, liposomal Santolina essential oil is non toxic in the range of the concentration tested.     

Preparation and Characterization of Gas-filled Liposomes: Can They Improve Oil Recovery?

Although well known for delivering various pharmaceutical agents, liposomes can be prepared to entrap gas rather than aqueous media and have the potential to be used as pressure probes in magnetic resonance imaging (MRI). Using these gas-filled liposomes (GFL) as tracers, MRI imaging of pressure regions of a fluid flowing through a porous medium could be established. This knowledge can be exploited to enhance recovery of oil from the porous rock regions within oil fields. In the preliminary studies, we have optimized the lipid composition of GFL prepared using a simple homogenization technique and investigated key physico-chemical characteristics (size and the physical stability) and their efficacy as pressure probes. In contrast to the liposomes possessing an aqueous core which are prepared at temperatures above their phase transition temperature (T_c), homogenization of the phospholipids such as 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-distearoyl-sn-glycero-3-phosphocoline (DSPC) in aqueous medium below their T_c was found to be crucial in formation of stable GFL. DSPC based preparations yielded a GFL volume of more than five times compared to their DPPC counter part. Although the initial vesicle sizes of both DSPC and DPPC based GFL were about 10 μm, after 7 days storage at 25°C, the vesicle sizes of both formulations significantly (p < 0.05) increased to 28.3 ± 0.3 μm and 12.3 ± 1.0 μm, respectively. When the DPPC preparation was supplemented with cholesterol at a 1:0.5 or 1:1 molar ratio, significantly (p < 0.05) larger vesicles were formed (12–13 μm), however, compared to DPPC only vesicles, both cholesterol supplemented formulations displayed enhanced stability on storage indicating a stabilizing effect of cholesterol on these gas-filled vesicles. In order to induce surface charge on the GFL, DPPC and cholesterol (1: 0.5 molar ratio) liposomes were supplemented with a cationic surfactant, stearylamine, at a molar ratio of 0.25 or 0.125. Interestingly, the ζ potential values remained around neutrality at both stearylamine ratios suggesting the cationic surfactant was not incorporated within the bilayers of the GFL. Microscopic analysis of GFL confirmed the presence of spherical structures with a size distribution between 1–8 μm. This study has identified that DSPC based GFL in aqueous medium dispersed in 2% w/v methyl cellulose although yielded higher vesicle sizes over time were most stable under high pressures exerted in MRI.     

Liposomes for (trans)dermal drug delivery: the skin-PVPA as a novel in vitro stratum corneum model in formulation development

Penetration potential of vesicles destined for trans(dermal) administration remains to be of great interests both in respect to drug therapy and cosmetic treatment. This study investigated the applicability of the phospholipid vesicle-based permeation assay (PVPA) as a novel in vitro skin barrier model for screening purposes in preformulation studies. Various classes of liposomes containing hydrophilic model drug were examined, including conventional liposomes (CLs), deformable liposomes (DLs) and propylene glycol liposomes (PGLs). The size, surface charge, membrane deformability and entrapment efficiency were found to be affected by the vesicle lipid concentration, the presence of the surfactant and propylene glycol. All liposomes exhibited prolonged drug release profiles with an initial burst effect followed by a slower release phase. The permeation of the drug from all of the tested liposomes, as assessed with the mimicked stratum corneum – PVPA model, was significantly enhanced as compared to the permeability of the drug in solution form. Although the DLs and the PGLs exhibited almost the same membrane elasticity, the permeability of the drug delivered by PGLs was higher (6.2?×?10^?6?cm/s) than DLs (5.5?×?10^?6?cm/s). Therefore, this study confirmed both the potential of liposomes as vesicles in trans(dermal) delivery and potential of the newly developed skin-PVPA for the screening and optimization of liposomes at the early preformulation stage.     

Stability of a liposomal formulation containing lipoyl or dihydrolipoyl acylglycerides

Context: The acylglycerides of lipoic and dihydrolipoic acids may serve as slow-release sources for cutaneous delivery of these antioxidants when formulated in a liposomal vehicle.

Objective: Testing was conducted to determine the storage stability of lipoyl glycerides in phospholipid-based liposomes.

Materials and methods: Lipoyl glycerides prepared by transesterification of lipoic acid with high oleic sunflower oil were incorporated into unilamellar liposomes comprised of soy phosphatidylcholine (soyPC) or dioleoylphosphatidylcholine (DOPC).

Results: Lipoyl glycerides were stable in soyPC at 4?°C (90% remaining after five weeks) and decayed with a half-life (t_½) of 14?d at 40?°C. In contrast, lipoyl glycerides embedded in DOPC were completely stable for four weeks at 40?°C. Dihydrolipoyl glycerides in soyPC converted to lipoyl glycerides at 4?°C (t_½?=?14?d) over four weeks, and much more rapidly so at 40?°C (t_½?=?1?d). A hydroperoxide accumulation analysis indicated that lipoyl glycerides and dihydrolipoyl glycerides were modified or degraded while suppressing autoxidation of the polyunsaturated fatty acids present in soyPC. Dynamic light scattering measurements found that liposomes containing lipoyl glycerides or dihydrolipoyl glycerides did not undergo significant size changes for at least 48?d, indicating that inclusion of the lipoic acid derivatives did not induce vesicle aggregation.

Discussion/Conclusion: Substitution of the soyPC with DOPC, which is not readily subject to autoxidation, provided a much more stable storage environment for lipoyl glycerides. These findings confirm the expectation that phospholipid liposomes need to be oxidatively stable vehicles for dermal delivery of lipoic acid derivatives.     

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.     

The Drosophila dopamine 2-like receptor D2R (Dop2R) is required in the blood brain barrier for male courtship

The blood brain barrier (BBB) has the essential function to protect the brain from potentially hazardous molecules while also enabling controlled selective uptake. How these processes and signaling inside BBB cells control neuronal function is an intense area of interest. Signaling in the adult Drosophila BBB is required for normal male courtship behavior and relies on male-specific molecules in the BBB. Here we show that the dopamine receptor D2R is expressed in the BBB and is required in mature males for normal mating behavior. Conditional adult male knockdown of D2R in BBB cells causes courtship defects. The courtship defects observed in genetic D2R mutants can be rescued by expression of normal D2R specifically in the BBB of adult males. Drosophila BBB cells are glial cells. Our findings thus identify a specific glial function for the DR2 receptor and dopamine signaling in the regulation of a complex behavior.     

Topical delivery and in vivo antileishmanial activity of paromomycin-loaded liposomes for treatment of cutaneous leishmaniasis

The present study aimed to evaluate the potential of liposomes loaded with paromomycin (PA), an aminoglycoside antibiotic associated with poor skin penetration, for the topical treatment of cutaneous leishmaniasis (CL). Fluid liposomes were prepared and characterized for particle size, zeta potential, and drug entrapment. Permeation studies were performed with two in vitro models: intact and stripped skin. The antileishmanial activity of free and liposomal PA was evaluated in BALB/c mice infected by Leishmania (L.) major. Drug entrapment ranged from 10 to 14%, and the type of vesicle had little influence on this parameter. Particle size and polydispersity index of the vesicles composed by phosphatidylcholine (PC) and PC/cholesterol (Chol) ranged from of 516 to 362?nm and 0.7 to 0.4, respectively. PA permeation across intact skin was low, regardless of the formulation tested, while drug penetration into skin (percent of the applied dose) from PC (7.2?±?0.2%) and PC/Chol (4.8?±?0.2%) liposomes was higher than solution (1.9?±?0.1%). PA-loaded liposomes enhanced in vitro drug permeation across stripped skin and improved the in vivo antileishmanial activity in experimentally infected mice. Our findings suggest that the liposomes represent a promising alternative for the topical treatment of CL using PA.     

Blood-brain barrier unlocked

The brain is protected by a physiological blood-brain barrier (BBB) against toxins and some metabolites circulating in the blood. At the same time, the BBB limits penetration into the brain of many neuroactive drugs. Efficient ways to increase BBB permeability for delivery of drugs of different chemical nature into the brain are unknown. This work deals with delivery into the brain of 10⁻² M dopamine, a substance that does not penetrate the BBB under normal circumstances. It was studied in two independent experiments: (i) penetration of ³H-labeled dopamine from its mixture with 10⁻⁵ M H₂O₂ into hypothalamus and striatum structures of intact rat brain, and (ii) effect of unlabeled dopamine from a mixture with H2O2 on the rat motor activity in a haloperidol catalepsy model. It was shown that (i) at the third minute after nasal application of the dopamine + H₂O₂ mixture, the dopamine level increases 45-fold in the hypothalamus and almost 30-fold in the striatum and (ii) motility of animals in the catalepsy haloperidol model is recovered 90 sec after intranasal introduction of dopamine. No such effects were observed after replacement of H₂O₂ by 0.9% NaCl solution. Thus, it was shown on the example of dopamine that its introduction into the nasal cavity simultaneously with H₂O₂ provides for rapid delivery of the drug into the brain. These results expand our knowledge concerning the biological role of exoROS in modulating BBB permeability and may contribute to the development of a new therapeutic strategy for neurological diseases.     

Drug-Carrier and Stability Properties of the Long-Lived Lipid Vesicles. Cryptosomes,in Vitro and in Vivo

Abstract

Lipid vesicles composed of phosphatidylcholine and suitable polyoxyethylene-derivat-ives of phosphatidylethanolamine (cryptosomes) remain in circulation 8–10 times longer than standard liposomes after an i.v. administration in mice. In contrast to previous belief, this longevity is not destroyed by the net charges on the lipid vesicle surface and is not a direct consequence of the high surface hydrophilicity; also bilayer fluidity is not an obstacle for the attainment of long circulation times. All these three factors, however, can affect the effectiveness of the drug encapsulation into lipid vesicles and the stability of the resulting carrier suspensions. Terminal head-group modifications, moreover, can affect the final carrier and drug distribution after vesicle applications in vivo and lead to accumulation in certain body subsites, such as the gut.     

Stability study of stavudine-loaded O-palmitoyl-anchored carbohydrate-coated liposomes

The purpose of this study was to evaluate the physicochemical stability of carbohydrate-anchored liposomes. In the present study, carbohydrate (galactose, fucose, and mannose) was palmitoylated and anchored on the surface of positively charged liposomes (PL). The stabilities of plain neutral liposomes (NL), PL, and O-palmitoyl carbohydrate-anchored liposomes were determined. The effects of storage conditions (4°C±2°C, 25°C±2°C/60%±5% relative humidity [RH], or 40°C±2°C/75%±5% RH for a period of 10, 20, and 30 days) were observed on the vesicle size, shape, zeta potential, drug content, and in vitro ligand agglutination assay by keeping the liposomal formulations in sealed ambercolored vials (10-mL capacity) after flushing with nitrogen. The stability of liposomal formulations was found to be temperature dependent. All the liposomal formulations were found to be stable at 4°C±2°C up to 1 month. Storage at 25°C±2°C/60%±5% RH and 40°C±2°C/75%±5% RH adversely affected uncoated liposomal formulations. Carbohydrate coating of the liposomes could enhance the stability of liposomes at 25°C±2°C/60%±5% RH and 40°C±2°C/75%±5% RH. Published: May 18, 2007     

Development of egg PC/cholesterol/α-tocopherol liposomes with ionic gradients to deliver ropivacaine

Context: Ropivacaine (RVC) is an aminoamide local anesthetic widely used in surgical procedures. Studies with RVC encapsulated in liposomes and complexed in cyclodextrins have shown good results, but in order to use RVC for lengthy procedures and during the postoperative period, a still more prolonged anesthetic effect is required.

Objective: This study therefore aimed to provide extended RVC release and increased upload using modified liposomes.

Materials and methods: Three types of vesicles were studied: (i) large multilamellar vesicle (LMV), (ii) large multivesicular vesicle (LMVV) and (iii) large unilamellar vesicle (LUV), prepared with egg phosphatidylcholine/cholesterol/α-tocopherol (4:3:0.07?mol%) at pH 7.4. Ionic gradient liposomes (inside: pH 5.5, pH 5.5?+?(NH₄)₂SO₄ and pH 7.4?+?(NH₄)₂SO₄) were prepared and showed improved RVC loading, compared to conventional liposomes (inside: pH 7.4).

Results and discussion: An high-performance liquid chromatography analytical method was validated for RVC quantification. The liposomes were characterized in terms of their size, zeta potential, polydispersion, morphology, RVC encapsulation efficiency (EE(%)) and in vitro RVC release. LMVV liposomes provided better performance than LMV or LUV. The best formulations were prepared using pH 5.5 (LMVV 5.5_in) or pH 7.4 with 250?mM (NH₄)₂SO₄ in the inner aqueous core (LMVV 7.4_in?+?ammonium sulfate), enabling encapsulation of as much as 2% RVC, with high uptake (EE(%) ～70%) and sustained release (～25?h).

Conclusion: The encapsulation of RVC in ionic gradient liposomes significantly extended the duration of release of the anesthetic, showing that this strategy could be a viable means of promoting longer-term anesthesia during surgical procedures and during the postoperative period.     

Development and Evaluation of Chitosan-Coated Liposomes for Oral DNA Vaccine: The Improvement of Peyer’s Patch Targeting Using a Polyplex-Loaded Liposomes

The aim of this study was to develop chitosan-coated and polyplex-loaded liposomes (PLLs) containing DNA vaccine for Peyer’s patch targeting. Plain liposomes carrying plasmid pRc/CMV-HBs were prepared by the reverse-phase evaporation method. Chitosan coating was carried out by incubation of the liposomal suspensions with chitosan solution. Main lipid components of liposomes were phosphatidylcholine/cholesterol. Sodium deoxycholate and dicetyl phosphate were used as negative charge inducers. The zeta potentials of plain liposomes were strongly affected by the pH of the medium. Coating with chitosan variably increased the surface charges of the liposomes. To increase the zeta potential and stability of the liposome, chitosan was also used as a DNA condensing agent to form a polyplex. The PLLs were coated with chitosan solution. In vivo study of PLLs was carried out in comparison with chitosan-coated liposomes using plasmid encoding green fluorescence protein as a reporter. A single dose of plasmid equal to 100 μg was intragastrically inoculated into BALB/c mice. The expression of green fluorescence protein (GFP) was detected after 24 h using a confocal laser scanning microscope. The signal of GFP was obtained from positively charged chitosan-coated liposomes but found only at the upper part of duodenum. With chitosan-coated PLL540, the signal of GFP was found throughout the intestine. Chitosan-coated PLL demonstrated a higher potential to deliver the DNA to the distal intestine than the chitosan-coated liposomes due to the increase in permanent positive surface charges and the decreased enzymatic degradation.     

Monensin intercalation in liposomes: effect on cytotoxicities of ricin,Pseudomonas exotoxin A and diphtheria toxin in CHO cells

Monensin, a car☐ylic ionophore was intercalated in liposomes (liposomal monensin) and its effect on cytotoxicities of ricin, Pseudomonas exotoxin A and diphtheria toxin in CHO cells was studied. Intercalation of monensin in liposomal bilayer is found to have no effect on its stability and interaction with cells. Liposomal monensin)(1 nM) substantially enhance the cytotoxicities of ricin (62-fold) and Pseudomonas exotoxin A (11.5-fold) while it has no effect on diphtheria toxin. This observed effect is highly dependent on the liposomal lipid composition. The potentiating ability of monensin (1 nM) in neutral vesicles is significantly higher (2.2-fold) as compared to negatively charged vesicles. This ability is drastically reduced by incorporation of stearylamine in liposomes and is found to be dependent on the density of stearylamine as well as on the concentration of serum in the medium. Monensin in liposomes containing 24 mol% stearylamine has a very marginal effect on the cytotoxicity of ricin (7.5-fold) which is further reduced (1.5-fold) in the presence of 20% serum. The uptake of ¹²⁵I-gelonin from neutral vesicles is significantly higher (∼ 2.0-fold) than that from the negative vesicles. The uptake from positive vesicles is highly dependent on the concentration of stearylamine. The reduction in the lag period (30 min) of ricin action by monensin in neutral and negative vesicle is comparable with free monensin. However, monensin in positive vesicle has no effect on it. These studies have suggested that liposomes could be used as a delivery vehicle for monensin for selective elimination of tumor cells in combination with hybrid toxins.