Radioprotective effect of transferrin targeted citicoline liposomes

The high level of expression of transferrin receptors (Tf-R) on the surface of endothelial cells of the blood–brain-barrier (BBB) had been widely utilized to deliver drugs to the brain. The primary aim of this study was to use transferrin receptor mediated endocytosis as a pathway for the rational development of holo-transferrin coupled liposomes for drug targeting to the brain. Citicoline is a neuroprotective agent used clinically to treat for instance Parkinson disease, stroke, Alzheimer's disease and brain ischemia. Citicoline does not readily cross the BBB because of its strong polar nature. Hence, citicoline was used as a model drug. (Citicoline liposomes have been prepared using dipalmitoylphosphatidylcholine (DPPC) or distearoylphosphatidylcholine (DSPC) by dry lipid film hydration–extrusion method). The effect of the use of liposomes composed of DPPC or DSPC on their citicoline encapsulation efficiency and their stability in vitro were studied. Transferrin was coupled to liposomes by a technique which involves the prevention of scavenging diferric iron atoms of transferrin. The coupling efficiency of transferrin to the liposomes was studied. In vitro evaluation of transferrin-coupled liposomes was performed for their radioprotective effect in radiation treated cell cultures. In this study, OVCAR-3 cells were used as a model cell type over-expressing the Tf-R and human umbilical vein endothelial cells (HUVEC) as BBB endothelial cell model. The average diameter of DPPC and DSPC liposomes were 138 ± 6.3 and 79.0 ± 3.2 nm, respectively. The citicoline encapsulation capacity of DPPC and DSPC liposomes was 81.8 ± 12.8 and 54.9 ± 0.04 μg/μmol of phospholipid, respectively. Liposomes prepared from DSPC showed relatively better stability than DPPC liposomes at 37°C and in the presence of serum. Hence, DSPC liposomes were used for transferrin coupling and an average of 46–55 molecules of transferrin were present per liposome. Free citicoline has shown radioprotective effect at higher doses tested. Interestingly, encapsulation of citicoline in pegylated liposomes significantly improved the radioprotective effect by 4-fold compared to free citicoline in OVCAR-3 but not in HUVEC. Further, citicoline encapsulation in transferrin-coupled liposomes has significantly improved the radioprotective effect by approximately 8-fold in OVCAR-3 and 2-fold in HUVEC cells with respect to the free drug. This is likely due to the entry of citicoline into cells via transferrin receptor mediated endocytosis. In conclusion, our results suggest that low concentrations of citicoline encapsulated in transferrin-coupled liposomes could offer therapeutic benefit in treating stroke compared to free citicoline.     

双配体修饰的阿霉素脂质体靶向于脑胶质瘤的体外研究

目的筛选和优化转铁蛋白、叶酸共同修饰的阿霉素脂质体的处方及制备工艺,以期得到具有良好的脑胶质瘤靶向治疗作用的给药系统。方法采用薄膜分散和硫酸铵梯度法制备阿霉素脂质体。将叶酸连接至二硬脂酸磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000-NH2)得到DSPE-PEG2000-Folic,考察不同磷脂种类、药脂比、水化介质和载药时间,对脂质体粒径、包封率和稳定性的影响,确定脂质体的处方工艺。以大鼠的脑毛细血管内皮细胞(bEnd3)和星形胶质细胞组成体外血脑屏障(blood-brain barrier,BBB),并结合大鼠胶质瘤C6细胞,构建体外模拟胶质瘤靶向治疗的复合BBB模型。考察阿霉素脂质体在bEnd3细胞中的摄取机制和透过BBB的转运速率及对C6细胞的毒性。结果确定了DSPC作为主要磷脂组分,并以120 mmol.L 1的硫酸铵作为水化介质,药脂比为1∶1 5,载药时间选择60 min,成功制备了高包封率和稳定性的双配体脂质体。其在bEnd3细胞中摄取远大于普通脂质体(P<0.05),摄取过程受网格蛋白和小窝内陷介导的细胞内吞作用,并受转铁蛋白和叶酸的影响;同时其在BBB模型中的药物透过速率、及其进一步透过BBB后对下层C6细胞的毒性,均显著高于其他脂质体组。结论转铁蛋白和叶酸共同修饰的阿霉素脂质体具有较好的体外脑胶质瘤靶向治疗作用。     

Transferrin-conjugated liposomal system for improved delivery of 5-fluorouracil to brain

The objective of this study is to achieve the enhanced delivery of 5-fluorouracil to brain through transferrin-coupled liposomes. 5-Fluorouracil-loaded liposomes were prepared by cast film method and characterized for particle size, shape, percent encapsulation efficiency and in vitro drug release. Biodistribution studies were carried out with the help of radiolabelled 5-fluorouracil. 5-Fluorouracil was labelled with (99m)Tc-DTPA by oxidation-reduction method using stannous chloride and optimized for labelling parameters to get a high labelling efficiency. The in vitro stability was determined to check the efficiency of a system to find out the suitability of the radiolabelled system for in vivo studies. (99m)Tc-DTPA-labelled 5-fluorouracil bearing non-coupled and coupled liposomes were administered intravenously and biodistribution studies were performed. The distribution of 5-fluorouracil via non-coupled and coupled liposomes was determined in various organs, such as lungs, liver, kidneys, spleen and brain, by measuring the radioactivity using a gamma scintillation unit. The results of in vivo studies confirmed a selective uptake of the transferrin-coupled liposomes from the brain capillary endothelial cells. An average of 10-fold increase in the brain uptake of the drug was observed after the liposomal delivery of 5-fluorouracil, while the transferrin-coupled liposomes caused a 17-fold increase in the brain uptake of 5-fluorouracil. Therefore, it can be concluded that transferrin-coupled liposomes enhance the brain uptake of the drug, like 5-fluorouracil.     

Transferrin coupled liposomes as drug delivery carriers for brain targeting of 5-florouracil

Diseases and disorders of the brain are extremely difficult to treat pharmacologically because most drugs are unable to pass across the blood--brain barriers. Complex multi-strand tight junctions between adjacent cerebral endothelial cells and between choroid plexus epithelial cells form a physical barrier and prevent the passage of water soluble drugs from the blood into the brain, whereas the inward passage of lipid soluble drugs is restricted by drug efflux pumps which act as a functional barrier. In the present work, a transferrin-coupled liposomal system for brain delivery of 5-florouracil has been investigated.5-florouracil and (99m)Tc-DTPA bearing non-coupled liposomes were prepared by cast film method, which were coupled with the transferrin by incubating these liposomes with transferrin in the presence of the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride in saline phosphate buffer (pH 7.4). These liposomal systems were characterized for vesicle size, percent drug entrapment, and in vitro drug release. The size of the liposomes was increased on coupling with transferrin while percent drug entrapment reduced. The results of the in vitro release profile demonstrated that non-coupled liposomal formulation releases a comparatively higher percent (i.e. 74.8+/-3.21%) of drug than coupled liposomes. Results of in vivo study suggested a selective uptake of the transferrin-coupled liposomes from the brain capillary endothelial cells. In case of coupled liposomes, the level of radioactivity was 17-fold more as compared to the free radioactive agent and 13 times more with the non-coupled liposomes. Therefore, it could be concluded that using transferrin coupled liposomes the brain uptake of the drug could be enhanced.     

Transferrin coupled liposomes as drug delivery carriers for brain targeting of 5-florouracil

Diseases and disorders of the brain are extremely difficult to treat pharmacologically because most drugs are unable to pass across the blood–brain barriers. Complex multi-strand tight junctions between adjacent cerebral endothelial cells and between choroid plexus epithelial cells form a physical barrier and prevent the passage of water soluble drugs from the blood into the brain, whereas the inward passage of lipid soluble drugs is restricted by drug efflux pumps which act as a functional barrier. In the present work, a transferrin-coupled liposomal system for brain delivery of 5-florouracil has been investigated.

5-florouracil and ^99mTc-DTPA bearing non-coupled liposomes were prepared by cast film method, which were coupled with the transferrin by incubating these liposomes with transferrin in the presence of the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride in saline phosphate buffer (pH 7.4). These liposomal systems were characterized for vesicle size, percent drug entrapment, and in vitro drug release. The size of the liposomes was increased on coupling with transferrin while percent drug entrapment reduced. The results of the in vitro release profile demonstrated that non-coupled liposomal formulation releases a comparatively higher percent (i.e. 74.8±3.21%) of drug than coupled liposomes. Results of in vivo study suggested a selective uptake of the transferrin-coupled liposomes from the brain capillary endothelial cells. In case of coupled liposomes, the level of radioactivity was 17-fold more as compared to the free radioactive agent and 13 times more with the non-coupled liposomes. Therefore, it could be concluded that using transferrin coupled liposomes the brain uptake of the drug could be enhanced.     

Effect of transferrin receptor-targeted liposomal doxorubicin in P-glycoprotein-mediated drug resistant tumor cells

The over-expression of P-glycoprotein (P-gp) has been associated with the development of multidrug resistance (MDR) in cancer cells. In this study, we examined whether transferrin receptor (Tf-R) targeted liposomes can efficiently deliver encapsulated doxorubicin (DXR) into MDR cells (SBC-3/ADM) via Tf-R-mediated endocytosis thus overcoming MDR by by-passing P-gp-mediated drug efflux. We prepared four types of liposome, i.e. untargeted and Tf-R-targeted, made of either egg-PC/cholesterol or hydrogenated egg PC/cholesterol. Only with the targeted EPC-liposome we achieved significant delivery of encapsulated DXR and increased cytotoxicity of encapsulated DXR on the MDR cells (3.5-fold higher than free DXR). Confocal microscopy and an intracellular drug-accumulation assay indicated that the targeted liposomes efficiently delivered DXR into cells where it readily accumulated in the nucleus, in both drug-sensitive and MDR cells. These findings suggest that the targeted liposomes are rapidly internalized via Tf-R-mediated endocytosis followed by release of their contents into the cytoplasm. The rapid internalization and content release, most likely facilitated by the higher fluidity of the EPC-based liposomes, may explain why only targeted EPC-liposomes were able to prevent drug efflux by P-gp and to consequently circumvent MDR. Our results indicate that in order to achieve MDR circumvention by means of liposomal encapsulation of DXR the liposomes not only need to be targeted, but also to have the proper physicochemical properties for adequate release of the drug. Furthermore, these in vitro results suggest that Tf-R targeted EPC-liposomes are a potentially useful drug delivery system to circumvent P-gp-mediated MDR of tumors.     

Liposomes for drug delivery to the lungs by nebulization.

Preparation of drug-loaded freeze-dried (FD) liposomes, designed for delivery to lungs after rehydration/nebulization was investigated. Rifampicin (RIF) incorporating multilamelar (MLV) and dried rehydrated vesicles (DRV); composed of phosphatidylcholine (PC), dipalmitoyloglycero-PC (DPPC) or distearoyloglycero-PC (DSPC), containing or not Cholesterol (Chol), were prepared. Vesicles were characterized for encapsulation efficiency (EE%), size distribution, zeta-potential, stability during freeze drying (FD) and nebulization (nebulization efficiency (NE%) and retention of RIF after nebulization (NER%)). Mucoadhesion and toxicity in A549 cells was measured. RIF EE% was not affected by liposome type but lipid composition was important; Synthetic lipid vesicles (DPPC and DSPC) had higher EE% compared to PC. As Chol increased EE% decreased. Freeze drying (FD) had no effect on EE%, however trehalose decreased EE% possibly due to RIF displacement. NER% was highly affected by lipid composition. Results of NE% and NER% for RIF-loaded liposomes show that DSPC/Chol (2:1) is the best composition for RIF delivery in vesicular form to lungs, by nebulization. Mucoadhesion and A549 cell toxicity studies were in line with this conclusion, however if mucoadhesion is required, improvement may be needed.     

Fusogenic pH sensitive liposomal formulation for rapamycin: Improvement of antiproliferative effect

Context: Liposomes are increasingly employed to deliver chemotherapeutic agents, antisense oligonucleotides, and genes to various therapeutic targets.

Objective: The present investigation evaluates the ability of fusogenic pH-sensitive liposomes of rapamycin in increasing its antiproliferative effect on human breast adenocarcinoma (MCF-7) cell line.

Materials and methods: Cholesterol (Chol) and dipalmitoylphosphatidylcholine (DPPC) (DPPC:Chol, 7:3) were used to prepare conventional rapamycin liposomes by a modified ethanol injection method. Dioleoylphosphatidylethanolamine (DOPE) was used to produce fusogenic and pH-sensitive properties in liposomes simultaneously (DPPC:Chol:DOPE, 7:3:4.2). The prepared liposomes were characterized by their size, zeta potential, encapsulation efficiency percent (EE%), and chemical stability during 6 months. The antiproliferative effects of both types of rapamycin liposomes (10, 25, and 50?nmol/L) with optimized formulations were assessed on MCF-7 cells, as cancerous cells, and human umbilical vein endothelial cells (HUVEC), as healthy cells, employing the diphenyltetrazolium bromide (MTT) assay for 72?h.

Results and discussion: The particle size, zeta potential, and EE% of the liposomes were 165?±?12.3 and 178?±?15.4?nm, ?39.6?±?1.3, and ?41.2?±?2.1?mV as well as 76.9?±?2.6 and 76.9?±?2.6% in conventional and fusogenic pH-sensitive liposomes, respectively. Physicochemical stability results indicated that both liposome types were relatively stable at 4?°C than 25?°C. In vitro antiproliferative evaluation showed that fusogenic pH-sensitive liposomes had better antiproliferative effects on MCF-7 cells compared to the conventional liposomes. Conversely, fusogenic pH-sensitive liposomes had less cytotoxicity on HUVEC cell line.     

Liposomes for drug delivery to the lungs by nebulization

Preparation of drug-loaded freeze-dried (FD) liposomes, designed for delivery to lungs after rehydration/nebulization was investigated. Rifampicin (RIF) incorporating multilamelar (MLV) and dried rehydrated vesicles (DRV); composed of phosphatidylcholine (PC), dipalmitoyloglycero-PC (DPPC) or distearoyloglycero-PC (DSPC), containing or not Cholesterol (Chol), were prepared. Vesicles were characterized for encapsulation efficiency (EE%), size distribution, zeta-potential, stability during freeze drying (FD) and nebulization (nebulization efficiency (NE%) and retention of RIF after nebulization (NER%)). Mucoadhesion and toxicity in A549 cells was measured. RIF EE% was not affected by liposome type but lipid composition was important; Synthetic lipid vesicles (DPPC and DSPC) had higher EE% compared to PC. As Chol increased EE% decreased. Freeze drying (FD) had no effect on EE%, however trehalose decreased EE% possibly due to RIF displacement. NER% was highly affected by lipid composition. Results of NE% and NER% for RIF-loaded liposomes show that DSPC/Chol (2:1) is the best composition for RIF delivery in vesicular form to lungs, by nebulization. Mucoadhesion and A549 cell toxicity studies were in line with this conclusion, however if mucoadhesion is required, improvement may be needed.     

Liposomes modified with cyclic RGD peptide for tumor targeting

Cyclic RGD peptide anchored sterically stabilized liposomes (RGD-SL) were investigated for selective and preferential presentation of carrier contents at angiogenic endothelial cells overexpressing alphavbeta3 integrins on and around tumor tissue and thus for assessing their targetabilty. Liposomes were prepared using distearoylphosphatidylcholine (DSPC), cholesterol and distearoylphosphatidylethanolamine-polyethyleneglycol-RGD peptide conjugate (DSPE-PEG-RGD) in a molar ratio 56:39:5. The control RAD peptide anchored sterically stabilized liposomes (RAD-SL) and liposome with 5 mol% PEG (SL) without peptide conjugate which had similar lipid composition were used for comparison. The average size of all liposome preparations prepared was approximately 105 nm and maximum drug entrapment was 10.5+/- 1.1%. In vitro endothelial cell binding of liposomes exhibited 7-fold higher binding of RGD-SL to HUVEC in comparison to the SL and RAD-SL. Spontaneous lung metastasis and angiogenesis assays show that RGD peptide anchored liposomes are significantly (p<0.01) effective in the prevention of lung metastasis and angiogenesis compared to free 5-FU, SL and RAD-SL. In therapeutic experiments, 5-FU, SL, RGD-SL and RAD-SL were administered intravenously on day 4 at the dose of 10 mg 5-FU/kg body weight to B16F10 tumor bearing BALB/c mice resulting in effective regression of tumors compared with free 5-FU, SL and RAD-SL. Results indicate that cyclic RGD peptide anchored sterically stabilized liposomes bearing 5-FU are significantly (p<0.01) active against primary tumor and metastasis than the non-targeted sterically stabilized liposomes and free drug. Thus cyclic RGD peptide anchored sterically stabilized liposomes hold potential of targeted cancer chemotherapeutics.     

Polyethylene glycol-coated liposomes for oral delivery of recombinant human epidermal growth factor

The present study was to investigate the feasibility of oral delivery of recombinant human epidermal growth factor (rhEGF). Polyethylene glycol (PEG)-coated liposomes containing rhEGF was prepared and evaluated for their stability and permeability in Caco-2 cells. In the animal study, we also determined plasma concentration and gastric ulcer healing effect after oral administration of rhEGF liposomes or the solution. Encapsulation of rhEGF into liposomes, suppressed the degradation in Caco-2 cell homogenate compared with the solution. The flux of rhEGF from dipalmitoylphosphatidylcholine (DPPC) liposome across Caco-2 cell monolayer from the apical to basolateral side was three times greater than that from phosphatidylcholine (PC) liposome or the solution. After oral administration of rhEGF liposomes or the solution in rats, the area under the concentration-time curve (AUC) of rhEGF increased 1.7- and 2.5-fold for PC and DPPC liposomes, respectively. The gastric ulcer healing effect was significantly increased in DPPC liposome compared with PC liposome and the solution. The enhanced curative ratio of rhEGF encapsulated into DPPC liposome may be due to the resistance to enzyme degradation, higher permeability and increased plasma AUC. Therefore, PEG-coated liposomes containing rhEGF could be used as an oral delivery formulation with enhanced encapsulation efficiency.     

Prolonged Blood Circulation of Methotrexate by Modulation of Liposomal Composition

Prolonged circulation by liposomal incorporation has been shown to enhance the therapeutic efficacy of drugs in many cases. The purpose of this study was to investigate whether the prolonged circulation of methotrexate (MTX) can be achieved by modulating the liposomal compositions. Various compositions of liposomes were prepared with 2:1 of phosphatidylcholine (PC) and cholesterol (CH) with or without distearoylphosphatidyl-ethanolamine-N-poly(ethyleneglycol) 2000 (DSPE-PEG). The MTX encapsulation efficiency depended on the type of PC used. It also appeared to increase by inclusion of DSPE-PEG. The size of liposomes decreased by the inclusion of DSPE-PEG. The inclusion of DSPE-PEG lowered the plasma-induced release of MTX from EggPC/CH and DPPC/CH liposomes, suggesting its enhancement effect on the liposomal stability. After intravenous injection to rats, the pharmaockinetics and biodistribution of MTX were significantly changed by liposomal incorporation and also by the composition of liposomes. The total body clearance of MTX incorporated in EggPC/CH, DPPC/CH, EggPC/CH/DSPE-PEG, and DPPC/CH/DSPE-PEG liposomes decreased 4.4-, 14.9-, 24.5-, and 53.1-fold, compared with that of free MTX. The ratio of MTX concentration in blood to liver and spleen after injection of DPPC/CH, EggPC/CH/DSPE-PEG, and DPPC/CH/DSPE-PEG liposomes was 5.4-, 8.5-, and 13.5-fold higher than that of EggPC/CH liposomes. Furthermore, the accumulation of MTX in the kidney, one of the organs in which MTX exhibits its toxicity, was significantly lowered by liposomal incorporation, especially by DSPE-PEG-containing liposomes. Taken together, DPPC/CH/DSPE-PEG liposomes most effectively prolonged the blood circulation, and reduced hepatosplenic and kidney uptake of MTX. DPPC/CH/DSPE-PEG liposomes may have potential as an efficient delivery system for MTX.     

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.     

核磁共振法测定喃氟啶温度敏感性脂质体的相转变温度

用核磁共振法测定了喃氟啶温度敏感性脂质体的相转变温度。此法与经典的差热分析法不同,有灵敏度高、准确性好、提供信息全面等优点。用该法测得的DPPC-脂质体和DSPC-脂质体的相转变温度分别为36℃和48℃;DPPC-DSPC-脂质体的相转变温度与DPPC和DSPC的含量有关,随DPPC含量的增加而降低,随DSPC含量的增加而增加;药物的加入及含量不影响相转变温度。同时,制得可供临床前研究用相转变温度为41℃的喃氟啶温度敏感性脂质体。     

Influence of cationic lipids on the stability and membrane properties of paclitaxel-containing liposomes

Paclitaxel (taxol) is a poorly soluble anticancer agent that is in widespread clinical use. Liposomes provide a less toxic vehicle for solubilizing the drug and increasing the therapeutic index of paclitaxel in model tumor systems. The role of liposome membrane composition in the stability of paclitaxel-containing formulations is understood partially for neutral and anionic liposomes, but poorly for other compositions. We investigated the effect of dialkyl cationic lipids on the stability and physical properties of paclitaxel-containing liposomes, using circular dichroism (CD), fluorescence spectroscopy, and differential interference contrast microscopy (DIC). DOTAP (1,2-dioleoyl-3-trimethylammonium propane), a cationic lipid used frequently for gene delivery, was combined at various ratios with dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), or distearoylphosphatidylcholine (DSPC). In the absence of DOTAP, the stability of liposomes containing > or =3 mol% paclitaxel was observed to follow the following rank order: DPPC >DSPC > DMPC. Increasing concentrations of DOTAP increased the physical stability of all compositions, and maximal stabilization was achieved at 30-50 mol% DOTAP, depending on the paclitaxel concentration and the acyl chain length of the phosphatidylcholine. The relationship between stability and mole fraction of DOTAP was complex for some compositions. DOTAP exerted a major fluidizing effect on DMPC, DPPC, and DSPC membranes, and the addition of paclitaxel at 3-8 mol% did not increase fluidity further. Studies of membrane phase domain behavior using the probe Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) indicated that both paclitaxel and DOTAP were miscible with the phosphatidylcholine phase. The physical events leading to destabilization of formulations are hypothesized to arise from concentration-dependent paclitaxel self-association rather than immiscibility of the membrane lipids. Given the increased incorporation and stability of paclitaxel in DOTAP-containing membranes and the potential for enhanced interaction with cells, cationic liposomes may provide a therapeutic advantage over previously described liposome formulations.     

Preparation, characterisation and entrapment of a non-glycosidic threitol ceramide into liposomes for presentation to invariant natural killer T cells

Dendritic cells (DCs) are able to present glycolipids to invariant natural killer T (iNKT) cells in vivo. Very few compounds have been found to stimulate iNKT cells, and of these, the best characterised is the glycolipid α-galactosylceramide, which stimulates the production of large quantities of interferon-gamma (IFN-γ) and interleukin-4 (IL-4). However, αGalCer leads to overstimulation of iNKT cells. It has been demonstrated that the αGalCer analogue, threitol ceramide (ThrCer 2), successfully activates iNKT cells and overcomes the problematic iNKT cell activation-induced anergy. In this study, ThrCer 2 has been inserted into the bilayers of liposomes composed of a neutral lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), or dimethyldioctadecylammonium bromide (DDA), a cationic lipid. Incorporation efficiencies of ThrCer within the liposomes was 96% for DSPC liposomes and 80% for DDA liposomes, with the vesicle size (large multilamellar vs. small unilamellar vesicles) making no significant difference. Langmuir-Blodgett studies suggest that both DSPC and DDA stack within the monolayer co-operatively with the ThrCer molecules with no condensing effect. In terms of cellular responses, IFN-γ secretion was higher for cells treated with small DDA liposomes compared with the other liposome formulations, suggesting that ThrCer encapsulation in this liposome formulation resulted in a higher uptake by DCs.     

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.     

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.     

Transdermal lontophoretic delivery of colchicine encapsulated in liposomes

Iontophoresis is useful for the transdermal delivery of charged drugs. However, nonionized drugs either have a low flux (due to electro-osmosis) or cannot be delivered using this technique. Because ionized or nonionized drugs can be encapsulated in charged liposomes, it was hypothesized that charged liposomes can deliver neutral or nonionized drug efficiently by iontophoresis. Colchicine, a neutral drug, was encapsulated in large unilamellar vesicles (LUVs), prepared with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) along with cholesterol (1:0.5 mole ratio). Multilamellar vesicles (MLVs) were prepared by the thin-film hydration method and LUVs were obtained by extruding MLVs through polycarbonate filters of 200 nm pore size. Positive charge was induced in the liposomes by adding stearylamine and negative charge by adding dicetyl phosphate. Nonencapsulated drug was separated from LUVs by the Ficoll density gradient method. Positively charged LUVs were delivered under the anode, negatively charged LUVs under the cathode, and neutral LUVs without current using Franz cells and human cadaver skin. Plain colchicine as well as colchicine encapsulated in positively charged LUVs was delivered better under the anode compared with the cathode and passive conditions. Delivery of colchicine encapsulated in positively charged DSPC liposomes was four to five times greater than that of plain colchicine and two to three times greater than that of colchicine encapsulated in DMPC or DPPC liposomes. Because LUVs prepared with DMPC and DPPC were fluid at 37°C, the encapsulated drug leaked during iontophoresis and therefore the delivery was less. Delivery of colchicine was lower under the cathode due to the change in pH during iontophoresis, which, as observed in high-performance liquid chromatographic analysis, caused degradation of the drug. Thus, it can be concluded that iontophoresis of colchicine encapsulated in positively charged DSPC liposomes can improve its delivery across human cadaver skin     

Kanamycin incorporation in lipid vesicles prepared by ethanol injection designed for tuberculosis treatment

The primary goal of this study was the production of liposomes encapsulating kanamycin for drug administration by inhalation. The selected drug is indicated for multiresistant tuberculosis, and administration through inhalation allows both local delivery of the drug to the lungs and systemic therapy. The ethanol injection method used for the liposome production is easily scaled up and is characterized by simplicity and low cost. Vesicles were prepared using different lipid compositions, including hydrogenated soybean phosphatidylcholine and cholesterol (SPC/Chol), egg phosphatidylcholine and cholesterol (EPC/Chol), distearoyl phosphatidylcholine and cholesterol (DSPC/Chol), distearoyl phosphatidylcholine, dimyristoyl phosphatidylethanolamine and cholesterol (DSPC/DMPE/Chol), dipalmitoyl phosphatidylcholine and cholesterol (DPPC/Chol) and dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidylglycerol and cholesterol (DPPC/DPPG/Chol). The effects of different operational conditions for vesicle production and drug encapsulation were evaluated, aiming at a compromise between final process cost and suitable vesicle characteristics. The best performance concerning drug incorporation was achieved with the DSPC/Chol system, although its production cost was considerably larger than that of the natural lipids formulations. Encapsulation efficiencies up to 63% and final drug to lipid molar ratios up to 0.1 were obtained for SPC/Chol vesicles presenting mean diameters of 132 nm incubated at 60 degrees C with the drug for 60 min at an initial drug-to-lipid molar ratio of 0.16.