Spectroscopic investigation of the molecular state of nystatin encapsulated in liposomes

The stability and spectral properties of nystatin-encapsulating liposomes, composed of various combinations of dipalmitoyl phosphatidylcholine (DPPC), cholesterol (CH) and distearoyl-N-(monomethoxy poly(ethylene glycol)succinyl) phosphatidylethanolamine (DSPE-PEG), were studied in order to elucidate the molecular state and localization of nystatin encapsulated in liposomes. Localization of nystatin at the surface region of the liposomal membrane was investigated by PEG/dextran two-phase partition and measurement of the fluorescence quenching of nystatin by p-xylene-bis-pyridinium bromide (DPX). In DPPC/DSPE-PEG liposomes and DPPC/CH/DSPE-PEG liposomes, containing 151 and 160 mcg nystatin per mg lipid, respectively, nystatin appeared to be present at the surface region of the liposomal membranes. Self-quenching of nystatin fluorescence was observed in DPPC/CH and DPPC/CH/DSPE-PEG liposomes even at low encapsulated amounts, suggesting the localization of nystatin in CH-incorporating membranes. In CH-free liposomes, nystatin molecules were at first delocalized in the membranes and then self-associated at a higher level of encapsulation. Absorption and circular dichroism (CD) spectra were also measured to examine the monomeric and aggregated states of nystatin in liposomes. High encapsulation efficacy was observed in DPPC and DPPC/DSPE-PEG liposomes, but the highest stability and retention of nystatin in liposomes were observed in DPPC/CH/DSPE-PEG liposomes, evaluated in terms of the nystatin and calcein release from nystatin-encapsulating liposomes in vitro. From the results, possible encapsulation mechanisms of nystatin in liposomes narrowed down to the following three points; interaction with lipid membrane, adsorption on the liposomal surface and complex formation with DSPE-PEG.     

Molecular localization and state of amphotericin B in PEG liposomes

We investigated the molecular localization and state of amphotericin B (AmB) encapsulated in polyethylene glycol (PEG)-coated liposomes. AmB-encapsulating PEG-liposomes composed of dipalmitoylphosphatidylcholine (DPPC), cholesterol (CH) and distearoyl-N-(monomethoxy poly(ethylene glycol)succinyl) phosphatidylethanolamine (DSPE-PEG, average MW of the PEG chain 2000) were prepared by hydration with 9% sucrose solution and extrusion. The amount of AmB encapsulated in the liposomes increased with incorporation of DSPE-PEG and decreased with that of CH. The molecular localization and state of AmB were investigated by PEG/dextran two-phase partition, potassium permeability measurement, fluorescence quenching measurement and circular dichroism (CD) spectroscopy. The results suggest that there are two types of AmB localization in PEG-liposomes, one of which corresponds to the complex of AmB with DSPE-PEG on the membrane surface, while the other corresponds to the pore form of AmB in the hydrophobic core of the liposomal membrane. AmB in PEG liposomes was present in both aggregated and monomeric states.     

pH-sensitive, serum-stable and long-circulating liposomes as a new drug delivery system

The lack of stability in blood and the short blood circulation time of pH-sensitive liposomes are major drawbacks for their application in-vivo. To develop pH-sensitive, serum-stable and long-circulating liposomes as drug delivery systems, the impact of polyethylene glycol-derived phosphatidylethanolamine (DSPE-PEG) on the properties of pH-sensitive liposomes was investigated. pH-sensitive liposomes were prepared with dioleoylphosphatidylethanolamine (DOPE) and oleic acid (DOPE/oleic acid liposome) or DOPE and 1,2-dipalmitoylsuccinylglycerol (DOPE/DPSG liposome). The inclusion of DSPE-PEG enhanced the serum stability of both DOPE/oleic acid and DOPE/DPSG liposomes, but also shifted the pH-response curve of pH-sensitive liposomes to more acidic regions and reduced the maximum leakage percentage. The impact of DSPE-PEG, however, was much lower in the DOPE/DPSG liposomes than in the DOPE/oleic acid liposomes. In tumour tissue homogenates, where the pH is lower than normal healthy tissues, the pH-sensitive DOPE/DPSG liposomes released the entrapped markers rapidly, in comparison with pH-insensitive dipalmitoylphosphatidylcholine/cholesterol/DSPE-PEG liposomes. Moreover, the release rate was not affected by the content of DSPE-PEG. The blood circulation time of methotrexate incorporated in DOPE/UDPSG liposomes was significantly prolonged with increasing content of DSPE-PEG. Taken together, the liposomes composed of DOPE, DPSG and DSPE-PEG (up to 5%) were pH sensitive, plasma stable and had a long circulation time in the blood. The complete destabilization of the liposomes at tumour tissues suggests that the liposomes might be useful for the targeted delivery of drugs such as anticancer agents.     

Stability,liposome interaction,and in vivo pharmacology of ghrelin in liposomal suspensions

Ghrelin is an appetite-stimulating peptide hormone. It is a pharmacologically interesting peptide because of its involvement in e.g. appetite and metabolism, but it has a very short half-life in the body. Ghrelin carries a Ser-3-octanoyl group, and it has previously been suggested that acylated peptides can bind to or be incorporated into liposomes. Therefore, neutral dipalmitoylphosphatidylcholine (DPPC) liposomes and phosphatidylcholine:cholesterol (PC:Chol) (70:30) liposomes as well as negatively charged dipalmitoylphosphatidylcholine:dipalmitoylphosphatidylserine (DPPC:DPPS) liposomes (70:30) were prepared, and ghrelin was added. The chemical and physical stability of ghrelin was examined. Affinity capillary electrophoresis (ACE) revealed an interaction between ghrelin and the negatively charged (DPPC:DPPS) liposomes, whereas only very small affinities were discerned in the other liposomal formulations of ghrelin. Differential scanning calorimetry showed no changes in phase transitions (T_m). In vivo pharmacokinetics following subcutaneous administration of ghrelin in buffer and in the liposomal formulations was examined in rats. The PC:Chol formulation had a longer-lasting effect as compared to the ghrelin buffer solution and the other two liposomal formulations. The prolonged effect of the PC:Chol formulation is suggested not to be caused by association between ghrelin and the liposome.     

Activity of mammalian secreted phospholipase A(2) from inflammatory peritoneal fluid towards PEG-liposomes. Early indications

Due to an increase in the activity of phospholipase A(2) (PLA(2)) in various inflammatory diseases, this enzyme may play a key role in the degradation of liposomes and the subsequent release of drug when PEG-liposomes passively target inflammatory tissue. The activity of mammalian secreted phospholipase A(2) (sPLA(2)) in casein stimulated peritoneal fluid was tested toward liposomes of different compositions. Early results indicate only a slight degradation of conventional dipalmitoylphosphatidylcholine (DPPC) liposomes as well as DPPC liposomes incorporated with different concentrations of PEG(2000). However, the DPPC degradation increased to 7% when inclusion of 30 mol% phosphatidylethanolamine (PE) in the lipid bilayer. The increase in degradation may be due to an improvement of the substrate - as it is well known, that PE is a better substrate for the mammalian sPLA(2) than PC. Incorporation of PE into the bilayer may increase the binding properties of the bilayer resulting in improved conditions for the enzymatic attack by sPLA(2). In addition, inhibitory zones of Staphylococcus aureus in an agar diffusion test showed that PLA(2) from Crotalus atrox venom was able to catalyze the release of gentamicin from PEG-liposomes. In conclusion, this study suggest that degradation of the lipid bilayer of PEG-liposomes by PLA(2) result in release of incapsulated drug, e.g. gentamicin and inclusion of PE in the liposomal bilayer, may enhance the activity of the mammalian sPLA(2) toward liposomes composed of DPPC.     

PEGylated liposomes loading palmitoyl prednisolone for prolonged blood concentration of prednisolone

We investigated the pharmacokinetic behavior of palmitoyl prednisolone (Pal-PLS) and its liposomes with L-alpha-distearoylphosphatidylcholine (DSPC) and cholesterol (Chol) with or without L-alpha-distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG 2000) after their intravenous administration in rats. Pal-PLS rapidly disappeared from the systemic circulation and prednisolone (PLS) was regenerated after the administration of DSPC/Chol liposomes. PEGylated liposomes including DSPE-PEG 2000, however, successfully maintained high blood concentrations of Pal-PLS and PLS. The blood profiles of drugs after the administration of liposomal Pal-PLS were analyzed according to a two-compartment model. The larger content of DSPE-PEG 2000 in DSPC/Chol liposomes showed a lower first order elimination rate constant from the central compartment (K(el)) and clearance (CL). The area under the concentration-time curve (AUC) of Pal-PLS and PLS in PEGylated liposomes was larger than DSPC/Chol liposomes. The mean resident time (MRT) of Pal-PLS and PLS was also prolonged by PEGylated liposomes. Although DSPC/Chol liposomes showed a high distribution of Pal-PLS in the liver and spleen, PEGylated liposomes significantly decreased the liver distribution of Pal-PLS. The biliary and urinary excretions of drugs for 240 min after drug administration were less than 1% of the administrated dose in any formulations. In conclusion, PEGylated liposomes, including Pal-PLS, are useful for maintain the PLS concentration in the blood after intravenous administration.     

Physico-chemical characteristics of methotrexate-entrapped oleic acid-containing deformable liposomes for in vitro transepidermal delivery targeting psoriasis treatment

This study aimed to investigate the physico-chemical characteristics and in vitro permeability of methotrexate (MTX)-entrapped deformable liposomes prepared from phosphatidylcholine (PC) and oleic acid (OA), comparing with those of MTX-entrapped conventional liposomes prepared from PC and cholesterol (CH). Two formulations of MTX-entrapped PC2:CH1 and PC9:CH1 liposomes and one formulation of MTX-entrapped PC2.5:OA1 liposomes were prepared. The size, size distribution, zeta potential, thermal properties, entrapment efficiency, stability, and in vitro permeability across a porcine skin of the MTX-entrapped liposomes were evaluated. All liposome formulations showed a narrow size distribution with the size range of 80-140 nm which is appropriate for the skin permeability. The percentage of MTX loading, entrapment efficiency and the stability of MTX-entrapped PC2:CH1 and PC9:CH1 liposomes were slightly higher than those of MTX-entrapped PC2.5:OA1 liposomes. However, the MTX-entrapped PC2.5:OA1 liposomes enhanced the skin permeability characterized by the higher concentration and flux of MTX diffused across or accumulated in the epidermis and dermis layers of porcine skin. The enhanced permeability of MTX-entrapped PC2.5:OA1 liposomes was explained by 2 mechanisms: (1) the deformable and elasticity characteristics of OA-containing liposomes and (2) a property as a skin penetration enhancer of OA. This suggested that the PC2.5:OA1 deformable liposome was one of promising candidates to enhance the permeability of MTX for the treatment of psoriasis.     

Pharmacodynamics of insulin in polyethylene glycol-coated liposomes

To reduce the injection frequency and toxicity of intravenously administered protein drugs, it is necessary to develop safe and sustained injectable delivery systems. In this study, to evaluate liposomes as safe and sustained injectable delivery systems of proteins, we chose insulin as a model protein drug and tested its incorporation efficiency and pharmacodynamics in various liposomes with and without polyethylene glycol (PEG)-derivatized phospholipid. The liposomes coated with PEG showed 3-fold higher efficiency of insulin incorporation than did the liposomes without PEG. Moreover, among the liposomes coated with PEG, dipalmitoylphosphocholine (DPPC) liposomes showed higher incorporation efficiency than did dimyristoylphosphocholine (DMPC) liposomes. For pharmacodynamic study, insulin (2 IU/kg) was administered in various formulations, such as insulin alone in phosphate-buffered saline and insulin in the DPPC liposomes with and without PEG, to streptozotocin-treated diabetic rats. The pharmacodynamics of insulin alone, however, could not be measured due to the immediate death of rats caused by hypoglycemic shock. In contrast, all the rats treated with liposomal insulin survived, probably by the sustained release of insulin from liposomes. Pharmacodynamics of liposomal insulin showed that PEG-coated liposomes induced the lowest level of blood glucose-the nadir-1 h later than did the liposomes without PEG. These results indicate that PEG-coated liposomes could be developed as a relatively safe and sustained injectable delivery system for insulin with improved incorporation efficiency. Moreover, it is suggested that the liposomes coated with PEG might have a potential as safe injectable delivery systems for other protein and peptide drugs.     

Encapsulation enhancement and stabilization of insulin in cationic liposomes

The purpose of this study was to enhance encapsulation efficiency and sustained-release delivery for parenteral administration of a protein drug. To reduce the administration frequency of protein drugs, it is necessary to develop sustained delivery systems. In this study, protein drug-loaded cationic liposomes were formulated with dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), dioleoyl-3-trimethylammonium-propane (DOTAP), and cholesterol (CH) at a molar ratio of DOPE/DOTAP/CH of 2/1.5/2. Five mol% of distearoylphosphatidyl ethanolamine polyethylene glycol (DSPE-PEG) was added prior to encapsulation of the drug into liposomes. Insulin was chosen as a model protein drug and encapsulation efficiency was evaluated in various liposomes with and without DSPE-PEG. Scanning electron microscopy was used to examine the insulin-loaded cationic liposomes. Structural analysis was performed using spectropolarimetry. Additionally, the stability and cytotoxicity of insulin-loaded cationic liposomes were evaluated. Liposomes coated with DSPE-PEG showed higher insulin encapsulation efficiency than did those without DSPE-PEG, but not significantly. Moreover, among the liposomes coated with DSPE-PEG, those hydrated with 10% sucrose showed higher encapsulation efficiency than did liposomes hydrated in either phosphate-buffered saline or 5% dextrose. In vitro release of insulin was prolonged by cationic liposomes. Our findings suggest that cationic liposomes may be a potential sustained-release delivery system for parenteral administration of protein and peptide drugs to prolong efficacy and improve bioavailability.     

Antimicrobial effectiveness of liposomal polymyxin B against resistant Gram-negative bacterial strains

Polymyxin B is a polycationic antibiotic effective in the treatment of Gram-negative bacterial infections. Systemic use of polymyxin B has been limited due to its toxicity, most notably nephrotoxicity, ototoxicity, and neuromuscular blockade. Entrapment of antibiotics in liposomes is known to enhance their antimicrobial activities while minimizing their toxic effects. In the present study, polymyxin B was incorporated into liposomes composed of either 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol (Chol) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and Chol. The entrapment efficiency of sonicated liposomes containing DPPC/Chol (32.1+/-2.43%) was six-fold higher than that of liposomes containing POPC/Chol (5.35+/-0.32%). On the other hand, the entrapment efficiency of extruded DPPC/Chol liposomes (3.23+/-0.46%) was about 30% less than that of liposomes composed of POPC/Chol (5.10+/-0.37%). Incubation of extruded DPPC/Chol liposomes containing polymyxin B in serum at 37 degrees C resulted in a complete release of the antibiotic into the supernatant after 3h as compared to 6h in the case of POPC/Chol liposomes. Spontaneous release of polymyxin B from DPPC/Chol liposomes incubated in saline was significantly higher (66%) than that from POPC/Chol liposomes (24%) after 48h at 37 degrees C. With respect to the antimicrobial activities of the liposomal polymyxin B formulations, the MICs of sonicated DPPC/Chol liposomes against Gram-negative strains were generally lower when compared to free polymyxin B. Immunocytochemistry and electron transmission microscopic studies revealed that the penetration of polymyxin B into a resistant strain of Pseudomonas aeruginosa was higher following its administration as a liposomal formulation as compared to its conventional form. The combination of free drug and plain liposomes had an antibacterial activity similar to that of free antibiotic. These data suggest that incorporation of polymyxin B in liposomes could be useful in the management of Gram-negative infections induced by these microorganisms.     

A folate receptor-targeted liposomal formulation for paclitaxel

A novel liposomal formulation of paclitaxel targeting the folate receptor (FR) was synthesized and characterized. This formulation was designed to overcome vehicle toxicity associated with the traditional Cremophor EL-based formulation and to provide the added advantages of prolonged systemic circulation time and selective targeting of the FR, which is frequently overexpressed on epithelial cancer cells. The formulation had the composition of dipalmitoyl phosphatidylcholine/dimyristoyl phosphatidylglycerol/monomethoxy-polyethylene glycol (PEG)2000-distearoyl phosphatidylethanolamine/folate-PEG3350-distearoyl phosphatidylethanolamine (DPPC/DMPG/mPEG-DSPE/folate-PEG-DSPE) at molar ratios of (85.5:9.5:4.5:0.5) and a drug-to-lipid molar ratio of 1:33. The liposomes were prepared by polycarbonate membrane extrusion. The mean particle size of the liposomes was 97.1 nm and remained stable for at least 72 h at 4 degrees C. FR-targeted liposomes of the same lipid composition entrapping calcein were shown to be efficiently taken up by KB oral carcinoma cells, which are highly FR+. FR-targeted liposomes containing paclitaxel showed 3.8-fold greater cytotoxicity compared to non-targeted control liposomes in KB cells. Plasma clearance profiles of paclitaxel in the liposomal formulations were then compared to paclitaxel in Cremophor EL formulation. The liposomal formulations showed much longer terminal half-lives (12.33 and 14.23 h for FR-targeted and non-targeted liposomes, respectively) than paclitaxel in Cremophor EL (1.78 h). In conclusion, the paclitaxel formulation described in this study has substantial stability and favorable pharmacokinetic properties. The FR-targeted paclitaxel formulation is potentially useful for treatment of FR+ tumors and warrants further investigation.     

Enhancement of polyethylene glycol (PEG)-modified cationic liposome-mediated gene deliveries: effects on serum stability and transfection efficiency

In this study, we modified cationic liposomes either by polyethylene glycol (PEG)-grafting or PEG-adding methods, and compared the physical properties of transfection complexes and transfection efficiency in-vitro and prolonged circulation in-vivo. The PEG-grafted transfection complexes were prepared by mixing plasmid DNA with PEG-grafted cationic liposomes, which were composed of DSPE-PEG 2000 and cationic lipids. The PEG-added transfection complexes were prepared by adding DSPE-PEG 2000 to the mixture of cationic liposomes and plasmid DNA. The particle sizes of the PEG-modified transfection complexes (approximately 200 nm) changed a little over 4 weeks compared with the conventional transfection complexes. In the presence of serum, the transfection efficiency of the conventional transfection complexes was lowered whereas the transfection efficiency of the PEG-modified transfection complexes was maintained. Moreover, the transfection efficiency of the conventional transfection complexes was significantly reduced when they were stored. However, the transfection efficiency was stable for the PEG-modified transfection complexes, even after two weeks of storage. Of the in-vitro transfection efficiencies, there was no difference between PEG-grafted and PEG-added transfection complexes. When the conventional, PEG-grafted, and PEG-added transfection complexes were administered into mice by the tail vein, the PEG-added transfection complexes showed a prolonged circulation of plasmid DNA compared with other transfection complexes. These results suggest that the PEG-added transfection complexes could be a useful non-viral vector because of their simplicity in preparation, enhanced stability and prolonged circulation compared with the conventional transfection complexes.     

Pharmacokinetics and organ distribution of cyclosporin A incorporated in liposomes and mixed micelles.

The commercially available intravenous dosage form of cyclosporin A (C-CsA) contains a solubilizing agent, polyoxyethylated castor oil, which has been reported to be toxic. To replace the toxic solubilizing agent present in C-CsA, liposomal and mixed micellar preparations were made to solubilize CsA by the proliposome method and characterized. Furthermore, pharmacokinetics and organ distributions of these preparations were evaluated in comparison to C-CsA, which is micellar. The mean size of liposomal preparation (L-CsA) composed of DPPC/PA (molar ratio 3/1) and CsA was 43.6 nm and that of mixed micellar preparation (M-CsA) composed of DMPC/DSPE-PEG (molar ratio 95/5) and CsA was 6.5 nm. The solubilization of CsA was 2-fold greater in mixed micellar solution than in liposomes (0.06 vs 0.03 mg of CsA/mg of lipid). L-CsA, M-CsA and C-CsA were intravenously administered into rats via the femoral vein to analyze pharmacokinetics and organ distribution of CsA. M-CsA was not significantly different from C-CsA in every pharmacokinetic parameter studied. However, L-CsA resulted in 30% decrease in AUC and 55% increase in Cl(t) compared with C-CsA (P<0. 05), without any significant differences in MRT, V(dss) and t(1/2). In addition, the distributions of M-CsA and L-CsA in different organs were not significantly different from those of C-CsA (0.05), except for a 51% decrease of M-CsA in the spleen at 4 h and a 33% increase of L-CsA in the liver at 4 h (P<0.05). These findings demonstrate that the liposomal preparation composed of DPPC/PA and CsA shows slightly different pharmacokinetics and organ distribution patterns from C-CsA, whereas the mixed micellar preparation composed of DMPC/DSPE-PEG and CsA exhibits similar patterns to C-CsA, as expected. Furthermore, these results suggest that those mixed micellar and liposomal preparations can replace C-CsA containing the toxic solubilizing agent, thus providing useful alternative dosage forms for intravenous administration of CsA.     

In vitro antimicrobial activity of liposomal meropenem against Pseudomonas aeruginosa strains

Twelve lipid formulations of liposomal meropenem were tested on six drug-susceptible and two drug-resistant Pseudomonas aeruginosa strains to determine their antibacterial activity. Cationic liposomes, especially PC/DOPE/SA 4:4:2 and PC/DOTAP/Chol 5:2:3, were more effective than anionic ones against sensitive isolates as the MICs of those formulations were two to four times lower than those of the free drug. None of the studied liposomal forms of meropenem exhibited bactericidal activity against isolates, which are drug-resistant due to low permeability. Even Fluidosomes (liposomes made of DPPC/DMPG 18:1), which demonstrated fusion with P. aeruginosa membranes, showed 4-16 times higher MICs for sensitive and resistant strains than did the free meropenem.     

Association of liposome-entrapped [3H]methotrexate with thioglycollate-elicited macrophages in-vitro

The association of free or liposome-entrapped [3H]methotrexate [( 3H]MTX) with thioglycollate-elicited macrophages was investigated in-vitro. [14C]Cholesteryl oleate was incorporated into the liposomes as a lipid marker. [3H]MTX association with the macrophages was 5 to 9-fold higher with liposome-entrapped [3H]MTX than with the free drug. Macrophage-liposome association was biphasic, temperature-dependent and saturable at high liposomal lipid concentration. A high liposome cholesterol (CH) content or the presence of 2,4-dinitrophenol or colchicine also reduced macrophage-liposome association.     

Enhanced Tumor Targeting of Doxorubicin by Ganglioside GMl-bearing Long-circulating Liposomes

Abstract

Doxorubicin (DXR) was encapsulated in long-circulating liposomes, composed of ganglioside GM1 (GM1)/distearoylphosphatidylcholine (DSPC)/cholesterol (CH) (0.13:1:1 in molar ratio) and sized to approximately 100 nm in mean diameter, with 98% entrapping efficiency by the transmembrane pH gradient method. Free DXR, DXR-DSPC/CH and DXR-GM1/DSPC/CH liposomes were injected intravenously into Colon 26 tumor-bearing Balb/c mice via the tail vein at a dose of 5.0 mg DXR/kg. DXR-GM1/DSPC/CH liposomes gave a higher blood level of the drug than did DXR-DSPC/CH liposomes or free DXR up to 24 hours after injection, and the area under the blood concentration-time curve (AUC) for DXR-GM1/DSPC/CH liposomes was 1.5 or 526 times higher than that for DXR-DSPC/CH liposomes or free DXR, respectively. DXR-GM1/DSPC/CH liposomes gave a decreased DXR concentration in the reticuloendothelial system (RES) of the liver and the spleen. Both liposomal formulations effectively reduced the DXR concentration in the heart as compared with that in the case of free DXR. At 6 hours after i.v. injection, DXR-GM1/DSPC/CH liposomes provided an approximately 3.3- or 9-fold higher peak DXR level in the tumor as compared with DXR-DSPC/CH liposomes or the free drug, respectively. These high tumor levels of DXR appear to reflect the prolonged residence time of the liposomes. The results suggest that encapsulation of DXR in GM1-bearing long-circulating liposomes will be useful for cancer chemotherapy.     

Safety and pharmacokinetic studies of liposomal antioxidant formulations containing N-acetylcysteine, α-tocopherol or γ-tocopherol in beagle dogs

Abstract

The safety and pharmacokinetic profile of liposomal formulations containing combinations of the antioxidants α-tocopherol, γ-tocopherol or N-acetylcysteine in beagle dogs was examined. Each group consisted of beagle dogs of both genders with a control group receiving empty dipalmitoylphosphatidylcholine (DPPC) liposomes (330?mg/kg DPPC, EL), and test groups receiving liposomes prepared from DPPC lipids with (i) N-acetylcysteine (NAC) (60?mg/kg NAC [L-NAC]); (ii) NAC and α-tocopherol (αT) (60?mg/kg NAC and 25?mg/kg α-tocopherol [L-αT-NAC]) and (iii) NAC and γ-tocopherol (60?mg/kg NAC and 25?mg/kg γ-tocopherol (γT) [L-γT-NAC]). The dogs in the control group (EL) and three test groups exhibited no signs of toxicity during the dosing period or day 15 post treatment. Weight gain, feed consumption and clinical pathology findings (hematology, coagulation, clinical chemistry, urinalysis) were unremarkable in all dogs and in all groups. Results from the pharmacokinetic study revealed that the inclusion of tocopherols in the liposomal formulation significantly increased the area under the curve (AUC) and β-half life for NAC; the tocopherols had greater impact on the clearance of NAC, where reductions of central compartment clearance (CL) ranged from 56% to 60% and reductions of tissue clearance (CL₂) ranged from 73% to 77%. In conclusion, there was no treatment-related toxicity in dogs at the maximum feasible dose level by a single bolus intravenous administration while the addition of tocopherols to the liposomal formulation prolonged the circulation of NAC in plasma largely due to a decreased clearance of NAC.     

Enhanced Delivery and Antitumor Activity of Doxorubicin Using Long-Circulating Thermosensitive Liposomes Containing Amphipathic Polyethylene Glycol in Combination with Local Hyperthermia

Enhanced delivery of doxorubicin (DXR) to a solid tumor subjected to local hyperthermia was achieved by using long-circulating, thermosensitive liposomes (TSL) composed of dipalmitoyl phosphatidylcholine (DPPC)/distearoyl phosphatidylcholine (DSPC) (9:1, m/m) and 3 mol% amphipathic polyethylene glycol (PEG) in colon 26-bearing mice. Inclusion of 3 mol% of distearoyl phosphatidylethanolamine derivatives of PEG (DSPE-PEG, amphipathic PEG) with a mean molecular weight of 1000 or 5000 in DPPC/DSPC liposomes resulted in decreased reticuloendothelial system (RES) uptake and a concomitant prolongation of circulation time, affording sustained increased blood levels of the liposomes. Concomitantly, DXR levels in blood were also kept high over a long period. The presence of amphipathic PEG did not interfere with the encapsulation of DXR by the pH gradient method (>90% trapping efficiency) or with the temperature-dependent drug release from the liposomes. The optimal size of these liposomes was 180 – 200 nm in mean diameter for thermosensitive drug release and prolonged circulation time. The DXR levels in the tumor after injection of long-circulating TSL (DXR-PEG1000TSL or DXR-PEG5000TSL, at a dose of 5 mg DXR/ kg) with local hyperthermia were much higher than after treatment with DXR-TSL lacking PEG or with free DXR, reaching 7.0 – 8.5 DXR µg/g tumor (approximately 2 times or 6 times higher than that of DXR-TSL or free DXR, respectively). Furthermore, the combination of DXR-PEGTSL and hyperthermia effectively retarded tumor growth and increased survival time. Our results indicate that the combination of drug-loaded, long-circulating, thermosensitive liposomes with local hyperthermia at the tumor site could be clinically useful for delivering a wide range of chemotherapeutic agents in the treatment of solid tumors.     

Long-circulating liposomes of indomethacin in arthritic rats--a biodisposition study

To improve the targeting efficiency of liposomes of indomethacin to the arthritic joints, circulation half-life of the liposomes was increased by grafting amphipathic polyethylene glycol-2000 to the bilayer surface. A comparative biodistribution study was performed between the conventional liposomes (PC:CH:PE--1:0.5:0.16) and long-circulating liposomes (PC:CH:PE-PEG--1:0.5:0.16) in arthritic rats. Pharmacokinetics of the drug changed significantly when administered in liposomal form. Pharmacokinetic parameters of the drug such as AUC0-t (trapezoidal), clearance and t1/2 (elimination half-life) changed significantly (p < 0.05) when encapsulated in liposomes. Significant difference in pharmacokinetics was observed in AUC0-t and clearance between the conventional liposomes and long-circulating liposomes. The increased AUC0-t and reduced clearance of the drug with long-circulating liposomes, increased the availability of the drug by reducing RES uptake, in turn localization in arthritic paw tissue was also increased. A concentration of 0.33 microgram of indomethacin/g of the tissue was achieved with S-liposomes after 24 h whereas it was only 0.26 microgram of drug/g of the tissue with conventional liposomes. From the study, in may be concluded that the targeting efficiency of the long-circulating liposomes was about four times more than the conventional liposomes.     

Preparation,Characterisation and Biodistribution of 99mTc-labeled Liposome Encapsulated Cyclosporine

The present study investigated the effect of charge (neutral, negative and positive) on liposomal membrane on the distribution of cyclosporine encapsulated in it to various organs. Liposomes were prepared by using different phospholipids by thin film hydration followed by sequential extrusion through polycarbonate membranes to achieve a desired particle size, with high entrapment efficiency and then lyophilised using sucrose as cryoprotectant. The possible in vivo distribution of cyclosporine and its liposomes after direct labeling with reduced technetium-99m has been studied in mice. The blood kinetics and biodistribution study of these labeled complexes shows prolonged circulation of positive and neutral charged liposomes in blood compared to free drug and negative charged liposomal formulation. The biodistribution of the tagged liposomes confirms that increased radioactivity was seen in liver and spleen, with minimal involvement of the kidney. At 4h post injection the biodistribution data in kidney reveals approximately 1-2% of the injected dose was present for cyclosporine loaded liposomes, which elicits the possibility of reducing the nephrotoxicity, generally seen in free cyclosporine. Interestingly, the biodistribution and -y imaging studies of the charged cyclosporine liposomes indicated that an appreciable amount of these labeled complexes goes to bone marrow when compared to the free cyclosporine. The findings demonstrate the distribution of these liposomes within various organs and proved that the positively charged liposomes experience increased bone uptake and prolonged circulation half-life. Hence this finding implies the possibility of using these formulations for liver and bone marrow transplantation.