Enhanced Delivery and Antitumor Effect of Doxorubicin Encapsulated in Long-Circulating Liposomes

Abstract

Long-circulating liposomes containing amphipathic polyethyleneglycol (PEG) or ganglioside GM1 (GM1) have been tested for their utility as enhanced delivery system of doxorubicin (DXR) in vivo. DXR was entrapped into liposomes by pH gradient method.

The long-circulating LUV (200 nm in size) composed of DSPC/CH (1:1, m/m) and either 6 mol% of DSPE-PEG1000 or GM1 entrapped DXR with >95% in trapping efficiency. DXR-long-circulating LUVs were administered to leukemic (LI210) mice via the tail vein at a dose of 5mg DXR/kg. The high blood concentration was kept for long time, and significantly increased survival time was observed as compared with free DXR and DXR-LUV. The data indicated that DXR was slowly released from long-circulating LUV during that stayed in bloodstream for long time. Administration of DXR-long-circulating SUV (100 nm) to the colon 26 bearing mice produced the increased DXR level in tumor compared with bear SUV or free drug did, respectively, and resulted in effective tumor growth retardation and increased survival time. DXR was delivered to tumor by accumulation of SUVs themselves.

Long-circulating thermosensitive liposomes (TSL) were prepared from DPPC /DSPC (9:1, m/m) and 3-6 mol% of PEG1000 or GM1. DXR was entrapped with >95% in trapping efficiency. Accumulation of DXR into tumor tissue by local hyperthermia after injection of DXR-long-circulating TSL to colon 26 bearing mice was significantly higher man that of DXR-bare TSL or free DXR, and resulted in effective tumor growth retardation and increased survival time. It was suggested that the entrapped DXR was efficiently released from long-circulating TSL by hyperthermia at the tumor site and entered the tumor tissue by simple diffusion.     

Liposomes containing alkylated methotrexate analogues for phospholipase A2 mediated tumor targeted drug delivery

Two lipophilic methotrexate analogues have been synthesized and evaluated for cytotoxicity against KATO III and HT-29 human colon cancer cells. Both analogues contained a C₁₆-alkyl chain attached to the γ-carboxylic acid and one of the analogues had an additional benzyl group attached to the α-carboxylic acid. The cytotoxicity of the γ-alkylated compound towards KATO III (IC₅₀ = 55 nM) and HT-29 (IC₅₀ = 400 nM) cell lines, was unaffected by the alkylation, whereas the additional benzyl group on the α-carboxyl group made the compound nontoxic. The γ-derivative with promising cytotoxicity was incorporated into liposomes that were designed to be particularly susceptible to a liposome degrading enzyme, secretory phospholipase A₂ (sPLA₂), which is found in high concentrations in tumors of several different cancer types. Liposome incorporation was investigated by differential scanning calorimetry (DSC), and sPLA₂ hydrolysis was examined by fluorescence spectroscopy and high performance liquid chromatography (HPLC). The results showed that the methotrexate (MTX)-analogue could be incorporated into liposomes that were degradable by sPLA₂. However, the in vitro cytotoxicity of the MTX-liposomes against KATO III and HT-29 cancer cells was found to be independent of sPLA₂ hydrolysis, indicating that the alkylated MTX-analogue was available for cancer cell uptake even in the absence of liposome hydrolysis. Using a DSC based method for assessing the anchoring stability of alkylated compounds in liposomes, it was demonstrated that the MTX-analogue partitioned into the water phase and thereby became available for cell uptake. It was concluded that liposomes containing alkylated MTX-analogues show promise as a drug delivery system, although the MTX-analogue needs to be more tightly anchored to the liposomal carrier. Also, the developed DSC-assay for studying the anchoring stability of alkylated drugs will be a useful tool in the development of liposomal drug delivery systems.     

Spectroscopic characterization of nanoErythrosomes in the absence and presence of conjugated polyethyleneglycols: an FTIR and P-NMR study

We have recently developed from red blood cells a new delivery system called nanoErythrosomes. These nanovesicles offer a high degree of versatility for the encapsulation of biological or nonbiological compounds and for the binding of targeting agents. In particular, polyethyleneglycols can be conjugated by a covalent link to the basic amino acid residues constitutive of the different proteins. The binding of polyethyleneglycols to the nanoErythrosome membrane could be interesting for the therapeutic use of this delivery system since it could overcome heterologous immunogenicity and reduce rapid clearance from circulation. In the present study, we have investigated the effect of temperature on the nanoErythrosome behavior in the absence and presence of conjugated polyethyleneglycols. More specifically, Fourier transform infrared (FTIR) spectroscopy has been used to evaluate the lipid order and dynamics, the hydration and the degree of protein aggregation of the nanoErythrosomes after covalent binding of polyethyleneglycols having molecular weights of 2000 and 5000 g mol⁻¹. The results indicate that the nanoErythrosome lipid chain order is not significantly affected by heating the nanoErythrosomes at temperatures up to 50 °C. They also indicate that the nanoErythrosome proteins aggregate irreversibly at temperatures above 37 °C, this effect being abolished in the presence of polyethyleneglycols. The presence of polyethyleneglycols decreases the accessibility of water to the lipid head groups. On the other hand, ³¹P-nuclear magnetic resonance (NMR) and electron microscopy results reveal that the presence of polyethyleneglycols prevents the aggregation of the nanoErythrosome structures.     

Secreted phospholipase A2 as a new enzymatic trigger mechanism for localised liposomal drug release and absorption in diseased tissue

Polymer-coated liposomes can act as versatile drug-delivery systems due to long vascular circulation time and passive targeting by leaky blood vessels in diseased tissue. We present an experimental model system illustrating a new principle for improved and programmable drug-delivery, which takes advantage of an elevated activity of secretory phospholipase A₂ (PLA₂) at the diseased target tissue. The secretory PLA₂ hydrolyses a lipid-based proenhancer in the carrier liposome, producing lyso-phospholipids and free fatty acids, which are shown in a synergistic way to lead to enhanced liposome destabilization and drug release at the same time as the permeability of the target membrane is enhanced. Moreover, the proposed system can be made thermosensitive and offers a rational way for developing smart liposome-based drug delivery systems. This can be achieved by incorporating specific lipid-based proenhancers or prodestabilisers into the liposome carrier, which automatically becomes activated by PLA₂ only at the diseased target sites, such as inflamed or cancerous tissue.     

Gravimetric antigen detection utilizing antibody-modified lipid bilayers

Lipid bilayers containing 5% nitrilotriacetic acid (NTA) lipids supported on SiO₂ have been used as a template for immobilization of oligohistidine-tagged single-chained antibody fragments (scFvs) directed against cholera toxin. It was demonstrated that histidine-tagged scFvs could be equally efficiently coupled to an NTA-Ni²⁺-containing lipid bilayer from a purified sample as from an expression supernatant, thereby providing a coupling method that eliminates time-consuming protein prepurification steps. Irrespective of whether the coupling was made from the unpurified or purified antibody preparation, the template proved to be efficient for antigen (cholera toxin) detection, verified using quartz crystal microbalance with dissipation monitoring. In addition, via a secondary amplification step using lipid vesicles containing G_M1 (the natural membrane receptor for cholera toxin), the detection limit of cholera toxin was less than 750 pM. To further strengthen the coupling of scFvs to the lipid bilayer, scFvs containing two histidine tags, instead of just one tag, were also evaluated. The increased coupling strength provided via the bivalent anchoring significantly reduced scFv displacement in complex solutions containing large amounts of histidine-containing proteins, verified via cholera toxin detection in serum.     

Inhibition of macroautophagy by bafilomycin A1 lowers proliferation and induces apoptosis in colon cancer cells

Macroautophagy is a process by which cytoplasmic content and organelles are sequestered by double-membrane bound vesicles and subsequently delivered to lysosomes for degradation. Macroautophagy serves as a major intracellular pathway for protein degradation and as a pro-survival mechanism in time of stress by generating nutrients. In the present study, bafilomycin A₁, a vacuolar type H⁺-ATPase inhibitor, suppresses macroautophagy by preventing acidification of lysosomes in colon cancer cells. Diminished macroautophagy was evidenced by the accumulation of undegraded LC3 protein. Suppression of macroautophagy by bafilomycin A₁ induced G₀/G₁ cell cycle arrest and apoptosis which were accompanied by the down-regulation of cyclin D₁ and cyclin E, the up-regulation of p21^Cip1 as well as cleavages of caspases-3, -7, -8, and -9 and PARP. Further investigation revealed that bafilomycin A₁ increased the phosphorylation of ERK, JNK, and p38. In this regard, p38 inhibitor partially reversed the anti-proliferative effect of bafilomycin A₁. To conclude, inhibition of macroautophagy by bafilomycin A₁ lowers G₁-S transition and induces apoptosis in colon cancer cells. Our results not only indicate that inhibitors of macroautophagy may be used therapeutically to inhibit cancer growth, but also delineate the relationship between macroautophagy and apoptosis.     

Involvement of the nadA gene in formation of G-group aflatoxins in Aspergillus parasiticus

The nadA gene is present at the end of the aflatoxin gene cluster in the genome of Aspergillus parasiticus as well as in Aspergillus flavus. RT-PCR analyses showed that the nadA gene was expressed in an aflatoxin-inducible YES medium, but not in an aflatoxin-non-inducible YEP medium. The nadA gene was not expressed in the aflR gene-deletion mutant, irrespective of the culture medium used. To clarify the nadA gene’s function, we disrupted the gene in aflatoxigenic A. parasiticus. The four nadA-deletion mutants that were isolated commonly accumulated a novel yellow-fluorescent pigment (named NADA) in mycelia as well as in culture medium. When the mutants and the wild-type strain were cultured for 3 days in YES medium, the mutants each produced about 50% of the amounts of G-group aflatoxins that the wild-type strain produced. In contrast, the amounts of B-group aflatoxins did not significantly differ between the mutants and the wild-type strain. The NADA pigment was so unstable that it could non-enzymatically change to aflatoxin G₁ (AFG₁). LC–MS measurement showed that the molecular mass of NADA was 360, which is 32 higher than that of AFG₁. We previously reported that at least one cytosol enzyme, together with two other microsome enzymes, is necessary for the formation of AFG₁ from O-methylsterigmatocystin (OMST) in the cell-free system of A. parasiticus. The present study confirmed that the cytosol fraction of the wild-type A. parasiticus strain significantly enhanced the AFG₁ formation from OMST, whereas the cytosol fraction of the nadA-deletion mutant did not show the same activity. Furthermore, the cytosol fraction of the wild-type strain showed the enzyme activity catalyzing the reaction from NADA to AFG₁, which required NADPH or NADH, indicating that NADA is a precursor of AFG₁; in contrast, the cytosol fraction of the nadA-deletion mutant did not show the same enzyme activity. These results demonstrated that the NadA protein is the cytosol enzyme required for G-aflatoxin biosynthesis from OMST, and that it catalyzes the reaction from NADA to AFG₁, the last step in G-aflatoxin biosynthesis.     

Entry into Mitosis in Vertebrate Somatic Cells Is Guarded by a Chromosome Damage Checkpoint That Reverses the Cell Cycle When Triggered during Early but Not Late Prophase

When vertebrate somatic cells are selectively irradiated in the nucleus during late prophase (<30 min before nuclear envelope breakdown) they progress normally through mitosis even if they contain broken chromosomes. However, if early prophase nuclei are similarly irradiated, chromosome condensation is reversed and the cells return to interphase. Thus, the G₂ checkpoint that prevents entry into mitosis in response to nuclear damage ceases to function in late prophase. If one nucleus in a cell containing two early prophase nuclei is selectively irradiated, both return to interphase, and prophase cells that have been induced to returned to interphase retain a normal cytoplasmic microtubule complex. Thus, damage to an early prophase nucleus is converted into a signal that not only reverses the nuclear events of prophase, but this signal also enters the cytoplasm where it inhibits e.g., centrosome maturation and the formation of asters. Immunofluorescent analyses reveal that the irradiation-induced reversion of prophase is correlated with the dephosphorylation of histone H1, histone H3, and the MPM2 epitopes. Together, these data reveal that a checkpoint control exists in early but not late prophase in vertebrate cells that, when triggered, reverses the cell cycle by apparently downregulating existing cyclin-dependent kinase (CDK1) activity.     

Enhanced autophagy and mitochondrial aberrations in murine G(M1)-gangliosidosis

G_M1-gangliosidosis is an autosomal recessive lysosomal lipid storage disorder, caused by mutations of the lysosomal β-galactosidase (β-gal) and results in the accumulation of G_M1. The underlying mechanisms of neurodegeneration are poorly understood. Here we demonstrate increased autophagy in β-gal-deficient (β-gal^−/−) mouse brains as evidenced by elevation of LC3-II and beclin-1 levels. Activation of autophagy in the β-gal^−/− brain was found to be accompanied with enhanced Akt-mTOR and Erk signaling. In addition, the mitochondrial cytochrome c oxidase activity was significantly decreased in brains and cultured astrocytes from β-gal^−/− mouse. Mitochondria isolated from β-gal^−/− astrocytes were morphologically abnormal and had a decreased membrane potential. These cells were more sensitive to oxidative stress than wild type cells and this sensitivity was suppressed by ATP, an autophagy inhibitor 3-methyladenine and a pan-caspase inhibitor z-VAD-fmk. These results suggest activation of autophagy leading to mitochondrial dysfunction in the brain of G_M1-gangliosidosis.     

Activation of MAD 2 checkprotein and persistence of cyclin B1/CDC 2 activity associate with paclitaxel-induced apoptosis in human nasopharyngeal carcinoma cells

Paclitaxel (Taxol) is a microtubule-interfering agent that induced persistent and transient G₂/M arrest before apoptosis in human nasopharyngeal carcinoma (NPC) cells at high and low concentrations, respectively. In this study, we intended to explore the underlying molecular events and found that cellular cyclin B1/CDC 2 kinase activity was increased and persisted for >6 h upon paclitaxel treatment both at high and low concentrations. Furthermore, activation of MAD 2 checkprotein could account for the loss of cyclin B1 ubiquitination and the persistence of cyclin B1/CDC 2 activation in the cases. To investigate the involvement of cyclin B1 and MAD 2 activation in paclitaxel-induced apoptosis, we introduced affinity-purified anti-cyclin B1 and MAD 2 antibodies into NPC cells by electroporation before the further paclitaxel treatment. The antibodies against cyclin B1 and MAD 2 indeed attenuated paclitaxel-induced cytotoxicity and DNA fragmentation. Our study suggests that activation of cyclin B1/CDC 2 and MAD 2 were the M-phase events required for paclitaxel-induced apoptosis in NPC cells. The dys-regulated cyclin B1/CDC 2 activation could enhance the prometaphase progression, but activation of MAD 2 rendered cells inable to exit from the metaphase. Under this circumstance, cells were probably going to mitotic catastrophe and ultimately, destined to apoptosis.     

Fluorescent BODIPY-labelled GM1 gangliosides designed for exploring lipid membrane properties and specific membrane-target interactions

New fluorophore-labelled G_M1 gangliosides have been synthesised and spectroscopically characterised. Spectroscopically different BODIPY groups were covalently linked, specifically to either the polar or the hydrophobic part of the ganglioside molecule. The absorption and fluorescence spectroscopic properties are reported for 564/571-BODIPY- and 581/591-BODIPY-labelled G_M1. Each of the different BODIPY groups is highly fluorescent and depolarisation experiments provide molecular information about the spatial distribution in lipid bilayers, as well as order and dynamics. From experiments performed on two spectroscopically different BODIPY:s, specific interactions can be revealed by monitoring the rate/efficiency of donor-acceptor electronic energy transfer. Systems of particular interest for applying these probes are e.g. mixtures of lipids, and peptides/proteins interacting with lipid membranes.     

Effects of electrical stimulation of ventral septal area on firing rates of pyrogen-treated thermosensitive neurons in preoptic anterior hypothalamus from rabbits

Although there is considerable evidence supporting that fever evolved as a host defense response, it is important that the rise in body temperature would not be too high. Many endogenous cryogens or antipyretics that limit the rise in body temperature have been identified. Endogenous antipyretics attenuate fever by influencing the thermoregulatory neurons in the preoptic anterior hypothalamus (POAH) and in adjacent septal areas including ventral septal area (VSA). Our previous study showed that intracerebroventricular (I.C.V.) injection of interleukin-1beta (IL-1beta) affected electrophysiological activities of thermosensitive neurons in VSA regions, and electrical stimulation of POAH reversed the effect of IL-1beta. To further investigate the functional electrophysiological connection between POAH and VSA and its mechanisms in thermoregulation, the firing rates of thermosensitive neurons in POAH of forty-seven unit discharge were recorded by using extracellular microelectrode technique in New Zealand white rabbits. Our results show that the firing rates of the warm-sensitive neurons decreased significantly and those of the cold-sensitive neurons increased in POAH when the pyrogen (IL-1beta) was injected I.C.V. The effects of IL-1beta on firing rates in thermosensitive neurons of POAH were reversed by electrical stimulation of VSA. An arginine vasopressin (AVP) V1 antagonist abolished the regulatory effects of VSA on the firing rates in thermosensitive neurons of POAH evoked by IL-1beta. However, an AVP V2 antagonist had no effects. These data indicated that VSA regulates the activities of the thermosensitive neurons of POAH through AVP V1 but not AVP V2 receptor.     

Enzyme-linked immunosorbent assay for the activator protein specific for the enzymic hydrolysis of GM1 in urine

We have established a sensitive and specific enzyme-linked immunosorbent assay (ELISA) for the detection of the activator protein which stimulates the enzymic hydrolysis of G_M1 (G_M1-activator) in human urine. The level of G_M1-activator in 19 normal, adult urine samples was estimated to be 370.7±33.2 ng/ml. The amounts of G_M1-activator excreted in 24 h were estimated to be between 0.28 and 1.1 mg. The coefficient of variation for this method is 4.3% for the intra-assay and 14.4% for the inter-assay. Urine samples, without purification, can be used directly for the ELISA.     

Accumulation of liposome with Sialyl Lewis X to inflammation and tumor region: application to in vivo bio-imaging

We prepared the liposome binding Sialyl Lewis X (SLX) on the surface in order to specifically and efficiently deliver substances (fluorescent materials, chemical substances, proteins, genes, etc.) to inflammation or tumor regions. The liposome with SLX (SLX-Lipo-Cy5.5), in which fluorescent substance Cy5.5 was included, was administered intravenously to arthritis or Ehrlich Ascites Tumor (EAT) bearing mouse, and the accumulation of liposome was observed using two types of in vivo fluorescent imaging equipment. The result was that the accumulation of SLX-Lipo-Cy5.5 to inflammation or tumor regions was significantly higher than the control liposome without sugar chain (Lipo-Cy5.5) at 24 and 48 h after administration. In addition, it was confirmed that this accumulation showed a shift of liposome from blood vessels to the surrounding tissues. Thus, it was proven that this liposome is useful not only as an in vivo bio-imaging reagent but also as a drug delivery system (DDS).     

Cholesterol-mediated membrane surface area dynamics in neuroendocrine cells

How cholesterol, a key membrane constituent, affects membrane surface area dynamics in secretory cells is unclear. Using methyl-β-cyclodextrin (MβCD) to deplete cholesterol, we imaged melanotrophs from male Wistar rats in real-time and monitored membrane capacitance (C_m), fluctuations of which reflect exocytosis and endocytosis. Treatment with MβCD reduced cellular cholesterol and caused a dose-dependent attenuation of the Ca²⁺-evoked increase in C_m (IC₅₀ = 5.3 mM) vs. untreated cells. Cytosol dialysis of MβCD enhanced the attenuation of C_m increase (IC₅₀ = 3.3 mM), suggesting cholesterol depletion at intracellular membrane sites was involved in attenuating exocytosis. Acute extracellular application of MβCD resulted in an immediate C_m decline, which correlated well with the cellular surface area decrease, indicating the involvement of cholesterol in the regulation of membrane surface area dynamics. This decline in C_m was three-fold slower than MβCD-mediated fluorescent cholesterol decay, implying that exocytosis is the likely physiological means for plasma membrane cholesterol replenishment. MβCD had no effect on the specific C_m and the blockade of endocytosis by Dyngo 4a, confirmed by inhibition of dextran uptake, also had no effect on the time-course of MβCD-induced C_m decline. Thus acute exposure to MβCD evokes a C_m decline linked to the removal of membrane cholesterol, which cannot be compensated for by exocytosis. We propose that the primary contribution of cholesterol to surface area dynamics is via its role in regulated exocytosis.     

Phenotypical changes of satellite glial cells in a murine model of GM1‐gangliosidosis

Satellite glial cells (SGCs) of dorsal root ganglia (DRG) react in response to various injuries in the nervous system. This study investigates reactive changes within SGCs in a murine model for G_M1‐gangliosidosis (G_M1). DRG of homozygous β‐galactosidase‐knockout mice and homozygous C57BL/6 wild‐type mice were investigated performing immunostaining on formalin‐fixed, paraffin‐embedded tissue. A marked upregulation of glial fibrillary acidic protein (GFAP), the progenitor marker nestin and Ki67 within SGCs of diseased mice, starting after 4 months at the earliest GFAP, along with intracytoplasmic accumulation of ganglioside within neurons and deterioration of clinical signs was identified. Interestingly, nestin‐positive SGCs were detected after 8 months only. No changes regarding inwardly rectifying potassium channel 4.1, 2, 3‐cyclic nucleotide 3‐phosphodiesterase, Sox2, doublecortin, periaxin and caspase3 were observed in SGCs. Iba1 was only detected in close vicinity of SGCs indicating infiltrating or tissue‐resident macrophages. These results indicate that SGCs of DRG show phenotypical changes during the course of G_M1, characterized by GFAP upregulation, proliferation and expression of a neural progenitor marker at a late time point. This points towards an important role of SGCs during neurodegenerative disorders and supports that SGCs represent a multipotent glial precursor cell line with high plasticity and functionality.