The Use of Liposomes as Carriers of Lipophilic Anthracycline Antibiotics

Abstract

Tumor drug resistance and lack of tumor selectivity are the two main limitations of current systemic anticancer therapy. Liposomes have been shown to decrease certain doxorubicin (Dox)-related toxicities. By modifying liposome size and composition, the tumor localization of liposome entrapped drugs can be greatly enhanced. Through extensive structure-activity studies aimed at identifying anthracycline antibiotics which combine an enhanced affinity for lipid membranes and an ability to overcome multidrug resistance (MDR), we have identified Annamycin (Ann), which is ideally suited for entrapment in liposomes of different size and composition and has shown remarkable in vivo antitumor activity in different tumor models that display natural or acquired resistance to Dox. The unprecedented liposome formulation flexibility offered by Ann is expected to facilitate current efforts aimed at developing pharmaceutically acceptable liposomal-Ann formulations with optimal tumor targeting properties.     

Encapsulation of Vincristine in Liposomes Reduces its Toxicity and Improves its Anti-Tumor Efficacy

Abstract

Vincristine is a potent therapeutic agent with activity against a variety of tumor types. It is a cell-cycle specific agent which has exhibited enhanced anti-tumor activity when delivered in liposomal form. Vincristine can be encapsulated into large unilamellar vesicles in response to a transmembrane pH gradient with trapping efficiencies approaching 100%. The extent of vincristine encapsulation, and the subsequent retention of the drug within the liposomes, both in vitro and in vivo, are strongly dependent on the lipid composition of the liposome and on the magnitude of the transmembrane pH gradient. Liposomal formulations of vincristine have been optimized for both liposome circulation longevity, drug retention characteristics and in vivo antitumor activity. When compared to free vincristine, these formulations significantly increase the levels of vincristine remaining in the plasma after i.v. administration. These formulations also significantly increase the delivery of vincristine to tumor sites. As a consequence of the improved accumulation of vincristine at tumor sites, liposomal formulations of vincristine exhibit dramatically improved efficacy against a variety of ascitic and solid murine and human tumors than does free vincristine. Liposomal vincristine is expected to be of wide utility in a variety of human malignancies.     

Interaction of alkylphospholipid liposomes with MT-3 breast-cancer cells depends critically on cholesterol concentration

We have investigated interaction of alkyphospholipid (APL) liposomes consisting of 1,1-dimethylpiperidin-1-ium-4-yl) octadecyl phosphate (OPP) and different concentrations of cholesterol (CH) with human MT-3 breast-cancer cells using electron paramagnetic resonance method (EPR) with advanced characterization of EPR spectra of spin labeled liposome membranes. After incubation of OPP liposomes with MT-3 cells, a reduction of liposome entrapped, water soluble spin-probe tempocholine (ASL) was observed, indicating that ASL is released from liposomes and is reduced by oxy-redoxy systems inside the cells. This process is fast if cholesterol content in the bilayer was 29 or 45 mol%, whereas at 56 mol% cholesterol the process is almost stopped. The rate of spin-probe reduction in first 10 min after incubation with cells is even faster as for the free ASL, indicating that liposomes with low amount of cholesterol accelerate penetration of ASL into the cells. A faster release of hydrophilic material from liposomes with low cholesterol content coincides with the presence of domains with highly disordered alkyl chain motion that disappears at 50 mol% of cholesterol. We propose that these highly fluid domains are responsible for interaction of OPP liposomes with cells and fast release of the entrapped material into the cells. These results suggest that micelles are not the only reason for cytotoxic effect of OPP liposome formulations, as it was suggested before. OPP in liposomes, containing 45 mol% cholesterol or less, also contributes to the cytotoxic effect, due to their fast interaction with breast-cancer cells.     

Novel camptothecin analogue (gimatecan)-containing liposomes prepared by the ethanol injection method

Small-sized liposomes have several advantages as drug delivery systems, and the ethanol injection method is a suitable technique to obtain the spontaneous formation of liposomes having a small average radius. In this paper, we show that liposomal drug formulations can be prepared in situ, by simply injecting a drug-containing lipid(s) organic solution into an aqueous solution. Several parameters should be optimized in order to obtain a final suitable formulation, and this paper is devoted to such an investigation. Firstly, we study the liposome size distributions determined by dynamic light scattering (DLS), as function of the lipid concentration and composition, as well as the organic and aqueous phases content. This was carried out, firstly, by focusing on POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) then on the novel L-carnitine derivative PUCE (palmitoyl-(R)-carnitine undecyl ester chloride), showing that it is possible to obtain monomodal size distributions of rather small vesicles. In particular, depending on the conditions, it was possible to achieve a population of liposomes with a mean size of 100 nm, when a 50 mM POPC ethanol solution was injected in pure water; in the case of 50 mM PUCE the mean size was around 30 nm, when injected in saline (0.9% NaCl). The novel anticancer drug Gimatecan, a camptothecin derivative, was used as an example of lipophilic drug loading by the injection method. Conditions could be found, under which the resultant liposome size distributions were not affected by the presence of Gimatecan, in the case of POPC as well as in the case of PUCE. To increase the overall camptothecin concentration in the final liposomal dispersion, the novel technique of "multiple injection method" was used, and up to a final 5 times larger amount of liposomal drug could be reached by maintaining approximately the same size distribution. Once prepared, the physical and chemical stability of the liposome formulations was satisfactory within 24, as judged by DLS analysis and HPLC quantitation of lipids and drug. The Gimatecan-containing liposomes formulations were also tested for in vitro and in vivo activity, against the human nonsmall cell lung carcinoma NCI-H460 and a murine Lewis lung carcinoma 3 LL cell lines. In the in vitro tests, we did not observe any improvement or reduction of the Gimatecan pharmacological effect by the liposomal delivery system. More interestingly, in the in vivo Lewis lung carcinoma model, the intravenously administration of liposomal Gimatecan formulation showed a mild but significant increase of Tumor Volume Inhibition with respect to the oral no-liposomal formulation (92% vs. 86 %, respectively; p < 0.05). Finally, our study showed that the liposomal formulation was able to realize a delivery system of a water-insoluble drug, providing a Gimatecan formulation for intravenous administration with a preserved antitumoral activity.     

Improvement of in vivo stability of phosphodiester oligonucleotide using anionic liposomes in mice

Antisense phosphodiester oligonucleotides (ODN) are unstable in biological fluids due to nuclease-mediated degradation and therefore cannot be used in most antisense therapeutic applications. We describe here an in vitro and in vivo stabilization of a 15 mer phosphodiester sequence using anionic liposomes. Two formulations have been studied: DOPC/OA/CHOL and DOPE/OA/CHOL (pH-sensitive liposomes). Our in vitro findings reveal the same stabilization effect in mouse plasma for both anionic liposomes. In vivo investigation showed a great protective effect for both formulations after intravenous administration to mice. By contrast with in vitro results, a higher protection of ODN was observed with DOPC/OA/CHOL liposomes compared to the DOPE/OA/CHOL formulation. The latter was degraded in blood (75% of the injected dose at 5 min) probably due to interactions with blood components, and the remaining (25% at 5 min) was distributed mostly to the liver and spleen. DOPC liposomes were remarkably stable in blood and were distributed more slowly to all studied organs (liver, spleen, kidneys and lungs). Intact ODN was still observed in some organs (liver, spleen, lungs), but not in blood, 24 hours after DOPC liposome administration. These results suggest that this antisense strategy using carrier systems may be applicable to the treatment of diseases involving the reticuloendothelial system.     

Synthesis and biological evaluation of aryl azide derivatives of combretastatin A-4 as molecular probes for tubulin

Two new aryl azides, (Z)-1-(3'-azido-4'-methoxyphenyl)-2-(3",4",5"-trimethoxyphenyl)ethene 9 and (Z)-1-(4'-azido-3'-methoxyphenyl)-2-(3",4",5"-trimethoxyphenyl)ethene 5, modeled after the potent antitumor, antimitotic agent combretastatin A-4 (CA-4), have been prepared by chemical synthesis as potentially useful photoaffinity labeling reagents for the colchicine site on beta-tubulin. Aryl azide 9, in which the 3'-hydroxyl group of CA-4 is replaced by an azido moiety, demonstrates excellent in vitro cytotoxicity against human cancer cell lines (NCI 60 cell line panel, average GI50 = 4.07 x 10(-8) M) and potent inhibition of tubulin polymerization (IC50 = 1.4+/-0.1 microM). The 4'-azido analogue 5 has lower activity (NCI 60 cell line panel, average GI50 = 2.28 x 10(-6) M, and IC50 = 5.2+/-0.2 microM for inhibition of tubulin polymerization), suggesting the importance of the 4'-methoxy moiety for interaction with the colchicine binding site on tubulin. These CA-4 aryl azide analogues also inhibit binding of colchicine to tubulin, as does the parent CA-4, and therefore these compounds are excellent candidates for photoaffinity labeling studies.     

A tumor vasculature targeted liposome delivery system for combretastatin A4: Design, characterization, and in vitro evaluation

The objective of this study was to develop an efficient tumor vasculature targeted liposome delivery system for combretastatin A4, a novel antivascular agent. Liposomes composed of hydrogenated soybean phosphatidylcholine (HSPC), cholesterol, distearoyl phosphoethanolamine-polyethylene-glycol-2000 conjugate (DSPE-PEG), and DSPE-PEG-maleimide were prepared by the lipid film hydration and extrusion process. Cyclic RGD (Arg-Gly-Asp) peptides with affinity for αvβ3-integrins expressed on tumor vascular endothelial cells were coupled to the distal end of PEG on the liposomes sterically stabilized with PEG (long circulating liposomes, LCL). The liposome delivery system was characterized in terms of size, lamellarity, ligand density, drug loading, and leakage properties. Targeting nature of the delivery system was evaluated in vitro using cultured human umbilical vein endothelial cells (HUVEC). Electron microscopic observations of the formulations revealed presence of small unilamellar liposomes of ∼120 nm in diameter. High performance liquid chromatography determination of ligand coupling to the liposome surface indicated that more than 99% of the RGD peptides were reacted with maleimide groups on the liposome surface. Up to 3 mg/mL of stable liposomal combretastatin A4 loading was achieved with ∼80% of this being entrapped within the liposomes. In the in vitro cell culture studies, targeted liposomes showed significantly higher binding to their target cells than non-targeted liposomes, presumably through specific interaction of the RGD with its receptors on the cell surface. It was concluded that the targeting properties of the prepared delivery system would potentially improve the therapeutic benefits of combretastatin A4 compared with nontargeted liposomes or solution dosage forms.     

Investigation of parameters influencing incorporation,retention and cellular cytotoxicity in liposomal formulations of poorly soluble camptothecin

Abstract

Improving tumor delivery of lipophilic drugs through identifying advanced drug carrier systems with efficient carrier potency is of high importance. We have performed an investigative approach to identify parameters that affect liposomes’ ability to effectively deliver lipophilic camptothecin (CPT) to target cells. CPT is a potent anticancer drug, but its undesired physiological properties are impairing its therapeutic use. In this study, we have identified parameters influencing incorporation and retention of lipophilic CPT in liposomes, evaluating the effect of lipid composition, lipid chemical structure (head and tail group variations, polymer inclusion), zeta potential and anisotropy. Polyethyleneglycol (PEG) surface decoration was included to avoid liposome fusing and increase the potential for prolonged in vivo circulation time. The in vitro effect of the different carrier formulations on cell cytotoxicity was compared and the effect of active targeting of one of the formulations was evaluated. We found that a combination of liposome surface charge, lipid headgroup and carbon chain unsaturation affect CPT incorporation. Retention in liposomes was highly dependent on the liposomal surroundings and liposome zeta potential. Inclusion of lipid tethered PEG provided stability and prevented liposome fusing. PEGylation negatively affected CPT incorporation while improving retention. In vitro cell culture testing demonstrated that all formulations increased CPT potency compared to free CPT, while cationic formulations proved significantly more toxic to cancer cells that healthy cells. Finally, antibody mediated targeting of one liposome formulation further enhanced the selectivity towards targeted cancer cells, rendering normal cells fully viable after 1 hour exposure to targeted liposomes.     

A method to determine the incorporation capacity of camptothecin in liposomes

The purpose of this study was to establish a new experimental approach to determine the maximum amount of campothecin (CPT) that can be incorporated in liposomes, and to use this method to compare the CPT-incorporation capacity of various liposome formulations. Small, CPT-saturated liposomes were prepared by dispersing freeze-dried blends of lipids and drug in phosphate buffer, and subsequent probe-sonication. Excess precipitated CPT could be separated from the liposomes by ultra-centrifugation. The small and homogeneous liposome size obtained gave a good and reproducible recovery of liposomes in the supernatant (>80%), whereas the acidic pH (pH 6.0) kept CPT in its hydrophobic lactone form, which is poorly soluble in the buffer. The maximum CPT-incorporation capacity of 12 different liposome formulations was investigated, using the described method, and was found to vary widely. With liposomes made of neutral and anionic phospholipids, the solubili ty of CPT in the buffer was improved by approximately a factor of 10 (from ∼2.7 to 15–50 μg/mL) as compared with buffer. With cationic liposomes containing 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), a maximum CPT-solubilization of ∼100-fold, the buffer solubility was reached, probably owing to an electrostatic interaction between the cationic lipids and the carboxylate-CPT isomer. Increasing DOTAP fractions within egg-phosphatidylcholine (EPC)/DOTAP liposomes reached a CPT-incorporation plateau at ∼20 mol% DOTAP. The presented approach appears suitable to study the incorporation capacity of any drug component within small vesicles as long as the liposome incorporation is high relative to the intrisic water solubility of the drug.     

Effect of cholesterol on molecular transport of organic cations across liposome bilayers probed by second harmonic generation

The effect of cholesterol on the molecular transport of an organic cation, malachite green (MG), across large unilamellar dioleolyphosphatidylglycerol (DOPG) liposome bilayers with 0-50 mol% cholesterol was studied by second harmonic generation (SHG). Because SHG is a surface-specific technique, it requires no labeled molecule, quencher, or shifting agent to distinguish the location of the solute molecules. An additional important feature of SHG is that it is sensitive only to the probe molecules bound to the liposome, whereas other methods can only differentiate between molecules that are outside and those inside the liposome. The transport kinetics of MG across the liposome bilayers was observed in real time, and the results show that cholesterol retards the rate of transport of MG across liposome bilayers. The rate was found to decrease by six times for 50 mol% cholesterol content compared with cholesterol-free liposomes. This demonstrates the applicability of SHG to investigation of the effect of liposome composition on the transport kinetics across the liposome bilayers.     

Preclinical and Clinical Experience with Liposome-Encapsulated Mitoxantrone

Abstract

Mitoxantrone (MTO) was encapsulated by different preparation techniques into liposomes of different lipid compositions. MTO complexed to liposomes containing phosphatidic acid (PA, PA/MTO-liposomes) had blood pharmacokinetics which were comparable to the free drug. Accumulation in liver and spleen was significantly higher with PA/MTO-liposomes. Acute toxicity was 2.5 fold lower and the liposomal preparation had better antitumor effects in the L1210 leukemia and in a large cell lung cancer model. In a phase I study in patients with advanced breast cancer the maximal tolerable dose of PA/MTO-liposomes was 18 mg/m². The PA/MTO-liposomes were well tolerated, granulocytopenia was the dose-limiting effect. Clinical responses were seen in soft-tissues, liver and bone metastases. The properties of new liposome formulations with MTO were evaluated MTO-liposomes prepared by the pH-gradient loading method and modified with polyethylene glycol(2000)-diphosphatidylethanolamine (PEG(2000)-DPPE) had pharmacokinetic properties which were significantly superior to the PAJMTO-liposomes. Compared to free MTO and PA/MTO-liposomes, ΔpH MTO-liposomes containing PEG(2000)-DPPE, prepared with a ΔpH of 5 across the liposome membrane produced a 40-fold increase of the area under the curve (AUC). The new MTO-liposome formulations have excellent antitumor activities in the LI210 leukemia model. These results and further preclinical evaluation of the ΔpH MTO-liposomes support the initiation of a phase I study.     

Intermembrane transfer of polyethylene glycol-modified phosphatidylethanolamine as a means to reveal surface-associated binding ligands on liposomes

In order to explore the use of exchangeable poly(ethylene glycol) (PEG)-modified diacylphosphatidylethanolamines (PE) to temporarily shield binding ligands attached to the surface of liposomes, a model reaction based on inhibition and subsequent recovery of biotinylated liposome binding to streptavidin immobilized on superparamagnetic iron oxide particles (SA magnetic particles) was developed. PEG-lipid incorporation into biotinylated liposomes decreased liposome binding to SA magnetic particles in a non-linear fashion, where as little as 0.1 mol% PEG-PE resulted in a 20% decrease in binding. Using an assay based on inhibition of binding, PEG(2000)-PE transfer from donor liposomes to biotinylated acceptor liposomes could be measured. The influence of temperature and acyl chain composition on the transfer of PEG-diacyl PEs from donor liposomes to acceptor liposomes, consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine, cholesterol and N-((6-biotinoyl)amino)hexanoyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (54.9:45:0.1 mole ratio), was measured. Donor liposomes were prepared using 1,2-distearoyl-sn-glycero-3-phosphocholine (50 mol%), cholesterol (45 mol%) and 5 mol% of either PEG-derivatized 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG(2000)), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG(2000)), or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG(2000)). Transfer of DSPE-PEG(2000) to the donor liposomes was not detected under the conditions employed. In contrast, DMPE-PEG(2000) was transferred efficiently even at 4 degrees C. Using an acceptor to donor liposome ratio of 1:4, the time required for DMPE-PEG(2000) to become evenly distributed between the two liposome populations (T(EQ)) at 4 degrees C and 37 degrees C was approx. 2 and <0.5 h, respectively. An increase in acyl chain length from C14:0 to C16:0 of the PEG-lipid resulted in a significant reduction in the rate of transfer as measured by this assay. The transfer of PEG-lipid out of biotinylated liposomes was also studied in mice following intravenous administration. The relative rates of transfer for the various PEG-lipids were found to be comparable under in vivo and in vitro conditions. These results suggest that it is possible to design targeted liposomes with the targeting ligand protected while in the circulation through the use of PEG-lipids that are selected on the basis of exchange characteristics which result in exposure of the shielded ligand following localization within a target tissue.     

Diallyl Disulfide Suppresses SRC/Ras/ERK Signaling-Mediated Proliferation and Metastasis in Human Breast Cancer by Up-Regulating miR-34a

Diallyl disulfide (DADS) is one of the major volatile components of garlic oil. DADS has various biological properties, including anticancer, antiangiogenic, and antioxidant effects. However, the anticancer mechanisms of DADS in human breast cancer have not been elucidated, particularly in vivo. In this study, we demonstrated that the expression of miR-34a was up-regulated in DADS-treated MDA-MB-231 cells. miR-34a not only inhibited breast cancer growth but also enhanced the antitumor effect of DADS, both in vitro and in vivo. Furthermore, Src was identified as a target of miR-34a, with miR-34a inhibiting SRC expression and consequently triggering the suppression of the SRC/Ras/ERK pathway. These results suggest that DADS could be a promising anticancer agent for breast cancer. miR-34a may also demonstrate a potential gene therapy agent that could enhance the antitumor effects of DADS.     

阿霉素脂质体的制备及其体外抗肿瘤活性的初步研究

目的:应用超声波分散法制备脂质体阿霉素,并比较脂质体阿霉素与游离性阿霉素抗肿瘤活性。方法:以卵磷脂和胆固醇为原料,将阿霉素包封于脂质体中,采用超声分散法制备脂质体阿霉素,对其在290-700nm范围内进行紫外扫描,用SephedexG-50柱分离脂质体阿霉素并计算其包封率。以昆明种小鼠为载体建立肿瘤模型(S180型肉瘤)和细胞荧光染色法研究脂质体阿霉素的抗肿瘤活性,以ZITA SIZER3000型表面电位与粒度测定仪测定其粒径分布。结果:脂质体阿霉素在480nm处有最大吸收峰值,包封率达91.3%,细胞荧光染色显示,脂质体及游离型阿霉素均对S180细胞有明显的抑制作用。结论:此法制备的脂质体阿霉素包封率高,粒径分布集中,脂质体阿霉素较游离型阿霉素有较强的抗肿瘤活性剂及较低的细胞毒作用,对阿霉素的临床应用有一定的参考价值。     

Novel Camptothecin Analogue (Gimatecan)‐Containing Liposomes Prepared by the Ethanol Injection Method

Small‐sized liposomes have several advantages as drug delivery systems, and the ethanol injection method is a suitable technique to obtain the spontaneous formation of liposomes having a small average radius. In this paper, we show that liposomal drug formulations can be prepared in situ, by simply injecting a drug‐containing lipid(s) organic solution into an aqueous solution. Several parameters should be optimized in order to obtain a final suitable formulation, and this paper is devoted to such an investigation. Firstly, we study the liposome size distributions determined by dynamic light scattering (DLS), as function of the lipid concentration and composition, as well as the organic and aqueous phases content. This was carried out, firstly, by focusing on POPC (1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine) then on the novel L‐carnitine derivative PUCE (palmitoyl‐(R)‐carnitine undecyl ester chloride), showing that it is possible to obtain monomodal size distributions of rather small vesicles. In particular, depending on the conditions, it was possible to achieve a population of liposomes with a mean size of 100 nm, when a 50 mM POPC ethanol solution was injected in pure water; in the case of 50 mM PUCE the mean size was around 30 nm, when injected in saline (0.9% NaCl). The novel anticancer drug Gimatecan, a camptothecin derivative, was used as an example of lipophilic drug loading by the injection method. Conditions could be found, under which the resultant liposome size distributions were not affected by the presence of Gimatecan, in the case of POPC as well as in the case of PUCE. To increase the overall camptothecin concentration in the final liposomal dispersion, the novel technique of “multiple injection method” was used, and up to a final 5 times larger amount of liposomal drug could be reached by maintaining approximately the same size distribution. Once prepared, the physical and chemical stability of the liposome formulations was satisfactory within 24, as judged by DLS analysis and HPLC quantitation of lipids and drug. The Gimatecan‐containing liposomes formulations were also tested for in vitro and in vivo activity, against the human nonsmall cell lung carcinoma NCI‐H460 and a murine Lewis lung carcinoma 3 LL cell lines. In the in vitro tests, we did not observe any improvement or reduction of the Gimatecan pharmacological effect by the liposomal delivery system. More interestingly, in the in vivo Lewis lung carcinoma model, the intravenously administration of liposomal Gimatecan formulation showed a mild but significant increase of Tumor Volume Inhibition with respect to the oral no‐liposomal formulation (92% vs. 86 %, respectively; p < 0.05). Finally, our study showed that the liposomal formulation was able to realize a delivery system of a water‐insoluble drug, providing a Gimatecan formulation for intravenous administration with a preserved antitumoral activity.     

Lipophilic prodrugs of a triazole-containing colchicine analogue in liposomes: Biological effects on human tumor cells

Colchicine site binders—blockers of tubulin polymerization—are potential antimitotic agents for anticancer therapy. To reduce their systemic toxicity and improve biodistribution, encapsulation in nanosized liposomes may be employed. Liposomes present a convenient means for preparation of injectable for-mulations of hydrophobic compounds, however colchicine as such is known to leak through the lipid bilayer. In this study, newly synthesized triazole-containing analogues of colchicine and allocolchicine, and their palmitic and oleic esters (lipophilic prodrugs) were tested for anti-proliferative activity and apoptosis-inducing potential. In contrast to colchicine conjugates, whose activities ranged with those of colchicine, allocolchicine derivatives exhibited drastically lower effects and were discarded. Liposomes of about 100 nm in diameter composed of egg phosphatidylcholine-yeast phosphatidylinositol-palmitic or oleic prodrug, 8: 1: 1, by mol, were prepared by standard extrusion technique and tested in a panel of four human tumor cell lines. Liposome formulations preserved the biological activities of the parent colchicinoid the most towards human epithelial tumor cells. Moreover, liposomal form of the oleoyl bearing colchicinoid inhibited cell proliferation more efficiently than free lipophilic prodrug. Due to substantial loading capacity of the liposomes, the dispersions contain sufficient concentration of the active agent to test wide dose range in experiments on systemic administration to animals.     

Surface-associated serum proteins inhibit the uptake of phosphatidylserine and poly(ethylene glycol) liposomes by mouse macrophages.

Serum proteins, acting as opsonins, are believed to contribute significantly to liposome-macrophage cell association and thus regulate liposome uptake by cells of the mononuclear phagocytic system (MPS). We studied the effect of serum protein on binding and uptake of phosphatidylglycerol-, phosphatidylserine-, cardiolipin-, and N,N-dioleyl-N,N-dimethylammonium chloride- (DODAC) containing as well as poly(ethylene glycol)- (PEG) containing liposomes by mouse bone marrow macrophages in vitro. Consistent with the postulated surface-shielding properties of PEG, protein-free uptake of liposomes containing 5 mol% PEG and either 20 mol% anionic phosphatidylserine or 20 mol% cationic DODAC was equivalent to uptake of neutral liposomes. In contrast to previous reports indicating that protein adsorption to liposomes increases uptake by macrophages, the presence of bound serum protein did not increase the uptake of these liposomes by cultured macrophages. Rather, we found that pre-incubating liposomes with serum reduced the uptake of liposomes containing phosphatidylserine. Surprisingly, serum treatment of PEG-containing liposomes also significantly reduced liposome uptake by macrophages. It is postulated that, in the case of phosphatidylserine liposomes, the bound serum protein can provide a non-specific surface-shielding property that reduces the charge-mediated interactions between liposomes and bone marrow macrophage cells. In addition, incubation of PEG-bearing liposomes with serum can result in a change in the properties of the PEG, resulting in a surface that is better protected against interactions with cells.     

Interaction of liposomes bearing a lipophilic doxorubicin prodrug with tumor cells

When used as nanosized carriers, liposomes enable targeted delivery and decrease systemic toxicity of antitumor agents significantly. However, slow unloading of liposomes inside cells diminishes the treatment efficiency. The problem could be overcome by the adoption of lipophilic prodrugs tailored for incorporation into lipid bilayer of liposomes. We prepared liposomes of egg yolk phosphatidylcholine and yeast phosphatidylinositol bearing a diglyceride conjugate of an antitumor antibiotic doxorubicin (a lipophilic prodrug, DOX-DG) in the membrane to study how these formulations interact with tumor cells. We also prepared liposomes of rigid bilayer-forming lipids, such as a mixture of dipalmitoylphosphatidylcholine and cholesterol, bearing DOX in the inner water volume, both pegylated (with polyethylene glycol (PEG) chains exposed to water phase) and non-pegylated. Efficiency of binding of free and liposomal doxorubicin with tumor cells was evaluated in vitro using spectrofluorimetry of cell extracts and flow cytometry. Intracellular traffic of the formulations was investigated by confocal microscopy; co-localization of DOX fluorescence with organelle trackers was estimated. All liposomal formulations of DOX were shown to distribute to organelles retarding its transport to nucleus. Intracellular distribution of liposomal DOX depended on liposome structure and pegylation. We conclude that the most probable mechanism of the lipophilic prodrug penetration into a cell is liposome-mediated endosomal pathway.     

Physicochemical and Safety Evaluation of 5-Aminolevulinic Acid in Novel Liposomes as Carrier for Skin Delivery

In this study we successfully entrapped 5-aminolevulinic acid (ALA) in liposome, although it exists as a zwitter ion. A molar ratio of 2:1:2.5 phosphatidyle-thanolamine (PE)/cholesterol/sodium stearate represented the best condition to achieve high entrapment efficiency (29.37 ± 1.21%), and the average vehicle size was 133.6 ± 2.8 nm. After 32 days of storage, the vehicle sizes of formulations with PE series were still approximately less than 200 nm. The safety of liposomes was tested and ensured both with regard to cellular cytotoxicity and erythrocyte hemolysis. Safety studies showed that liposome formulations did not affect cell viability except when both potassium stearate and sodium oleate were added. Moreover, PE and PE/cholesterol did not damage human erythrocytes in this study. The range of the hemolytic effect caused by liposomes was 5 to 37% and the effect was dependent on the amount of sodium stearate added to the formulation. According to the release rates and skin penetration of ALA liposomes in vitro, PE/cholesterol/sodium stearate liposomes might increase skin penetration, and it was shown that penetration across the stratum–corneum (sc) layer was the rate-limiting process. Images from confocal laser scanning microscopy (CLSM) confirmed the great potency of liposomes for delivering ALA into skin.     

Effect of human interferon β gene transfer upon human glioma, transplanted into nude mouse brain, involves induced natural killer cells

We investigated the immunological responses induced by human interferon β (IFNβ) gene transfer in human gliomas produced in the brains of nude mice. A suspension of human glioma U251-SP cells was injected into the brains of nude mice. The IFNβ gene was transferred by intratumoral injection with cationic liposomes or cationic liposomes associated with anti-glioma monoclonal antibody (immunoliposomes). When intratumoral injection of liposomes or immunoliposomes containing the human IFNβ gene was performed every second day for a total of six injections, starting 7 days after tumor transplantation, complete disappearance of the tumor was observed in six of seven mice that had received liposomes and in all seven mice receiving immunoliposomes. In addition, experimental gliomas injected with immunoliposomes were much smaller than those injected with ordinary liposomes following delayed injections beginning 14 days after transplantation. An immunohistochemical study of the treated nude mouse brains revealed a remarkable induction of natural killer (NK) cells expressing asialoGM1 antigen. To investigate the significance of NK cells in the antitumor effect, we injected liposomes or immunoliposomes containing the human IFNβ gene into tumors in nude mice depleted of NK cells by irradiation and anti-asialoGM1 antibody administration. The antitumor effect of the liposomes or immunoliposomes was abolished. Subsequent subcutaneous glioma challenge of the nude mice after intracerebral tumor implantation and gene transfer resulted in no subcutaneous tumor growth. These results suggest that the induction of NK cells is important in the cytocidal effect of liposomes or immunoliposomes containing the human IFNβ gene upon experimental gliomas. Received: 10 February 1998 / Accepted: 1 September 1998