Gadolinium-based cancer therapeutic liposomes for chemotherapeutics and diagnostics

Gadolinium (Gd)-based cancer therapeutic liposomes can be used for chemotherapeutics and diagnostics. In this study, dual functional liposomes co-encapsulating doxorubicin (Dox) and Gd were prepared by Dox-transition metal complexation. Preparation conditions were optimized to obtain liposomes containing high concentrations of Dox and Gd. The optimized liposomes Gd250 co-encapsulated 3.6 mM of Dox and 1.9 mM of Gd. The magnetic resonance (MR) properties of Gd250 liposomes were determined using a 4.7 T MR system. Cellular uptake of Dox was determined using a flow cytometer and a confocal microscopy and that of Gd was measured using an inductively coupled plasma-atomic emission spectrometer. Although encapsulated Gd exhibited lower relaxivity than MRbester?, which is widely used for clinical diagnosis, because of limited diffusion across the liposome membrane, Gd250 liposomes showed much higher cellular uptake than that of MRbester?. In Gd250 liposomes, Gd was highly accumulated in B16F10 cells, which could provide improved contrast sensitivity for molecular imaging. Additionally, in Gd250 liposomes, Dox was highly internalized, which could enhance its cancer therapeutic effects. Consequently, we suggest that dual functional liposomes can be used as therapeutic and diagnostic carriers.     

Maleimide-Functionalized Liposomes: Prolonged Retention and Enhanced Efficacy of Doxorubicin in Breast Cancer with Low Systemic Toxicity

Cell surface thiols can be targeted by thiol-reactive groups of various materials such as peptides, nanoparticles, and polymers. Here, we used the maleimide group, which can rapidly and covalently conjugate with thiol groups, to prepare surface-modified liposomes (M-Lip) that prolong retention of doxorubicin (Dox) at tumor sites, enhancing its efficacy. Surface modification with the maleimide moiety had no effect on the drug loading efficiency or drug release properties. Compared to unmodified Lip/Dox, M-Lip/Dox was retained longer at the tumor site, it was taken up by 4T1 cells to a significantly greater extent, and exhibited stronger inhibitory effect against 4T1 cells. The in vivo imaging results showed that the retention time of M-Lip at the tumor was significantly longer than that of Lip. In addition, M-Lip/Dox also showed significantly higher anticancer efficacy and lower cardiotoxicity than Lip/Dox in mice bearing 4T1 tumor xenografts. Thus, the modification strategy with maleimide may be useful for achieving higher efficient liposome for tumor therapy.     

Albumin-Albumin/Lactosylated Core-Shell Nanoparticles: Therapy to Treat Hepatocellular Carcinoma for Controlled Delivery of Doxorubicin

Doxorubicin (Dox) is the most widely used chemotherapeutic agent and is considered a highly powerful and broad-spectrum for cancer treatment. However, its application is compromised by the cumulative side effect of dose-dependent cardiotoxicity. Because of this, targeted drug delivery systems (DDS) are currently being explored in an attempt to reduce Dox systemic side-effects. In this study, DDS targeting hepatocellular carcinoma (HCC) has been designed, specifically to the asialoglycoprotein receptor (ASGPR). Dox-loaded albumin-albumin/lactosylated (core-shell) nanoparticles (tBSA/BSALac NPs) with low (LC) and high (HC) crosslink using glutaraldehyde were synthesized. Nanoparticles presented spherical shapes with a size distribution of 257 ± 14 nm and 254 ± 14 nm, as well as an estimated surface charge of −28.0 ± 0.1 mV and −26.0 ± 0.2 mV, respectively. The encapsulation efficiency of Dox for the two types of nanoparticles was higher than 80%. The in vitro drug release results showed a sustained and controlled release profile. Additionally, the nanoparticles were revealed to be biocompatible with red blood cells (RBCs) and human liver cancer cells (HepG2 cells). In cytotoxicity assays, Dox-loaded nanoparticles decrease cell viability more efficiently than free Dox. Specific biorecognition assays confirmed the interaction between nanoparticles and HepG2 cells, especially with ASGPRs. Both types of nanoparticles may be possible DDS specifically targeting HCC, thus reducing side effects, mainly cardiotoxicity. Therefore, improving the quality of life from patients during chemotherapy.     

Preparation and application of mesoporous core-shell nanosilica using leucine derivative as template in effective drug delivery

Core-shell structured mesoporous silica nanoparticles have been firstly synthesized with the new template from L-leucine methyl ester hydrochloride (H-Leu-OMe·HCl). LMSNs were characterized by transmission electron microscopy (TEM), nitrogen adsorption/desorption, and small-angle X-ray diffraction (SAXRD), demonstrating a well-ordered mesostructure. After loading doxorubicin hydrochloride (Dox) into pores, considerable loading capacity of 30.5% and favorable cumulative release amount were obtained. MTT assay suggested that Dox-loaded LMSNs demonstrated great promise to anti-tumor. The use of MSNs with the synthesized template, as a drug delivery carrier, will extend the pharmaceutical applications of silica materials.     

Development of a thermal neutron-sensitive liposome for a novel drug delivery system aiming for radio-chemo-concurrent cancer therapy

A thermal neutron-sensitive liposome has been developed focusing on irradiation site-specific-controlled release of a medicine. We recommended a ¹⁰B-borocaptate dimer as a detonator of liposome in expectation of effects of low-energy heavy ions produced by ¹⁰B(n, α)⁷Li reaction. As a result, an X-ray-sensitive liposome incorporating the detonator was vulnerable enough to release an anticancer agent, carboplatin, at a dose/dose–rate near clinical use. These results reveal, moreover, that the liposomes are also applicable to a heavy-ion beam.     

Reversal of Multidrug Resistance by Apolipoprotein A1-Modified Doxorubicin Liposome for Breast Cancer Treatment

Multidrug resistance (MDR) remains a major problem in cancer therapy and is characterized by the overexpression of p-glycoprotein (P-gp) efflux pump, upregulation of anti-apoptotic proteins or downregulation of pro-apoptotic proteins. In this study, an Apolipoprotein A1 (ApoA1)-modified cationic liposome containing a synthetic cationic lipid and cholesterol was developed for the delivery of a small-molecule chemotherapeutic drug, doxorubicin (Dox) to treat MDR tumor. The liposome-modified by ApoA1 was found to promote drug uptake and elicit better therapeutic effects than free Dox and liposome in MCF-7/ADR cells. Further, loading Dox into the present ApoA1-liposome systems enabled a burst release at the tumor location, resulting in enhanced anti-tumor effects and reduced off-target effects. More importantly, ApoA1-lip/Dox caused fewer adverse effects on cardiac function and other organs in 4T1 subcutaneous xenograft models. These features indicate that the designed liposomes represent a promising strategy for the reversal of MDR in cancer treatment.     

Fluorescence studies on radiation oxidative damage to membranes with implications to cellular radiosensitivity

Radiation oxidative damage to plasma membrane and its consequences to cellular radiosensitivity have received increasing attention in the past few years. This review gives a brief account of radiation oxidative damage in model and cellular membranes with particular emphasis on results from our laboratory. Fluorescence and ESR spin probes have been employed to investigate the structural and functional alterations in membranes after y-irradiation. Changes in the lipid bilayer in irradiated unilamellar liposomes prepared from egg yolk lecithin (EYL) were measured by using diphenylhexatriene (DPH) as a probe. The observed increase in DPH polarization and decrease in fluorescence intensity after γ-irradiation of liposomes imply radiation-induced decrease in bilayer fluidity. Inclusion of cholesterol in liposome was found to protect lipids against radiation damage, possibly by modulation of bilayer organization e.g. lipid packing. Measurements on dipalmitoyl phosphatidylcholine (DPPC) liposomes loaded with 6-carboxyfluorescein (CF) showed radiation dose-dependent release of the probe indicating radiation-induced increased permeability. Changes in plasma membrane permeability of thymocytes were monitored by fluorescein diacetate (FDA) and induced intracellular reactive oxygen species (ROS) were determined by 2,7-dichlorodihydro fluorescein diacetate (DCH-FDA). Results suggest a correlation between ROS generation and membrane permeability changes induced by radiation within therapeutic doses (0-10 Gy). It is concluded that increase in membrane permeability was the result of ROS-mediated oxidative reactions, which might trigger processes leading to apoptotic cell death after radiation exposure.     

Size Dependence of Protein-Induced Flocculation of Phosphatidylcholine Liposomes

The adsorption of lysozyme and cytochrome C on phosphatidylcholine liposomes essentially changes the physical properties of the phospholipid membranes and under certain circumstances greatly affects the stability of the colloid dispersion by inducing bridging liposome flocculation. This study was designed to examine experimentally the influence of liposome size on two kinetic parameters of the flocculation, its rate constant and activation energy. As the liposome radius increased in the range 50-500 nm, the activation energy tended to decrease, resulting in an increased flocculation rate, except for the flocculation of 400-nm liposomes, which was greatly impeded. The pronounced influence of the liposome size on the flocculation rate constant was evident, since a well-defined minimum in the kinetic rate of flocculation of 400-nm liposomes was detected experimentally. The obtained nonlinear radius dependencies of the flocculation rates and activation energies are interpreted in terms of the bridging mechanism of the protein-induced liposome flocculation and the supplementary concept of the stability of thin liquid films formed between approaching protein-adsorbed liposomes. Copyright 2000 Academic Press.     

The interactions between doxorubicin and amphiphilic and acidic β-sheet peptides towards drug delivery hydrogels

Amphiphilic β-sheet peptides decorated by acidic amino acids may spontaneously assemble into ordered monolayers at interfaces as well as form hydrogels at near physiological pH values. Here we monitored interactions between the peptide Pro-Asp-(Phe-Asp)(5)-Pro and the mildly amphiphilic chemotherapeutic drug doxorubicin (Dox). The peptide in the form of monolayers at the air water interface was found to enhance Dox adsorption, pointing to favorable interactions between the amphiphilic peptide and Dox. In solutions the fluorescence emission of Dox which was fitted to the Stern-Volmer quenching models suggested the formation of Dox associated forms >25 μM and larger forms at >100 μM. In presence of the peptide these larger associated forms appeared already at Dox concentrations >50 μM, indicating enhanced interactions between Dox and the peptide in the β-sheet conformation. Peptide hydrogels loaded with the drug showed sustained release profiles over several days. Smaller fractions of the drug were released with increase in either peptide or initially loaded drug concentrations. The released Dox was found to retain its cytotoxic activity in vitro. This study provides insights on the interactions between the amphiphilic and acidic peptide and Dox that are useful for the bottom up development of Dox-loaded peptide hydrogels for local drug delivery applications.     

Evaluation of fusion between liposomes and erythrocytes for intracellular derivatization of amino acids in cells

Erythrocytes were fused with liposome for intracellular derivatization of amino acids in cells. The fusion efficiency was evaluated with capillary electrophoresis (CE) and laser-induced fluorescence (LIF) detection. Reagent fluorescein isothiocyanate (FITC) was enveloped in liposomes and introduced into erythrocytes by fusion between liposomes and erythrocytes. The amino acids in the fused cells were derivated by the introduced FITC and the derivated amino acids were extracted for detection by capillary electrophoresis equipped with laser-induced fluorescence detector. The fusion conditions were investigated. It was found that incubation of liposome and erythrocytes in the presence of 13% polyethylene glycol 6000 (PEG 6000) for 15min produced the highest fusion efficiency and kept the erythrocytes stability.     

A study on the characterization and stability of rhenium(III) chloride-incorporated liposomes

Nanoliposomes are important carriers capable of packaging drugs for various delivery applications through passive targeting tumor sites by enhancing permeability and retention effect. Radiolabeled liposomes have potential applications in radiotherapy and diagnostic imaging. However, the physico-chemical instability of liposomes during manufacturing and storage limits their extensive application. Therefore, considerable numbers of studies have been made on the stability of liposomes over the last few years in order to overcome this problem. In this study, we attempted to prepare polymer-coated liposomes using water-soluble chitosan in order to enhance the stability of rhenium(III) chloride-incorporated liposomes. They were characterized by an electrophoretic light-scattering spectrophotometer, Fourier transform infrared spectroscopy (FT-IR), UV–Vis spectrometer, and phase-contrast microscopy. The chitosan-coated liposomes are spherical and the particle size is about 800–850 nm. Incorporation of chitosan into the liposome bilayer decreased rhenium(III) chloride release from the liposome due to an increased rigidity of the liposome membrane structure. Chitosan-coated liposomes showed a higher stability compared with the stability of non-coated liposomes. The release characteristics of rhenium(III) chloride encapsulated in the liposome were taken as a measure of stability of the liposome membrane.     

Optimization and characterization of liposome formulation by mixture design

This study presents the application of the mixture design technique to develop an optimal liposome formulation by using the different lipids in type and percentage (DOPC, POPC and DPPC) in liposome composition. Ten lipid mixtures were generated by the simplex-centroid design technique and liposomes were prepared by the extrusion method. Liposomes were characterized with respect to size, phase transition temperature, ζ-potential, lamellarity, fluidity and efficiency in loading calcein. The results were then applied to estimate the coefficients of mixture design model and to find the optimal lipid composition with improved entrapment efficiency, size, transition temperature, fluidity and ζ-potential of liposomes. The response optimization of experiments was the liposome formulation with DOPC: 46%, POPC: 12% and DPPC: 42%. The optimal liposome formulation had an average diameter of 127.5 nm, a phase-transition temperature of 11.43 °C, a ζ-potential of -7.24 mV, fluidity (1/P)(TMA-DPH)((?)) value of 2.87 and an encapsulation efficiency of 20.24%. The experimental results of characterization of optimal liposome formulation were in good agreement with those predicted by the mixture design technique.     

Effect of water-soluble polymers on fusion of egg yolk and soybean phospholipid liposomes

The efficiencies of polyelectrolytes, i.e., polycations and polyanions, and several kinds of water-soluble polymers as fusogens on soybean phospholipid liposome (SL) and egg yolk phospholipid liposome (EL) were investigated by the fluorescence quenching method. There were optimal concentrations for the induction of fusion in every system. Polycations induced fusion of liposomes at very low concentration in comparison with other polymers. Poly(carboxylic acid)s induced fusion at relatively high concentration. A strong acidic polyanion with high molecular weight also induced fusion of liposomes. The induction efficiency of poly(ethylene glycol) on fusion was higher than other nonionic polymers. The efficiency of fusion of EL was lower than that of SL in all systems because of the higher stability of EL membrane. It was found that electrostatic interactions, hydrogen bonding and/or hydrophobic interaction between these water-soluble polymers and liposomal membranes played an important role on aggregation and fusion of liposomes.     

Polyelectrolyte/Gold Nanoparticle Nanotubes Incorporating Doxorubicin-Loaded Liposomes

Anticancer agent doxorubicin-loaded liposomes (DoxLs) were drawn spontaneously into nanotubes comprising multilayers of polyelectrolytes and gold nanoparticles (PAuNTs). We describe a unique structure of PAuNTs incorporating DoxLs (DoxL-PAuNT). The number of DoxLs adsorbed on the tube interior surface was ascertained as 2.4×10³. Heating stimuli engendered gel-liquid crystal phase transition of DoxL in the capillary, subsequently inducing efficient release of Dox without structural changes of the tubule or liposome. Furthermore, antitumor activity of the DoxL-PAuNTs on cancer cells was elucidated.     

Unilamellar liposomes covalently coupled on silica gel for liquid chromatography

Silica gel was used as a support for the covalent coupling of liposomes, which could overcome drawbacks of soft gel beads in column efficiency and separation speed. The influences of the concentration of added dimethylaminopyridine and reaction time on the chloroformate activation reaction of silica gel were investigated. Temperature and pH for covalent coupling of liposomes on the activated silica gel were also optimized. Experimental results indicated that the stability of the covalently coupled liposome columns was obviously superior to that of the noncovalently coated liposome columns but the selectivity of both columns was basically identical. Separation and analysis of a crude extract of a traditional Chinese medicine Ligusticum Wallichii and a mixture of small peptides on both columns further support this conclusion.     

Chemophototherapeutic Ablation of Doxorubicin-Resistant Human Ovarian Tumor Cells†

Porphyrin-phospholipid (PoP) liposomes loaded with Doxorubicin (Dox) have been demonstrated to be an efficient vehicle for chemophototherapy (CPT). Multidrug resistance (MDR) of cancer cells is a problematic phenomenon in which tumor cells develop resistance to chemotherapy. Herein, we report that Dox-resistant tumor cells can be ablated using our previously described formulation termed long-circulating Dox loaded in PoP liposomes (LC-Dox-PoP), which is a PEGylated formulation containing 2 mol. % of the PoP photosensitizer. In vitro studies using free Dox and LC-Dox-PoP showed that human ovarian carcinoma A2780 cells were more susceptible to Dox compared to the corresponding Dox-resistant A2780-R cells. When CPT was applied with LC-Dox-PoP liposomes, effective killing of both nonresistant and resistant A2780 cell lines was observed. An in vivo study to assess the efficiency of LC-Dox-PoP showed effective tumor shrinkage and prolonged survival of athymic nude mice bearing subcutaneous Dox-resistant A2780-R tumor xenografts when they were irradiated with a red laser. Biodistribution analysis demonstrated enhanced tumoral drug uptake in Dox-resistant tumors with CPT, suggesting that increased drug delivery was sufficient to induce ablation of resistant tumor cells.     

不同电性脂质体对金黄色葡萄球菌组氨酸激酶AgrC跨膜镶嵌效率的影响

通过改变脂质体中磷脂成分, 构建了不同电性的脂质体. 利用表面活性剂介导方法, 将截短的金黄色葡萄球菌细胞膜上的组氨酸激酶AgrC(AgrC_TM6-7C)蛋白重构到不同电性的脂质体上. 结果表明, 阴离子脂质体对AgrC_TM6-7C蛋白的镶嵌效率明显高于阳离子脂质体, 约60%~70%镶嵌至阴离子脂质体中的AgrC_TM6-7C蛋白的细胞质域朝向脂质体囊泡的外部, 并保持较高活性. 利用圆二色光谱比较了AgrC_TM6-7C蛋白在表面活性剂胶束和脂质体中的二级结构稳定性, 发现阴离子脂质体对AgrC_TM6-7C蛋白的二级结构具有一定的保护作用, 可明显提高蛋白的热稳定性.     

Novel polymeric vesicles with pH-induced deformation character for advanced drug delivery

pH-responsive amphiphilic graft macromolecules consisting of a polyphosphazene backbone, hydrophilic PEG branches and pH-sensitive DPA were successfully synthesized and characterized. The copolymer can self-assemble into vesicles in an aqueous solution with unique inner structure and homogeneously encapsulate both lipophilic and hydrophilic molecules. The pH-dependent structure change of vesicles was also observed by DLS and TEM. Dox-loaded vesicles exhibit a sharp pH-responsive drug release profile and dramatically enhance the cytotoxicity of Dox against Dox-resistant MCF-7/adr cells. These results suggest such vesicles based on pH-responsive polyphosphazene hold great potential for specific drug therapy.     

Electrochemical Analysis of Liposome-encapsulated Colistimethate Sodium

In this study, the neutral and cationic liposomal formulations of Colistimethate sodium (CMS), an antibiotic for multi-drug resistant gram-negative bacteria, were prepared and electrochemical quantification of CMS from these liposomes were achieved. This is the first study of the electrochemical detection of CMS released from liposomes. First, the electrochemical properties of CMS were analysed, then the encapsulation efficiency, and the release kinetic of CMS from liposomes were determined with Differential Pulse Voltammetry (DPV) measurements. In addition, Cyclic Voltammetry were applied to determine oxidation signal of CMS. A higher encapsulation efficiency was found in the cationic liposome compared to the neutral liposome. Moreover, CMS was controlled released from liposomes with zero-order drug release kinetics.     

Thermodynamics of Chitinase Partitioning in Soy Lecithin Liposomes and Their Storage Stability

The goal of this study was to define the partitioning behavior of chitinase from Trichoderma spp. in soy lecithin liposomes, using a thermodynamic approach based on the partitioning variation with temperature. An effort has been made to define the liposomes, as well as free and immobilized enzyme stability during storage at 4 and 25 °C. The partition coefficients (K (o/w)) were greater than 1; therefore, the standard free energies of the enzyme transfer were negative, indicating an affinity of the enzymes for encapsulation in liposomes. The enthalpy calculation led to the conclusion that the process is exothermic. The presence of enzyme decreased the liposome storage stability from 70 days to an approximately 20 days at 25 °C and 30 days at 4 °C. Monitoring of the liposome's diameter demonstrated that their size and concentration decreased during storage. The liposome's diameters ranged from 1.06 to 3.30 μm. The higher percentage of liposome corresponded to a diameter range from 1.06 to 1.34 μm. This percentage increased during storage. There were no evidences for liposome fusion process. The stability of immobilized enzyme was increased in comparison with free chitinase.