Synergistic Effect Between Size and Cholesterol Content in the Enhanced Hepatic Uptake Clearance of Liposomes Through Complement Activation in Rats

Purpose. The effect of liposome size and cholesterol (CH) content on the pharmacokinetics of liposomes was investigated in rats. Methods. The pharmacokinetics of liposomes was examined using 5(6)-carboxyfluorescein (CF) as an aqueous phase marker. The extent of complement activation (ECA) was also measured by the release of CF from liposomes in serum. Results. Both the size and the CH content influenced the mean residence time, total body clearance, and the hepatic uptake clearance (CLh) of liposomes. The increase of the size of liposomes increased the CLh at each CH content. There was no CH dependency of CLh in small liposomes (200 nm in diameter), although the CLh increased with the increase in the CH content in large (800 nm) and medium (400 nm) liposomes. A significant interaction effect was observed between liposome size and the CH content on CLh according to the analysis of variance. The good correlation between CLh and ECA indicated the role of complements as opsonins in enhancing the hepatic uptake of liposomes. The interaction effect between the size and CH content on CLh was explained principally by the product of the size and CH content. Conclusions. A synergistic effect was observed between the size and the CH content on CLh. An underlying hypothesis of the synergistic effect was postulated based on the size dependent recognition of liposomes by complement system.     

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.     

PEG修饰鳖甲肽HGRFG脂质体的药动学研究

目的 制备鳖甲肽HGRFG脂质体以及聚乙二醇（polyethylene glycol,PEG）修饰后的长循环脂质体,考察经修饰的脂质体较未修饰脂质体在动物体内分布及滞留情况。方法 采用BLB/c裸鼠荧光活体成像实验,进行脂质体体内分布及代谢研究;采用药动学实验,初步探究2种载药脂质体在血浆内的滞留时间。结果 2种载药脂质体均能广泛分布于实验动物体内。与未经修饰的脂质体载药组相比,经PEG修饰的长循环脂质体载药组中裸鼠荧光全部消失的时间明显延长,药物在血浆滞留时间也明显延长。结论 经PEG修饰后的长循环脂质体可以延长药物在实验动物体内的滞留时间,延长药物半衰期。     

Analysis of Hepatic Disposition of Galactosylated Cationic Liposome/Plasmid DNA Complexes in Perfused Rat Liver

Purpose. To determine the intrahepatic disposition characteristics of galactosylated liposome/plasmid DNA (pDNA) complexes in perfused rat liver. Methods. Galactosylated liposomes containing N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), cholesterol (Chol), and cholesten-5-yloxy-N-{4-[(1-imino-2-D-thiogalactosylethyl)amino]butyl} formamide (Gal-C4-Chol) were prepared. The liposome/[³²P]-labeled pDNA complexes were administered to perfused liver, and the venous outflow patterns were analyzed based on a two-compartment dispersion model. Results. The single-pass hepatic extraction of pDNA complexed with DOTMA/Chol/Gal-C4-Chol liposomes was greater than that with control DOTMA/Chol liposomes. A two-compartment dispersion model revealed that both the tissue binding and cellular internalization rate were higher for the DOTMA/Chol/Gal-C4-Chol liposome complexes compared with the control liposome complexes. The tissue binding was significantly reduced by the presence of 20 mM galactose. When their cellular localization in the perfused liver at 30 min postinjection was investigated, it was found that the parenchymal uptake of the DOTMA/Chol/Gal-C4-Chol liposome complexes was greater than that of the control liposome complexes. The parenchymal cell/nonparenchymal cell uptake ratio was as high as unity. Conclusion. Galactosylation of the liposome/pDNA complexes increases the tissue binding and internalization rate via an asialoglycoprotein receptor-mediated process. Because of the large particle size of the complexes (150 nm), however, penetration across the fenestrated sinusoidal endothelium appears to be limited.     

以叶酸受体为靶向的阳离子脂质体的制备与性质考察

为了研制一种能通过叶酸受体途径靶向肿瘤细胞的叶酸受体靶向脂质体，将叶酸(folate，folic acid，F)、 聚乙二醇二胺(polyoxyethylene-bis-amine，NH₂-PEG-NH₂)、 琥珀酸酐(succinic anhydride，SUC)和二硬脂酰磷脂酰乙醇胺(distearoylphosphatidylethanolamine，DSPE)按序共价连接， 并使用薄层色谱和飞行时间质谱确证合成产物为叶酸-聚乙二醇-二硬脂酰磷脂酰乙醇胺(folate-polyethyleneglycol-distearoylphosphatidylethanolamine，F-PEG-DSPE)。膜材选用二棕榈酰磷脂酰胆碱(dipalmitoylphosphatidylcholine，DPPC)， 3β-［N-(N′,N′-二甲基胺乙基)胺基甲酰基］胆固醇(3β-［N(N′,N′-dimethylaminoethane) carbamoyl］ cholesterol，DC-Chol)和F-PEG-DSPE，以10∶10∶0.75(摩尔比)的配比，以荧光素标记的阴离子葡聚糖(dextran fluorescein anionic，DFA)为模型，用薄膜分散法制备含DFA的叶酸受体靶向脂质体，其包封率较高(>55%)、稳定性好，平均粒径为144 nm，体外释放慢。MTT法考察其对细胞的毒性结果表明该阳离子脂质体具有一定的细胞毒性，在低浓度时(0.012 5～0.1 μmol·L^{-1)脂质体的细胞毒性与DC-chol浓度成正比。流式细胞技术检测KB细胞和HepG2细胞对DFA脂质体的摄取，结果表明叶酸受体靶向的长循环阳离子脂质体能提高细胞对脂质体的摄取。该研究为进一步研究叶酸受体靶向阳离子脂质体在肿瘤基因治疗中的应用提供了理论基础。}     

LDL induced association of anionic liposomes with cells and delivery of contents as shown by the increase in potency of liposome dependent drugs

Purpose. To establish whether anionic liposomes interact with the low-density lipoprotein (LDL) receptor, to determine the role of lipoproteins in this interaction, and whether the association causes functional delivery of encapsulated drugs. Methods. The cell lines used were CV1-P and CHO wild type, both of which express the LDL receptor, and CHOldlA7, which lacks the LDL receptor. Cellular association of encapsulated methotrexate and fluorescein, labeled phosphatidylethanolamine in the lipid bilayer, was measured. Potency of three liposome dependent drugs (N-phosphonacetyl-L-aspartic acid, fluoroorotic acid, and methotrexate--aspartate) was also measured by growth inhibition. Results. Association of liposomes containing at least 75 mol egg phosphatidylglycerol (ePG)/100 mol phospholipid with cells grown in defined medium supplemented with 1.0 mg/ml LDL was up to 30-fold higher with CV1-P or CHO wild type cells than with CHOldlA7, and 5-fold higher than association in defined medium lacking LDL. The addition of LDL did not yield any elevation of cellular association of distearoylphosphatidylglycerol liposomes. Increased association was paralleled by a corresponding increase in potency of all three liposome dependent drugs tested. Conclusions. ePG liposomes interact with the LDL receptor in an LDL-dependent fashion, and the interaction results in the delivery of contents to cells.     

地塞米松脂质体的制备及其对乳腺癌作用的体内外研究

目的制备地塞米松脂质体,探讨地塞米松对乳腺癌4T1细胞的生长抑制作用及对荷瘤鼠的抗肿瘤药效。方法采用薄膜分散–超声法,以粒径和多分散指数(PDI)为指标进行单因素实验考察了大豆磷脂(SPC)与甲氧基聚乙二醇磷脂(DSPE-mPEG2000)的质量比、SPC与地塞米松的质量比、超声时间对地塞米松脂质体粒径的影响从而筛选得到最佳处方和最佳工艺条件。采用MTT法比较地塞米松注射液和地塞米松脂质体对4T1细胞的作用。建立4T1 BAL B/c荷瘤小鼠模型,研究地塞米松脂质体对4T1荷瘤小鼠的体内抗肿瘤作用。结果当SPC与DSPE-mPEG2000质量比为5∶1、SPC与地塞米松质量比为50∶3、超声时间为20 min时制备得到的脂质体粒径最小,粒径分布最窄,室温放置15 d稳定,于生理介质中稳定。MTT测定结果显示地塞米松注射液和脂质体对4T1细胞生长抑制作用均较弱,但在4T1荷瘤鼠的体内实验中,在5mg/kg的给药剂量下,地塞米松脂质体的抑瘤率却高达78.9%,显著高于地塞米松注射液(33.4%,P<0.05)和8mg/kg紫杉醇注射液(55%,P<0.05)。结论制备的地塞米松脂质体放置于生理介质中均能稳定存在,能口服能静脉给药。地塞米松脂质体对4T1荷瘤小鼠肿瘤生长有较强的抑制作用,但体外对4T1细胞抑制抑制作用并不强,推测地塞米松脂质体是通过调节肿瘤微环境来抑制肿瘤生长。     

Partitioning and thermodynamics of dipyridamole in the n-octanol/buffer and liposome systems

Abstract— The thermodynamics of partitioning (K) of dipyridamole has been determined in n-octanol/buffer and liposome-buffer systems at pH 7·4. Dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) were used to prepare multilamellar liposomes. Partitioning of dipyridamole did not depend on the amount of n-octanol employed, however, partitioning was dependent upon the quantity of DMPC employed to prepare liposomes. Plots of log K vs 1/T were linear in the n-octanol and liposome systems. Partitioning was generally greater in liposomes than in the n-octanol/buffer system. Among liposomes, the partitioning was greater in DMPC liposomes at all temperatures. The values of enthalpy (ΔH) and entropy (ΔS) were positive in both the n-octanol and liposome systems. These values were lower in DMPC liposomes and were comparable in the n-octanol and DPPC liposomes. Thus, the interaction of dipyridamole depends on the rigidity of lipid bilayers and liposomes constitute a more selective partitioning system than the n-octanol/buffer system.     

Kinetic modelling of liposome degradation in serum: Effect of size and concentration of liposomes in vitro

The purpose of this study is to propose a new method for quantitative evaluation of liposome degradation in serum. The time course of liposome degradation in rat serum was monitored continuously, using 6(5)-carboxyfluorescein as an aqueous phase marker. The degradation curves exhibited three characteristic phases: lag time, degradation, and plateau. This curve was described by a kinetic model with three parameters: lag time (τ), first-order degradation rate constant (k), and maximum degradation (α). The rate and extent of the degradation of liposomes were evaluated separately in terms of k and α, respectively. The effects of size and concentration of liposomes on their degradation kinetics were examined using this method. Both k and α increased with increasing liposomal size. The increased affinity of larger liposomes for complement was suggested to increase both k and α. On the other hand, α decreased with increasing liposomal concentration without altering k. The decreased extent of degradation was considered to result from the depletion of complement components. There was no significant effect of size and concentration of liposomes on τ. Quantitative evaluation of the rate and extent of degradation of liposomes will provide deeper insights into the interaction between liposomes and serum components, and basic information on liposomes as potential drug carriers.     

Interaction of Differently Designed Immunoliposomes with Colon Cancer Cells and Kupffer Cells. An in Vitro Comparison

Purpose. Evaluate the effectiveness of distal-end coupling of a tumor-specific antibody to liposomal polyethylene glycol (PEG) chains to improve target binding and reduce interference by macrophage uptake. Methods. Monoclonal antibody CC52, specific for CC531 rat colon carcinoma, was coupled to the bilayer of PEG-liposomes (type I) or to the distal end of bilayer-anchored PEG-chains (type II). Uptake of both (radiolabeled)liposome types by CC531 cells and rat liver macrophages was determined. Results. With increasing antibody density, both immunoliposome types showed increased binding to target cells, but type II liposomes displayed better target recognition than type I. Uptake by macrophages increased with antibody density for both liposome types. Lowest uptake by macrophages was found for type II liposomes at low antibody densities. Unexpectedly, not only for type I but also for type II liposomes, in which the antibody is coupled via its Fc moiety, uptake by macrophages was inhibited by aggregated IgG, indicating involvement of Fc receptors. Also polyinosinic acid, an inhibitor of scavenger receptors, reduced uptake of type II liposomes. Conclusion. Although distal end coupling of antibodies to bilayer-anchored PEG chains in liposomes through the Fc moiety enhances target cell binding, it does not prevent the recognition by Fc receptors on macrophages.     

In Vitro-In Vivo Myotoxicity of Intramuscular Liposomal Formulations

Purpose. The first objective was to study the in vitro myotoxicity of empty liposomes and to examine whether liposome size, charge and fluidity affect liposome myotoxicity. The second objective was to investigate the effect of liposomal encapsulation on the in vitro and in vivo myotoxicity of loxapine compared to the loxapine commercial preparation (Loxitane^®). Methods. The in vitro myotoxicity of empty liposomes and loxapine liposomes was evaluated by the cumulative efflux of the cytosolic enzyme creatine kinase (CK) from the isolated rat extensor digitorum longus (EDL) muscle over a 2 hour period. In the in vivo studies, the area under plasma CK curve over 12 hours was used to evaluate muscle damage. Results. The in vitro myotoxicity for all empty liposomal formulations was not statistically different from negative controls (untreated control muscles and normal saline injected muscles). However, these empty liposomal formulations were significantly less myotoxic than the positive controls (muscles injected with phenytoin and muscle sliced in half). In vitro-in vivo studies showed that the liposomal encapsulation of loxapine resulted in significant (P < 0.05) reduction in myotoxicity (80% in vitro and 60% in vivo) compared to the commercially available formulation which contains propylene glycol (70% V/V) and polysor-bate 80 (5% W/V) prepared at equal concentration. Conclusions. Results indicate that empty liposomes do not induce myotoxicity. Furthermore, liposomal size, charge and fluidity do not affect myotoxicity. In addition, in vitro and in vivo studies have demonstrated that liposomal encapsulation of loxapine can reduce myotoxicity compared to a formulation containing organic cosolvents.     

Pharmacokinetic and Pharmacological Profiles of Free and Liposomal Recombinant Human Erythropoietin After Intravenous and Subcutaneous Administrations in Rats

Purpose. Recombinant human erythropoietin (Epo) is used frequently through intravenous (i.v.) and subcutaneous (s.c.) administration for the clinical treatment of the last stage of renal anemia. We encapsulated Epo in liposomes to develop an alternative administration route. The purpose of our study was to evaluate the pharmacokinetics and the pharmacological effects of liposomal Epo in comparison with the Epo after i.v. and s.c. administration to rats. Methods. Epo was encapsulated in liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and soybean-derived sterol mixture (SS) prepared by the reversed-phase evaporation vesicle method. After filtration through a 0.1 m polycarbonate membrane, liposomes were gel filtered (Epo/liposomes). Results. Epo/liposomes showed higher pharmacological activity than Epo/liposomes before gel filtration after i.v. administration to rats. Non-encapsulated Epo lost its activity, whereas encapsulated Epo in liposomes retained it. The pharmacological effects of Epo/liposomes were greater than those of Epo after i.v. administration. Epo/liposomes afforded 3–9 times higher AUC, lower clearance and lower steady-state volume of distribution than Epo after both i.v. and s.c. administrations. Epo/liposomes had an improved pharmacokinetic profile compared with Epo. S.c. administration of Epo/liposomes at 7 h may penetrate primarily (40% of dose) through the blood as a liposome and partly (7% of dose) in lymph. Conclusions. Epo/liposomes may reduce the frequency of injections required for a certain reticulocyte effect in comparison to Epo. The lower clearance of Epo/liposomes may increase the plasma concentrations of Epo, which increases the efficacy.     

Interaction of Basic Drugs with Lipid Bilayers Using Liposome Electrokinetic Chromatography

No HeadingPurpose. This study explores factors influencing the interactions of positively charged drugs with liposomes using liposome electrokinetic chromatography (LEKC) for the development of LEKC as a rapid screening method for drug-membrane interactions.Methods. Liposomes were prepared and the retention factors were measured for a series of basic drugs under a variety of buffer conditions, including various buffer types, concentrations, and ionic strengths as well as using different phospholipids and liposome compositions. LEKC retention is compared with octanol-water partitioning.Results. The interaction of ionizable solutes with liposomes decreased with increasing ionic strength of the aqueous buffer. The type of buffer also influences positively charged drug partitioning into liposomes. Varying the surface charge on the liposomes by the selection of phospholipids influences the electrostatic interactions, causing an increase in retention with increasing percentages of anionic lipids in the membrane. Poor correlations are observed between LEKC retention and octanol-water partitioning.Conclusions. These studies demonstrate the overall buffer ionic strength at a given pH is more important than buffer type and concentration. The interaction of positively charged drugs with charged lipid bilayer membranes is selectively influenced by the pK_a of the drug. Liposomes are more biologically relevant in vitro models for cell membranes than octanol, and LEKC provides a unique combination of advantages for rapid screening of drug-membrane interactions.     

Effect of Dose and Release Rate on Pulmonary Targeting of Liposomal Triamcinolone Acetonide Phosphate

Purpose. To demonstrate the importance of dose and drug release rate for pulmonary targeting of inhaled glucocorticoids using an animal model of intrapulmonary drug deposition. Methods. Liposomes composed of 1,2-distearoyl phosphatidylcholine (DSPC), 1,2-distearoyl phosphatidylglycerol (DSPG) and triamcinolone acetonide phosphate (TAP) or liposomes containing triamcinolone acetonide (TA) were prepared by a mechanical dispersion method followed by extrusion through polycarbonate membranes. Encapsulation efficiency was assessed after size exclusion gel chromatography by reverse phase HPLC. The effect of liposome size (200 nm and 800 nm) on the release kinetics of water-soluble encapsulated material was determined in vitro at 37°C using 6-carboxyfluorescein as a marker and Triton X-100 (0.03%) as a leakage inducer. To investigate the relationship between drug release and pulmonary targeting, 100 g/kg of TAP in 800 nm liposomes was delivered to male rats by intratracheal instillation (IT) and the results compared to data for 100 g/kg TA liposomes (recently shown to exhibit a rapid drug release under sink conditions) and to previous studies reported for an equal dose of TAP in solution and TAP in 200 nm (1). Pulmonary targeting was assessed by simultaneously monitoring glucocorticoid receptor occupancy over time in lung and liver using an ex vivo receptor binding assay as a pharmacodynamic measure of glucocorticoid action. To assess the effect of dose on pulmonary targeting experiments were performed using 2.5, 7.5, 25, 100, and 450 g/kg of TAP in 800 nm liposomes. Results. The in vitro efflux of 6-carboxyfluorescein from (DSPC:DSPG) liposomes after exposure to Triton-X was biexponential. The terminal half-lives of 3.7 h and 9.0 h for the 200 nm and 800 nm liposomes, respectively, demonstrated that larger liposomes promote slower release of encapsulated water-soluble solute while previous results already indicated that encapsulation of lipophilic TA does not result in sustained release. Pulmonary targeting, defined as the difference between cumulative lung and liver receptor occupancies was most pronounced for the 800 nm liposomes (370%*h), followed by the 200 nm preparation (150%*h). No targeting was observed for TAP in solution (30%*h) or the rapid releasing TA liposome preparation. Correspondingly, the mean pulmonary effect time (MET) increased from 2.4–3.0 hr for TA liposomes or TAP in solution to 5.7 h and >6.2 h for TAP in 200 nm and in 800 nm liposomes, respectively. Escalating doses of TAP encapsulated in 800 nm liposomes revealed a distinct bell shaped relationship between the TAP dose and pulmonary targeting with a maximum occurring at 100 g/kg (370%*h). Conclusions. The in vivo data presented here confirm that pulmonary residence time and dose affect the extent of lung targeting of glucocorticoids delivered via the lung.     

Liposomes with Incorporated MHC Class Il/Peptide Complexes as Antigen Presenting Vesicles for Specific T Cell Activation

Purpose. The purpose of this study was to design a well-characterized liposomal carrier system for multivalent antigen presentation in order to activate T cells. Methods. MHC class II molecules were loaded with peptide and subsequently reconstituted into liposomes. A FACS assay was developed to monitor peptide loading and MHC class II incorporation in the liposomes. For in vitro testing of the resulting MHC class II/peptide liposomes, a T cell hybridoma assay was employed. Results. The FACS assay provided a qualitative means to visualize the amount of incorporated MHC class II and peptide molecules that were oriented in the appropriate way for antigen presentation to the T cells. Interestingly, when MHC class II molecules were loaded with the appropriate peptide prior to liposome incorporation, such liposomes were fully capable of inducing IL-2 production of a T cell hybridoma. Conclusions. This is the first article showing that MHC class II/peptide liposomes can serve as 'artificial antigen presenting cells' for activation of a CD4+ T cell hybridoma. As compared to soluble MHC class II/ peptide complexes, the multivalency of liposomal complexes may be an important advantage when studying possible applications in immunotherapy.     

Towards the Predictability of Drug-Lipid Membrane Interactions: The pH-Dependent Affinity of Propranolol to Phosphatidylinositol Containing Liposomes

Purpose. Prediction of the pH-dependent affinity of (RS)-[³H]propranolol to mixed phosphatidylcholine (PhC)/phosphatidylinositol(Phl) membranes from the partitioning in the single lipid liposome/buffer systems. Methods. Partition studies in liposome/buffer systems were performed by means of equilibrium dialysis at 37°C between pH 2 and 11 at a molar propranolol to lipid ratio of 10^–6 to 10^–5 in the membrane. Results. The Phl membrane more strongly attracts the protonated (RS)-[³H]propranolol than the neutral solute, i.e. the partition coefficient of the protonated base (P_i) is 17430 ± 1320, P of the neutral compound (P_n) is 3110 ± 1650. In the PhC-liposome system P_i is 580 ± 17, P_n 1860 ± 20. The partition coefficients show an exponential dependence on the molar Phl fraction in mixed liposomes. The partitioning in mixed PhC/Phl membranes is predictable from P_n and P_i in the single lipid liposome systems. Conclusions. The negative charge of biological lipid membranes causes strong electrostatic interactions with positively charged solutes. This strong attraction is not predictable from the octanol/buffer partition system, but it is important regarding drug accumulation in the tissue and drug attraction by certain lipids in the vicinity of membrane proteins.     

Transfer of lipophilic markers from PLGA and polystyrene nanoparticles to caco-2 monolayers mimics particle uptake

Purpose. The objective of this study was to evaluate nanoparticle uptake by the Caco-2 monolayer model in vitro. Special emphasis was placed on the localization and the quantification of the uptake of fluorescently labeled polystyrene and poly(lactic-co-glycolic acid) (PLGA) nanoparticles. Methods. Intracellular fluorescence was localized by fluorescence and confocal laser scanning microscopy. Particle uptake was quantified either directly, by counting internalized nanoparticles after separation from the Caco-2 monolayers, or indirectly, by extraction of the lipophilic fluorescence marker. In vitro release studies of lipophilic markers from nanoparticles were performed in standard buffer systems and buffer systems supplemented with liposomes. Results. Instead of uptake of polystyrene and PLGA nanoparticles by Caco-2 monolayers an efficient transfer of lipophilic fluorescence markers from nanoparticles into Caco-2 cells with subsequent staining of intracellular lipophilic compartments was observed. Whereas in standard buffer no release of fluorescent marker from polystyrene and PLGA nanoparticles was observed, the release studies using liposome dispersions as receiver revealed an efficient transfer of fluorescent marker into the liposome dispersion. Conclusions. The results suggest that the deceptive particle uptake is caused by a collision-induced process facilitating the transfer of lipophilic fluorescent marker by formation of a complex between the nanoparticles and the biomembranes. Diffusion of the marker within this complex into lipophilic compartments of the cell strongly affects quantitative evaluation of particle uptake.     

Enhancing effect of cholesterol on the elimination of liposomes from circulation is mediated by complement activation

The effects of cholesterol (Chol) content on the biodistribution of liposomes as well as the interaction of liposomes with plasma proteins, primarily complement (C) components, were examined in this study. The elimination of liposomes from blood circulation was enhanced by increasing the Chol content in liposomes. Furthermore, included Chol augmented the rate of liposome degradation as measured by the urinary excretion of ³H-inulin encapsulated in liposomes. We have also examined the effect of liposomal Chol on organ clearance (CL) and renal CL (CL_rel). The values of organ CL and CL_rel reflect the affinity of liposomes for the organ and the degree of liposome degradation in the blood, respectively. Hepatic CL and CL_rel, but not splenic CL, increased with the rise of Chol content in liposomes. The amount of liposome degradation in vitro, which reflects the extent of C activation, was correlated with degradation observed in vivo (CL_rel). However, the amount of plasma proteins bound to the liposomes was inversely proportional to the extent of in vitro liposome degradation. We have investigated the role of C activating factor (CAF) (Funato et al., 1994, Plasma factor triggering alternative complement pathway activation by liposomes, Pharm. Res., 11, 372–376) on Chol-dependent-C activation. Our results showed that binding of CAF to the liposomes is directly proportional to the amount of Chol present in the liposome. Thus, C activation by Chol in liposomes may proceed via a mechanism involving CAF. Taken together, these results suggest that increasing the Chol content of liposomes enhances the binding of CAF to the liposomes, which in turn, mediates Chol dependent-C activation, resulting in the augmentation of both degradation in blood and hepatic uptake of the liposomes.     

Gamma-Irradiation of Non-Frozen,Frozen, and Freeze-Dried Liposomes

Purpose. The aim of this work was to investigate the possibilities and limitations of gamma-irradiation as a sterilisation method for non-frozen, frozen, and freeze-dried liposomes. Methods. Liposomes with an average size of 0.2 µm were irradiated with doses up to about 5 × 10⁴ Gy in a nitrogen atmosphere. Results. Phospholipids in dipalmitoylphosphatidycholine/dipalmitoylphosphatidylglycerol (DPPC/DPPG) 10/1-liposomes and egg phosphatidylcholine/egg phosphatidylglycerol (EPC/EPG) 10/1-liposomes in 10 mM phosphate buffer (pH 7.4) without trehalose degraded considerably upon gamma-irradiation. Irradiation damage was reduced in the presence of 10% trehalose added as a cryoprotectant, but trehalose reacted with species induced by gamma-irradiation as demonstrated by large decreases in pH. Both pH decrease and oxidative damage of EPC/EPG 10/1-liposomes were strongly dependent on the physical state during irradiation (non-frozen, frozen or freeze-dried). No changes in liposomal size were found upon gamma-irradiation, and hardly any change was seen in bilayer rigidity. Differences in the gel-to-liquid phase transition of DPPC/DPPG 10/1-liposome dispersions before and after gamma-irradiation were small in the presence of 10% trehalose, but larger in the absence of trehalose. Conclusions. The degradation of trehalose limits the use of freezing or freeze-drying liposome dispersions as a way to minimise irradiation damage.     

紫草素钠脂质体的制备及性能研究

目的 研究紫草素钠脂质体的制备工艺并考察其性质,旨在开发一种安全高效的紫草新型制剂。方法 利用碱溶酸提法纯化紫草提取物,并采用乙醇注入法制备紫草素钠脂质体,测定其形貌、粒径和Zeta电位。利用高效液相建立左旋紫草素含量测定方法,并考察了脂质体包封率和体外释放性能。结果 紫草素钠脂质体平均粒径为104.2 nm左右,且大小均一,成正态分布;Zeta电位为-14.8 mV;左旋紫草素在5~50 mg/L范围与峰面积呈良好的线性关系,相关系数为0.999 9;紫草素钠脂质体的包封率为43.67%,药物累积释放量为65.82%。结论 成功制备了紫草素钠脂质体,具有较高的包封率和体外释放率。