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.     

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.     

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 microg/micromol of phospholipid, respectively. Liposomes prepared from DSPC showed relatively better stability than DPPC liposomes at 37 degrees 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.     

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.     

Brain-specific delivery of rifampin from lactyl stearate-coupled liposomes via monocarboxylic acid transporters

Background

The blood-brain barrier (BBB) is an obstacle for pharmacologists wishing to find treatments for patients with brain disorders. The BBB restricts the uptake of many valuable hydrophilic drugs and limits their efficacy because of the presence of tight junctions, a high metabolic capacity, low pinocytic vesicular traffic, and efficient efflux mechanisms.

Aim

The present study aimed to characterize lactyl stearate-coupled liposomes and their potential for the brain targeting of rifampin (rifampicin).

Method

A liposomal delivery system was prepared for achieving the brain-targeted delivery of rifampin in 21 albino rats utilizing the monocarboxylic acid transport system. Liposomes were prepared by the cast-film method using phosphatidylcholine and cholesterol. Similarly, lactyl stearate-coupled liposomal systems were prepared by casting lactyl stearate film with lipids. These liposomal formulations were characterized for entrapment efficiency, vesicle size, in vitro drug release (using dialysis membrane), and in vivo drug accumulation in various tissues.

Results

Coupling of lactyl stearate to liposomes had a profound influence on entrapment efficiency. Entrapment efficiency was reduced from 41.28 ± 2.02% in uncoupled liposomes to 34.23 ± 1.60% in coupled liposomes. The vesicle size was increased after coupling with lactyl stearate. The in vitro drug release for the uncoupled formulation LIPO-3 was 62.9 ± 3.01% after 24 hours, whereas that of the coupled formulation LIPO-3-Ls-III was 44.5 ± 2.09%. The percentage of rifampin dose recovered from the brain following administration of lactyl stearate-coupled liposomes to albino rats at different time intervals was about 6–8 times higher than with uncoupled liposomes and about 10–12 times higher than with the plain drug solution.

Conclusion

Lactyl stearate-coupled liposomes were better localized within the brain compared to uncoupled liposomes. Lactyl stearate-coupled liposomes could be an excellent carrier system for brain targeting of the hydrophilic drug rifampin.     

Dual Functionalized 5-Fluorouracil Liposomes as Highly Efficient Nanomedicine for Glioblastoma Treatment as Assessed in an In Vitro Brain Tumor Model

Drug delivery to the brain has been a major challenge due to the presence of the blood-brain barrier, which limits the uptake of most chemotherapeutics into brain. We developed a dual-functionalized liposomal delivery system, conjugating cell penetrating peptide penetratin to transferrin-liposomes (Tf-Pen–conjugated liposomes) to enhance the transport of an anticancer chemotherapeutic drug, 5-fluorouracil (5-FU), across the blood-brain barrier into the tumor cells. The in vitro cellular uptake study showed that the dual-functionalized liposomes are capable of higher cellular uptake in glioblastoma (U87) and brain endothelial (bEnd.3) cells monolayer. In addition, dual-functionalized liposomes demonstrated significantly higher apoptosis in U87 cells. The liposomal nanoparticles showed excellent blood compatibility and in vitro cell viability, as studied by hemolysis and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, respectively. The 5-FU–loaded dual-functionalized liposomes demonstrated higher transport across the brain endothelial barrier and delivered 5-FU to tumor cells inside poly(lactic-co-glycolic acid)-chitosan scaffold (an in vitro brain tumor model), resulting in significant tumor regression.     

Targeting of daunomycin using biotinylated immunoliposomes: pharmacokinetics, tissue distribution and in vitro pharmacological effects

Biotinylated immunoliposomes were prepared by a non-covalent (biotin-streptavidin) coupling procedure and conjugated to the OX26 monoclonal antibody directed against the rat transferrin receptor. In vitro, these biotinylated immunoliposomes were used to by-pass P-glycoprotein in multidrug-resistant RBE4 brain capillary endothelial cells and thereby to achieve 2- to 3-fold higher intracellular accumulation of liposomal daunomycin as compared to free drug. The extent of cellular uptake of liposomal daunomycin was dose- and time-dependent, was inhibited by competition with unbound OX26 and was associated with a pharmacological (i.e. cytotoxic) effect. Cytotoxic effects of liposomal formulations of daunomycin, in contrast to the free drug, were apparent only after prolonged incubation periods being indicative of a slow intracellular unpacking and release of liposomal daunomycin. Pharmacokinetics and tissue distribution studies in the rat revealed brain accumulation of daunomycin in OX26-immunoliposomes to higher levels as compared to brain uptake of free daunomycin, or daunomycin incorporated within pegylated liposomes or within unspecific IgG(2a) isotype control immunoliposomes. Such OX26-mediated effects were not observed in other tissues such as spleen, liver, muscle or kidney.     

Design and development of folate appended liposomes for enhanced delivery of 5-FU to tumor cells

Folate appended sterically-stabilized liposomes (FA-SL) were investigated for tumor targeting. Liposomes were prepared using HSPC, cholesterol and FA-polyethylene glycol (PEG)-SA. The liposomes with polyethylene glycol (PEG) without folic acid which has similar lipid composition were used for comparison. Liposomal preparations were characterized for shape, size and percent entrapment. The average size of liposomes was found to be in range 124-163 nm and maximum drug entrapment was found to be 34.2-40.3%. In vitro drug release from the formulations is obeying fickian release kinetics. Cellular uptake and IC(50) values of the FR-targeted formulation were determined in vitro in FR (+) B16F10 melanoma cells. In vitro cell binding of FA-SL exhibits 11-folds higher binding to B16F10 melanoma cells in comparison to SL. In vivo cytotoxicy assay on FR targeted liposomes gave IC(50) of 1.87 microM and non-targeted liposomes gave IC(50) of 4.02 microM. In therapeutic experiments 5-fluorouracil (5-FU), SL and FA-SL were administered at the dose of 10 mg 5-FU/kg body weight to B16F10 tumor bearing Balb/c mice. Administration of FA-SL formulation results in effective reduction in tumor growth as compared with free 5-FU and SL. Results indicate that folic acid appended SL bearing 5-FU are significantly (P < 0.01) active against primary tumor and metastasis than non-targeted sterically-SL. Thus, it could be concluded that folate coupled liposomal formulations enhanced drug uptake by tumor cells.     

Targeting of daunomycin using biotinylated immunoliposomes: Pharmacokinetics,tissue distribution and in vitro pharmacological effects

Biotinylated immunoliposomes were prepared by a non-covalent (biotin-streptavidin) coupling procedure and conjugated to the OX26 monoclonal antibody directed against the rat transferrin receptor. In vitro, these biotinylated immunoliposomes were used to by-pass P-glycoprotein in multidrug-resistant RBE4 brain capillary endothelial cells and thereby to achieve 2- to 3-fold higher intracellular accumulation of liposomal daunomycin as compared to free drug. The extent of cellular uptake of liposomal daunomycin was dose- and time-dependent, was inhibited by competition with unbound OX26 and was associated with a pharmacological (i.e. cytotoxic) effect. Cytotoxic effects of liposomal formulations of daunomycin, in contrast to the free drug, were apparent only after prolonged incubation periods being indicative of a slow intracellular unpacking and release of liposomal daunomycin. Pharmacokinetics and tissue distribution studies in the rat revealed brain accumulation of daunomycin in OX26-immunoliposomes to higher levels as compared to brain uptake of free daunomycin, or daunomycin incorporated within pegylated liposomes or within unspecific IgG_2a isotype control immunoliposomes. Such OX26-mediated effects were not observed in other tissues such as spleen, liver, muscle or kidney.     

Reduced hepatic toxicity, enhanced cellular uptake and altered pharmacokinetics of stavudine loaded galactosylated liposomes.

The aim of the present investigation was to reduce the hepatic toxicity, enhance the cellular uptake and alter the pharmacokinetics of stavudine using galactosylated liposomes. beta-D-1-Thiogalactopyranoside residues were covalently coupled with dimyristoyl phosphatidylethanolamine, which was then used to form liposomes. The galactosylated liposomal system was assessed for in vitro ligand-specific activity. The drug release from liposomes was studied by dialysis method. Ex vivo cellular uptake study was performed using liver parenchymal cells harvested from male albino rats. Changes in hematological parameters, hepatic enzymes, hepatomegaly, plasma and tissue distribution of the formulations (free stavudine solution, uncoated liposomal and galactosylated liposomes) were determined using albino rats. Percent cumulative drug release in 24h was low (34.8+/-2.6%). Enhanced hepatic cellular d4T uptake (27.96+/-2.41pg d4T/million cells) was seen in case of galactosylated liposomal d4T. Galactosylated liposomes maintained a significant level of d4T in tissues rich in galactose specific receptors and had a prolonged residence (11.44+/-1.25h) in the body resulting in enhanced half-life of d4T (23.07+/-1.25h). This formulation did not show either hematological or hepatic toxicity. Galactosylation of liposomes alter the biodistribution of encapsulated drug thereby delivering the drug to cells bearing galactose specific receptors.     

Elastic liposomes mediated transdermal delivery of an anti-hypertensive agent: propranolol hydrochloride

One major problem encountered in transdermal drug delivery is the low permeability of drugs through the skin barrier. In the present investigation ultradeformable lipid vesicles, that is, elastic liposomes were prepared incorporating propranolol hydrochloride for enhanced transdermal delivery. Elastic liposomes bearing propranolol hydrochloride were prepared by conventional rotary evaporation method and characterized for various parameters including vesicles shape and surface morphology, size and size distribution, entrapment efficiency, elasticity, turbidity, and in vitro drug release. In vitro flux, enhancement ratio (ER), and release pattern of propranolol hydrochloride were calculated for transdermal delivery. In vivo study conducted on male albino rats (Sprague Dawley) was also taken as a measure of performance of elastic liposomal, liposomal, and plain drug solution. The better permeation through the skin was confirmed by confocal laser scanning microscopy (CLSM). Results indicate that the elastic liposomal formulation for transdermal delivery of propranolol hydrochloride provides better transdermal flux, higher entrapment efficiency, ability as a self-penetration enhancer and effectiveness for transdermal delivery as compared to liposomes.     

Uptake of apolipoprotein E fragment coupled liposomes by cultured brain microvessel endothelial cells and intact brain capillaries

The suitability of surface modified liposomes as drug carriers for brain-specific targeting was investigated using apolipoprotein E fragments as brain-directed vectors. Liposomes coated with polyethylene glycol-2000 (sterically stabilized, PEGylated liposomes) were prepared from hydrogenated egg phosphatidylcholine, cholesterol, and a PEG-derivatized phospholipid. Liposomes were covalently coupled to a peptide of 26 amino acids length, derived from the binding site of human apolipoprotein E4 (ApoE4) and a peptide of random amino acid sequence, respectively. Rhodamine-labeled dipalmitoylphosphatidylethanolamine was incorporated into the lipid bilayer in order to visualize the liposomal interaction with brain capillary endothelial cell monolayers. The interaction of the liposomes with monolayers of porcine brain capillary endothelial cells (BCEC), the rodent cell line RBE4, and freshly isolated porcine brain capillaries was studied by means of confocal laser scanning fluorescence microscopy. In contrast to random peptide coupled liposomes, the ApoE4-fragment coupled liposomes were rapidly taken up by cultured BCECs and RBE4 cells. Uptake could be inhibited by ApoE4, free peptide, and antibodies against the LDL receptor in a concentration-dependent manner. The results indicate that the liposomes are internalized via the LDL receptor, which is expressed at the blood?brain barrier. In conclusion, liposomes coupled to ApoE4 fragments are taken up into brain endothelium via an endocytotic pathway and may therefore be a suitable carrier for drug delivery to the brain.     

Preparation and in vitro evaluation of anti-VCAM-1-Fab'-conjugated liposomes for the targeted delivery of the poorly water-soluble drug celecoxib

When an inflammatory stimulus is given, vascular endothelial cells express various cell adhesion molecules including the vascular cell adhesion molecule (VCAM)-1. In this study, the possibility of specifically delivering anti-inflammatory drugs to activated endothelial cells by utilizing VCAM-1 as a target receptor was explored by loading celecoxib, a selective cyclooxygenase-2 inhibitor, into liposomes coupled to the Fab' fragment against VCAM-1. Anti-VCAM-1-Fab'-conjugated liposomes were prepared by forming an amide linkage between amino groups of Fab' and the carboxylic group of glutaryl-N-phosphatidylethanolamine in liposomes using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide as a cross-linker in the presence of sulpho-N-hydroxysuccinimide. The coupling of Fab' to phospholipids constituting liposomes was confirmed by SDS-PAGE analysis. Under our optimized conjugation conditions, 130.0?μg Fab' was coupled to 1?μmol liposomes. Immunoblotting analysis showed that VCAM-1 protein expression could be induced by incubating human umbilical vein endothelial cells (HUVEC) with TNF-α. Confocal laser microsopy analysis revealed that Fab' conjugation to liposomes selectively increased liposomal uptake in TNF-α-pre-stimulated (VCAM-1-expressed) HUVECs, but not in cells without VCAM-1 expression. The concentration of celecoxib loaded in Fab'-conjugated liposomes was 281.1?±?29?μg/mL, suggesting that liposomal loading also helped to overcome the limitations in celecoxib administration caused by its poor water solubility. Celecoxib loaded in Fab'-conjugated liposomes inhibited prostaglandin E? (PGE?) production induced by TNF-α-pre-stimulation more efficiently than when loaded in conventional liposomes. Therefore, Fab'-conjugated liposomes served as a drug delivery system with dual functions: targeted delivery and solubilizing capacity.     

Grafting of cell-penetrating peptide to receptor-targeted liposomes improves their transfection efficiency and transport across blood-brain barrier model

We report bifunctional liposomal delivery system, combining transferrin (Tf)-mediated receptor targeting and poly-L-arginine (PR)-facilitated cell penetration, which overcomes the drawback of saturation of delivery. PR was conjugated to the distal end of distearoyl phosphoethanolamine-polyethylene glycol (PEG) 2000 and was incorporated with other phospholipids in chloroform/methanol (2:1) to form PR liposomes using thin-film hydration technique. Tf-PEG phospholipid micelles were incorporated into PR liposomes using postinsertion technique to form Tf-PR liposomes. The bifunctional liposomes demonstrated significantly (p < 0.05) higher cellular uptake by brain endothelial cells (bEnd.3) and about eightfold higher transfection in primary culture of glial cells as compared with the Tf liposomes. Cell viabilities of Tf-conjugated and bifunctional liposomes were not markedly different; however, transport across in vitro blood-brain barrier model improved considerably after dual modification. The study underlines the potential of bifunctional liposomes as high-efficiency and low-toxicity gene delivery system for the treatment of central nervous system disorders.     

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。     

Translocation of liposomes into cancer cells by cell-penetrating peptides penetratin and tat: a kinetic and efficacy study

Unlike conventional liposomes, sterically stabilized liposomes, with their smaller volume of distribution and reduced clearance, preferentially convey encapsulated drugs into tumor sites. Despite these improvements, intracellular delivery is hampered by the stable drug retention of the liposomes, which diminishes the efficacy of the liposomal drug. To facilitate uptake of liposomal drugs into cells, two cell-penetrating peptides, penetratin (PEN) and TAT, derived from the HIV-1 TAT protein, were studied. In contrast to control peptides, both TAT and PEN enhanced the translocation efficiency of liposomes in proportion to the number of peptides attached to the liposomal surface. A peptide number of as few as five could enhance the intracellular delivery of liposomes. The kinetics of uptake was peptide- and cell-type dependent. Intracellular accumulation of TAT-liposomes increased with incubation time, but PEN-liposomes peaked at 1 h and then declined gradually. After treatment with 1 microg/ml doxorubicin equivalents of liposome for 2 h, TAT increased the doxorubicin uptake of A431 cells by 12-fold. However, the improvement of uptake of liposomal doxorubicin was not reflected by cytotoxicity in vitro or tumor control in vivo. Our results demonstrated that merely adding CPP to a liposome encapsulating anticancer drug was inadequate in improving its antitumor activity. An additional approach to enhance the intracellular release of the encapsulated drug is obviously necessary.     

Effect of dendrimer on entrapment and release of bioactive from liposomes.

An active encapsulation method to obtain high entrapment in liposomes is described. The method harnesses the ability of dendrimer to interact with oppositely charged phospholipid and solubilize acidic drugs in their interior. The high drug entrapment in liposomes is due to the enhanced entrapment of dendrimer, which creates sink in the liposomal aqueous compartment where the methotrexate (MTX) molecules are fluxed in. The encapsulation increases with dendrimer generation. The release of bioactive was also decreased by this method. The method may be useful to entrap drugs with relatively high therapeutic dose.     

Influence of Short-Chain Cell-Penetrating Peptides on Transport of Doxorubicin Encapsulating Receptor-Targeted Liposomes Across Brain Endothelial Barrier

Purpose

To investigate the influence of different cell penetrating peptides (CPPs-TAT, Penetratin and Mastoparan), on the transport of doxorubicin encapsulating transferrin (Tf)-liposomes across brain endothelial barrier, in vitro and in vivo.

Methods

The cellular uptake of dual-functionalized, (Tf-CPP), liposomes into various tumor cells was assessed using HPLC. The transport of liposomes was also measured across a robust 3D brain tumor model constructed using chitosan-PLGA scaffolds. The growth of tumor cells was monitored using H&E staining and the fully grown tumor scaffolds were visualized using SEM. The tumor scaffolds were combined with the culture inserts carrying tightly packed brain endothelial cells. The in vitro and in vivo transport of drug (using Tf-CPP-liposomes) across the brain endothelial barrier was determined by extraction of the drug from cells and tissues followed by analysis using HPLC.

Results

The results demonstrated improved delivery of doxorubicin using dual-functionalized liposomes versus the single ligand or unmodified liposomes. Among different Tf-CPP-liposomes, the Tf-Penetratin liposomes showed efficient cellular transport of the encapsulated drug (approximately 90–98%) and maximum translocation of the drug across the brain endothelial barrier (approximately 15% across in vitro and 4% across in vivo BBB). The Tf-Penetratin and Tf-TAT liposomes demonstrated excellent cellular biocompatibility and no hemolytic activity upto 200nM phospholipid concentration.

Conclusions

The Tf-CPP liposomes showed efficient translocation of the anticancer drug across the brain endothelial barrier. In addition, an absolute and robust in vitro brain tumor model was successfully constructed to overcome the practical intricacies of developing a successful in vivo orthotopic brain tumor model.     

Factors governing the in vivo tissue uptake of transferrin-coupled polyethylene glycol liposomes in vivo

Liposomes, coated with transferrin (Tf)-coupled polyethylene glycol are considered to be potent carriers for drug delivery to various organs via receptor-mediated endocytosis. Since Tf receptors were ubiquitously expressed in various organs, additional perturbation of the liposomes such as regulation of the size may be required to exhibit the tissue selectivity. In the present study, the effect of size on the uptake of transferrin-coupled polyethylene glycol liposomes (Tf-PEG-L) to various organs was investigated. In liver and brain, Tf-dependent uptake was found to be dependent on the size of the liposomes used. In small liposomes with a diameter of 60-80 nm, Tf-PEG-L was taken up to these organs more efficiently than PEG-L. This Tf-dependent uptake for small liposomes decreased by the high dose administration, suggested that Tf-PEG-L is taken up via Tf receptor-mediated endocytosis even under the physiological condition, in which plasma concentration of endogenous Tf remains high. On the other hand, Tf receptor-mediated uptake was also observed in the heart, but size-dependency was not observed in this case. Collectively, these results indicate that size dependency in the uptake of Tf-PEG-L is tissue-dependent and therefore, controlling the size of Tf-PEG-L may be useful for the success of tissue targeting.     

Stavudine-loaded mannosylated liposomes: in-vitro anti-HIV-I activity, tissue distribution and pharmacokinetics

Cells of the mononuclear phagocyte system (MPS) are important hosts for human immunodeficiency virus (HIV). Lectin receptors, which act as molecular targets for sugar molecules, are found on the surface of these cells of the MPS. Stavudine-loaded mannosylated liposomal formulations were developed for targeting to HIV-infected cells. The mannose-binding protein concanavalin A was employed as model system for the determination of in-vitro ligand-binding capacity. Antiretroviral activity was determined using MT-2 cell line. Haematological changes, tissue distribution and pharmacokinetic studies of free, liposomal and mannosylated liposomal drug were performed following a bolus intravenous injection in Sprague-Dawley rats. The entrapment efficiency of mannosylated liposomes was found to be 47.2 +/- 1.57%. Protein-carbohydrate interaction has been utilized for the effective delivery of mannosylated formulations. Cellular drug uptake was maximal when mannosylated liposomes were used. MT2 cells treated continuously with uncoated liposomal formulation had p24 levels 8-12 times lower than the level of free drug solution. Further, the mannosylated liposomes have shown p24 levels that were 14-20 and 1.4-2.3 times lower than the level of free drug and uncoated liposomal formulation treatment, respectively. Similar results were observed when infected MT2 cells were treated overnight. Stavudine, either given plain or incorporated in liposomes, led to development of anaemia and leucocytopenia while mannosylated liposomes overcame these drawbacks. These systems maintained a significant level of stavudine in the liver, spleen and lungs up to 12 h and had greater systemic clearance as compared with free drug or the uncoated liposomal formulation. Mannosylated liposomes have shown potential for the site-specific and ligand-directed delivery systems with desired therapeutics and better pharmacological activity.