Coupling of metal containing homing devices to liposomes via a maleimide linker: use of TCEP to stabilize thiol-groups without scavenging metals

Liposomes for drug delivery are often prepared with maleimide groups on the distal end of PEG to enable coupling of homing devices, such as antibodies, or other proteins. EDTA is used to stabilize the thiol group in the homing device for attachment to the maleimide. However, when using a homing device that contains a metal, EDTA inactivates this by scavenging of the metal. Holo-transferrin (Tf) containing two iron atoms (Fe(3+)), has a much higher affinity for the Tf receptor than apo-Tf (which does not contain any Fe(3+)). To couple Tf to a liposome, the introduction of a thiol group is necessary. During this process, by using N-succinimidyl S-acetylthioacetate (SATA), followed by 2-3 h coupling to the liposomes, Fe(3+) is scavenged by EDTA. This causes a decreased affinity of Tf for its receptor, resulting in a decreased targeting efficiency of the liposomes. Tris(2-carboxyethyl)phosphine (TCEP) hydrochloride is a sulfhydryl reductant that is often used in protein biochemistry. We found that TCEP (0.01 mM) does not scavenge Fe(3+) from Tf and is able to protect thiol groups for the coupling to maleimide. Furthermore, TCEP does not interfere with the maleimide coupling itself. In this communication, we describe the preparation of liposomes, focussing on the coupling of Tf to the maleimide linker at the distal end of PEG, without loosing Fe(3+) from Tf. This method can be applied to other metal-containing homing devices as well.     

Inhibition of NF-κB–DNA binding by mercuric ion: utility of the non-thiol reductant,tris(2-carboxyethyl)phosphine hydrochloride (TCEP), on detection of impaired NF-κB–DNA binding by thiol-directed agents

Mercuric ion (Hg²⁺), a potent thiol inhibitor, prevents expression of nuclear factor κB (NF-κB) by mercaptide bond formation with a critical cysteine moiety (cys₆₂) on the p50 subunit required for DNA binding. NF-κB–DNA binding is typically measured in reaction mixtures in which dithiothreitol (DTT) or other thiol reductants are used to maintain cys₆₂ in the reduced state. However, the presence of thiol reductants prevents accurate assessment of the Hg²⁺ concentration required to prevent NF-κB–DNA binding because of competitive mercaptide bond formation. In the present studies we evaluated the efficacy of tris(2-carboxyethyl)phosphine-HCl (TCEP), a non-thiol reducing agent which does not bind Hg²⁺, on NF-κB–DNA binding in vitro, using recombinant p50 protein and a ³²P-labelled κB oligonucleotide. We also measured the minimal Hg²⁺ concentration required to prevent this interaction in the presence of either reagent. DTT promoted NF-κB–DNA binding in concentrations from 0.25 to 2.6 mm in binding reactions. However, in the presence of 0.25 mm DTT, inhibition of NF-κB binding was seen only at Hg²⁺ concentrations greater than 100 μm and results were highly variable. In contrast, TCEP promoted NF-κB–DNA binding in a dose-related manner in concentrations from 0.25 to 6 mm. In the presence of even 6 mm TCEP, Hg²⁺ prevented NF-κB–DNA binding at concentrations as low as 20 μm in binding reactions. Similar findings were observed with regard to the thiol alkylating agent N-ethylmaleimide (NEM). These findings demonstrate the utility of TCEP as reductant in nuclear binding reaction assays involving the interaction of thiol constituents. They also demonstrate inhibition of NF-κB–DNA binding at Hg²⁺ concentrations comparable to those known to initiate toxicity and apoptotic cell death in vivo.     

Phosphoenolpyruvate carboxykinase: Effects of the hyperglycaemic agent 3-aminopicolinic acid

Chelation by 3-aminopicolinic acid of Fe²⁺, Co²⁺ and Mn²⁺ has been measured spectrophotometrically. With Fe²⁺ or Co²⁺, 3-aminopicolinic acid inhibited phosphoenolpyruvate carboxykinase at low metal-ion concentrations. Activation was not observed. The enzyme appears not to bind 3-aminopicolinic acid. 3-Aminopicolinic acid protects phosphoenolpyruvate carboxykinase from inactivation by ferrous ions. Two models are suggested, which could account for activation of gluconeogenesis due to chelation of metal-ions by 3-aminopicolinic acid: (a) phosphoenolpyruvate carboxykinase may be in dynamic equilibrium between inactivation by ferrous ions and reactivation by thiol compounds; and (b) metal-ions may promote product-inhibition by phosphoenolpyruvate, which chelation could alleviate.     

Tracking of antibody reduction fragments by capillary gel electrophoresis during the coupling to microparticles surface

Due to their high specificity and efficiency, antibodies are ideal ligands for target-specific ultrasound contrast agents. The present study focuses on the chemical stability of antibodies during functionalisation with sulfosuccinimidyl-pyridyldithiopropionamidohexanoate (SPDP), a heterobifunctional linker, which exposes free thiol groups upon treatment with a reducing agent. Thiolated antibodies can then react with thiol-reactive group, such as maleimide present on the microbubble surface to form stable covalent complexes. The immunoglobulin structure relies on several intra- and inter-chain disulfide bridges which might be affected by reducing agents. A capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) method with UV detection was applied to address the effect of the functionalisation process on the structural integrity of the antibodies and revealed that antibody disulfide bonds are prone to reduction as function of the reducing agents. Depending on the coupling conditions, various IgG fragments were identified reflecting different combinations between the light and heavy chains. Furthermore, two commonly used reducing agents, namely triscarboxyethylphosphine (TCEP) and 1,4-dithiothreitol (DTT) were compared under various preparation conditions. Results showed that reduction conditions based on DTT as a reducing agent under acidic pH were more appropriate to preserve intra- and inter-disulfide bridges of SPDP-modified antibodies.     

Targeting liposomes with protein drugs to the blood-brain barrier in vitro.

In this study, we aim to target pegylated liposomes loaded with horseradish peroxidase (HRP) and tagged with transferrin (Tf) to the BBB in vitro. Liposomes were prepared with the post-insertion technique: micelles of polyethylene glycol (PEG) and PEG-Tf were inserted into pre-formed liposomes containing HRP. Tf was measured indirectly by measuring iron via atomic absorption spectroscopy. All liposomes were around 100 nm in diameter, contained 5-13 microg HRP per mumol phospholipid and 63-74 Tf molecules per liposome (lipo Tf) or no Tf (lipo C). Brain capillary endothelial cells (BCEC) were incubated with liposomes at 4 degrees C (to determine binding) or at 37 degrees C (to determine association, i.e. binding+endocytosis) and the HRP activity, rather than the HRP amount was determined in cell lysates. Association of lipo Tf was two- to three-fold higher than association of lipo C. Surprisingly, the binding of lipo Tf at 4 degrees C was four-fold higher than the association of at 37 degrees C. Most likely this high binding and low endocytosis is explained by intracellular degradation of endocytosed HRP. In conclusion, we have shown targeting of liposomes loaded with protein or peptide drugs to the BCEC and more specifically to the lysosomes. This is an advantage for the treatment of lysosomal storage disease. However, drug targeting to other intracellular targets also results in intracellular degradation of the drug. Our experiments suggest that liposomes release some of their content within the BBB, making targeting of liposomes to the TfR on BCEC an attractive approach for brain drug delivery.     

Enhancement of cytotoxicity of artemisinin toward cancer cells by transferrin-mediated carbon nanotubes nanoparticles

Artemisinin (ART) is a kind of drug with an endoperoxide bridge which tends to react with Fe²⁺ to generate radicals for killing cancer cells. However, simultaneous delivery of hydrophobic ART and Fe²⁺ ions into cancer cells remains a major challenge. In this study, a multi-functional tumor-targeting drug delivery system employing hyaluronic acid-derivatized multi-walled carbon nanotubes (HA-MWCNTs) as drug carriers, transferrin (Tf) as targeting ligand and ART as a model drug for cancer treatment was constructed. This delivery system (HA-MWCNTs/Tf@ART) not only retained optical property of MWCNTs and cytotoxicity of ART but also demonstrated synergistic anti-tumor effect using ART and Tf. Compared with free ART, remarkably enhanced anti-tumor efficacy of this drug vehicle was realized both in cultured MCF-7 cells in vitro and in a tumor-bearing murine model in vivo, due to increased intracellular accumulation of ART and co-delivery of Tf and ART analogs. HA-MWCNTs/Tf@ART with laser irradiation demonstrated the highest inhibition effect compared to the other groups. This result may provide a new way of using promising natural drugs for cancer therapy.     

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

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

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.     

Validation of the Transferrin Receptor for Drug Targeting to Brain Capillary Endothelial Cells In Vitro

Recently, we have shown that transferrin (Tf) is actively endocytosed by the Tf R on primary cultured bovine brain capillary endothelial cells (BCEC). The objective of this investigation is to determine whether the Tf R can facilitate endocytosis of a (protein) model drug, using Tf as a targeting vector. Secondly, the mechanism of endocytosis was investigated. Horseradish peroxidase (HRP, 40 kDa) was chosen as a model drug, since it normally does not cross the blood–brain barrier (BBB) and its concentration in biological media can be easily quantified.

Tf-HRP conjugates (1:1) are actively and specifically endocytosed by BCEC in vitro in a concentration and time-dependent manner. At an applied concentration of 3 μg/ml, association (a combination of binding and endocytosis) of Tf-HRP reached equilibrium at a concentration of 2 ng/mg cell protein after 1 h of incubation at 37°C. This was approximately 3-fold higher compared to binding at 4°C (0.6 ng/mg cell protein). Association of Tf-HRP was compared to BSA-HRP. After 2 h of incubation at 37°C association levels were 5.2 and 2.5 ng/mg cell protein, for Tf-HRP and BSA-HRP, respectively. Under those conditions, association of Tf-HRP could be inhibited to approximately 30% of total association by an excess of non-conjugated Tf, but not with BSA, while association of BSA-HRP could be inhibited by both proteins. Furthermore, by using specific inhibitors of endocytotic processes, it was shown that association of Tf-HRP is via clathrin-coated vesicles. Association of Tf-HRP is inhibited by phenylarsine oxide (an inhibitor of clathrin-mediated endocytosis) to 0.4 ng/mg cell protein, but not by indomethacin, which inhibits formation of caveolae. Finally, following iron scavenging by deferoxamine mesylate (DFO, resulting in a higher Tf R expression) a 5-fold increase in association of Tf-HRP to 15.8 ng/mg cell protein was observed.

In conclusion, the Tf R is potentially suitable for targeting of a (protein) cargo to the BBB and to facilitate its endocytosis by the BCEC.     

Transferrin and octaarginine modified dual-functional liposomes with improved cancer cell targeting and enhanced intracellular delivery for the treatment of ovarian cancer

Off-target effects of drugs severely limit cancer therapy. Targeted nanocarriers are promising to enhance the delivery of therapeutics to tumors. Among many approaches for active tumor-targeting, arginine-rich cell penetrating peptides (AR-CPP) and ligands specific to target over-expressed receptors on cancer-cell surfaces, are popular. Earlier, we showed that the attachment of an AR-CPP octaarginine (R8) to the surface of DOXIL^® (Doxorubicin encapsulated PEGylated liposomes) improved cytoplasmic and nuclear DOX delivery that enhanced the cytotoxic effect in vitro and improved therapeutic efficacy in vivo. Here, we report on DOX-loaded liposomes, surface-modified with, R8 and transferrin (Tf) (Dual DOX-L), to improve targeting of A2780 ovarian carcinoma cells via the over-expressed transferrin receptors (TfRs) with R8-mediated intracellular DOX delivery. Flow cytometry analysis with fluorescently labeled DualL (without DOX) showed two-fold higher cancer-cell association than other treatments after 4?h treatment. Blocking entry pathways of R8 (macropinocytosis) and Tf (receptor-mediated endocytosis, RME) resulted in a decreased cancer-cell association of DualL. Confocal microscopy confirmed involvement of both entry pathways and cytoplasmic liposome accumulation with nuclear DOX delivery for Dual DOX-L. Dual DOX-L exhibited enhanced cytotoxicity in vitro and was most effective in controlling tumor growth in vivo in an A2780 ovarian xenograft model compared to other treatments. A pilot biodistribution study showed improved DOX accumulation in tumors after Dual DOX-L treatment. All results collectively presented a clear advantage of the R8 and Tf combination to elevate the therapeutic potential of DOX-L by exploiting TfR over-expression imparting specificity followed by endosomal escape and intracellular delivery via R8.     

Effect of lactoferrin- and transferrin-conjugated polymersomes in brain targeting： in vitro and in vivo evaluations

Aim:

To evaluate the effect of lactoferrin (Lf) and transferrin (Tf) in brain targeting.

Methods:

Polymersomes (PSs), employed as vectors, were conjugated with Lf or Tf and were characterized by morphology, particle size, zeta potential, and surface densities of the Lf or Tf molecules. In vitro uptake of Lf-PS and Tf-PS by bEnd.3 cells was investigated using coumarin-6 as a fluorescent probe. In vivo tissue distribution and pharmacokinetics of ¹²⁵I-Lf-PS and ¹²⁵I-Tf-PS were also examined.

Results:

The mean particle size of PS, Lf-PS, and Tf-PS was around 150 nm and the zeta potential of the PSs was about -20 mV. Less than 0.12% of the coumarin was released from coumarin-6-loaded PS in 84 h indicating that coumarin-6 was an accurate probe for the PSs'' behavior in vitro. It was shown that the uptake of Lf-PS and Tf-PS by bEnd.3 cells was time-, temperature-, and concentration-dependent. Both Lf and Tf could increase the cell uptake of PSs at 37°C, but the uptake of Tf-PS was significantly greater than that of Lf-PS. In vivo tissue distribution and pharmacokinetics in mice revealed higher brain uptake and distribution of Tf-PS than Lf-PS, which was in accordance with in vitro uptake results. The drug targeting index (DTI) of Tf-PS with regard to Lf-PS was 1.51.

Conclusion:

Using a PS as the delivery vector and bEnd.3 cells as the model of the blood-brain barrier (BBB), Tf was more effective than Lf in brain targeting.     

A mechanistic study of the effect of transferrin conjugation on cytotoxicity of targeted liposomes

Abstract

This study was performed to prepare 5-fluorouracil (5FU) containing targeted liposomes for the safety and efficacy enhancement. Liposomes were prepared using thin layer method and transferrin (Tf) was employed as the targeting ligand. Morphology of 5FU-loaded liposomes was assessed by transmission electron microscopy (TEM). The in vitro cytotoxicity was investigated via MTT assay on HT-29, CT26 and fibroblast cells. Mitochondrial membrane and cell death evaluations were also investigated. Resulted showed that the encapsulation efficiency (EE%) and particle size of the liposomes were 40.12% and 130?nm, respectively. TEM image implied that liposomes were spherical in shape. In cancer cells, targeted liposomes triggered the mitochondrial apoptotic pathway by lower production of reactive oxygen species (ROS) (63.58 vs 84.95 fluorescence intensity), reduced mitochondrial membrane potential and releasing of cytochrome c (68.66 vs 51.13?ng/mL). The results of this study indicated that Tf-targeted 5FU liposomes can be employed as promising nanocarrier for the delivery of drugs to cancer cells.     

Mercuric ions inhibit mitogen-activated protein kinase dephosphorylation by inducing reactive oxygen species

Mercury intoxication profoundly affects the immune system, in particular, signal transduction of immune cells. However, the mechanism of the interaction of mercury with cellular signaling pathways, such as mitogen activated protein kinases (MAPK), remains elusive. Therefore, the objective of this study is to investigate three potential ways in which Hg²⁺ ions could inhibit MAPK dephosphorylation in the human T-cell line Jurkat: (1) by direct binding to phosphatases; (2) by releasing cellular zinc (Zn²⁺); and (3) by inducing reactive oxygen species (ROS). Hg²⁺ causes production of ROS, measured by dihydrorhodamine 123, and triggers ROS-mediated Zn²⁺ release, detected with FluoZin-3. Yet, phosphatase-inhibition is not mediated by binding of Zn²⁺ or Hg²⁺. Rather, phosphatases are inactivated by at least two forms of thiol oxidation; initial inhibition is reversible with reducing agents such as Tris(2-carboxyethyl)phosphine. Prolonged inhibition leads to non-reversible phosphatase oxidation, presumably oxidizing the cysteine thiol to sulfinic- or sulfonic acid. Notably, phosphatases are a particularly sensitive target for Hg²⁺-induced oxidation, because phosphatase activity is inhibited at concentrations of Hg²⁺ that have only minor impact on over all thiol oxidation. This phosphatase inhibition results in augmented, ROS-dependent MAPK phosphorylation. MAPK are important regulators of T-cell function, and MAPK-activation by inhibition of phosphatases seems to be one of the molecular mechanisms by which mercury affects the immune system.     

Efficient synthesis of [18F]FPyME: A new approach for the preparation of maleimide‐containing prosthetic groups for the conjugation with thiols

An improved high yielding radiosynthesis of the known thiol‐reactive maleimide‐containing prosthetic group1‐[3‐(2‐[¹⁸F]fluoropyridine‐3‐yloxy)propyl]pyrrole‐2,5‐dione ([¹⁸F]FPyME) is described. The target compound was obtained by a two‐step one‐pot procedure starting from a maleimide‐containing nitro‐precursor that was protected as a Diels‐Alder adduct with 2,5‐dimethylfurane. Nucleophilic radiofluorination followed by heat induced deprotection through a Retro Diels Alder reaction yielded, after chromatographic isolation, [¹⁸F]FPyME with a radiochemical yield of 20% in about 60 min overall synthesis time. A variety of other [¹⁸F]fluoropyridine based maleimide‐containing prosthetic groups should be accessible via the described synthetic strategy.     

The role of EDTA in phytoextraction of hexavalent and trivalent chromium by two willow trees

Effects of the synthetic chelator ethylenediamine tetraacetate (EDTA) on uptake and internal translocation of hexavalent and trivalent chromium by plants were investigated. Two different concentrations of EDTA were studied for enhancing the uptake and translocation of Cr from the hydroponic solution spiked with K₂CrO₄ or CrCl₃ maintained at 24.0 ± 1°C. Faster removal of Cr³⁺ than Cr⁶⁺ by hybrid willows (Salix matsudana Koidz × Salix alba L.) from the plant growth media was observed. Negligible effect of EDTA on the uptake of Cr⁶⁺ was found, but significant decrease of the Cr concentration in roots was measured. Although the translocation of Cr⁶⁺ within plant materials was detected in response to EDTA concentration, the amount of Cr⁶⁺ translocated to the lower stems was considerably small. EDTA in the nutrient media showed a negative effect on the uptake of Cr³⁺ by hybrid willows; the removal rates of Cr³⁺ were significantly decreased. Translocation of Cr³⁺ into the stems and leaves was undetectable, but roots were the exclusive sink for Cr³⁺ accumulation. Weeping willows (Salix babylonica L.) showed lower removal rates for both chemical forms of Cr than hybrid willows. Although EDTA had a minor effect on Cr⁶⁺ uptake by weeping willows, positive effect on Cr⁶⁺ translocation within plant materials was observed. It was also determined that EDTA in plant growth media significantly decreased the amount of Cr³⁺ taken up by plants, but significantly increased Cr³⁺ mobilization from roots to stems. Results indicated that EDTA was unable to increase the uptake of Cr⁶⁺ by both plant species, but translocation of Cr⁶⁺–EDTA within plant materials was possible. Addition of EDTA in the nutrient media showed a strong influence on the uptake and translocation of Cr³⁺ in both willows. Cr³⁺–EDTA in tissues of weeping willows was more mobile than that in hybrid willows. The information has important implications for the use of metal chelator in plant nutritional research.     

Carvacrol protects neuroblastoma SH-SY5Y cells against Fe2+-induced apoptosis by suppressing activation of MAPK/JNK-NF-κB signaling pathway

Aim:

Carvacrol (2-methyl-5-isopropylphenol), a phenolic monoterpene in the essential oils of the genera Origanum and Thymus, has been shown to exert a variety of therapeutic effects. Here we examined whether carvacrol protected neuroblastoma SH-SY5Y cells against Fe²⁺-induced apoptosis and explored the underlying mechanisms.

Methods:

Neuroblastoma SH-SY5Y cells were incubated with Fe²⁺ for 24 h, and the cell viability was assessed with CCK-8 assay. TUNEL assay and flow cytometric analysis were performed to evaluate cell apoptosis. The mRNA levels of pro-inflammatory cytokines and NF-κB p65 were determined using qPCR. The expression of relevant proteins was determined using Western blot analysis or immunofluorescence staining.

Results:

Treatment of SH-SY5Y cells with Fe²⁺ (50–200 μmol/L) dose-dependently decreased the cell viability, which was significantly attenuated by pretreatment with carvacrol (164 and 333 μmol/L). Treatment with Fe²⁺ increased the Bax level and caspase-3 activity, and decreased the Bcl-2 level, resulting in cell apoptosis. Furthermore, treatment with Fe²⁺ significantly increased the gene expression of IL-1β, IL-6 and TNF-α, and induced the nuclear translocation of NF-κB. Treatment with Fe²⁺ also significantly increased the phosphorylation of p38, ERK, JNK and IKK in the cells. Pretreatment with carvacrol significantly inhibited Fe²⁺-induced activation of NF-κB, expression of the pro-inflammatory cytokines, and cell apoptosis. Moreover, pretreatment with carvacrol inhibited Fe²⁺-induced phosphorylation of JNK and IKK, but not p38 and ERK in the cells.

Conclusion:

Carvacrol protects neuroblastoma SH-SY5Y cells against Fe²⁺-induced apoptosis, which may result from suppressing the MAPK/JNK-NF-κB signaling pathways.     

双配体修饰的阿霉素脂质体靶向于脑胶质瘤的体外研究

目的筛选和优化转铁蛋白、叶酸共同修饰的阿霉素脂质体的处方及制备工艺,以期得到具有良好的脑胶质瘤靶向治疗作用的给药系统。方法采用薄膜分散和硫酸铵梯度法制备阿霉素脂质体。将叶酸连接至二硬脂酸磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000-NH2)得到DSPE-PEG2000-Folic,考察不同磷脂种类、药脂比、水化介质和载药时间,对脂质体粒径、包封率和稳定性的影响,确定脂质体的处方工艺。以大鼠的脑毛细血管内皮细胞(bEnd3)和星形胶质细胞组成体外血脑屏障(blood-brain barrier,BBB),并结合大鼠胶质瘤C6细胞,构建体外模拟胶质瘤靶向治疗的复合BBB模型。考察阿霉素脂质体在bEnd3细胞中的摄取机制和透过BBB的转运速率及对C6细胞的毒性。结果确定了DSPC作为主要磷脂组分,并以120 mmol.L 1的硫酸铵作为水化介质,药脂比为1∶1 5,载药时间选择60 min,成功制备了高包封率和稳定性的双配体脂质体。其在bEnd3细胞中摄取远大于普通脂质体(P<0.05),摄取过程受网格蛋白和小窝内陷介导的细胞内吞作用,并受转铁蛋白和叶酸的影响;同时其在BBB模型中的药物透过速率、及其进一步透过BBB后对下层C6细胞的毒性,均显著高于其他脂质体组。结论转铁蛋白和叶酸共同修饰的阿霉素脂质体具有较好的体外脑胶质瘤靶向治疗作用。     

IN VIVO TRAFFICKING OF LONG-CIRCULATING LIPOSOMES IN TUMOUR-BEARING MICE DETERMINED BY POSITRON EMISSION TOMOGRAPHY

Various kinds of long-circulating liposome, such as ganglioside GM1-, polyethyleneglycol- (PEG-), and glucuronide-modified liposomes, have been developed for passive targeting of liposomal drugs to tumours. To evaluate the in vivo behaviour of such long-circulating liposomes, we investigated the liposomal trafficking, especially early trafficking just after injection of liposomes, by a non-invasive method using positron emission tomography (PET). Liposomes composed of dipalmitoylphosphatidylcholine, cholesterol, and modifier, namely, GM1, distearoylphosphatidylethanolamine (DSPE)–PEG or palmityl-D -glucuronide (PGlcUA), were labelled with [2-¹⁸F]-2-fluoro-2-deoxy-D -glucose ([2-¹⁸F]FDG), and administered to mice bearing Meth A sarcoma after having been sized to 100 nm. A PET scan was started immediately after injection of liposomes and continued for 120 min. PET images and time–activity curves indicated that PEG liposomes and PGlcUA liposomes were efficiently accumulated in tumour tissues time dependently from immediately after injection. In contrast, GM1 liposomes accumulated less in the tumour as was also the case for control liposomes that contained dipalmitoylphosphatidylglycerol (DPPG) instead of a modifier. Long-circulating liposomes including GM1 liposomes, however, remained in the blood circulation and avoided liver trapping compared with control DPPG liposomes. These data suggest that PGlcUA and PEG liposomes start to accumulate in the tumour just after injection, whereas GM1 liposomes may accumulate in the tumour after a longer period of circulation.     

Cyclosporine Binds to the Neutral Lipid and Potentially Other Binding Sites of Lipid Transfer Protein I

Purpose. The objective of this study was to determine if cyclosporine (CSA) binds directly to the neutral lipid-binding site of lipid transfer protein I (LTP I). Methods. This was accomplished by determining LTP I concentrations and cholesteryl esters (CE) and CSA radioactivity of eluted fast protein liquid chromatography (FPLC) fractions following an injection of different treatment groups (i.e., LTP I alone, ³H-CE liposomes alone, ³H-CSA liposomes alone, ³H-CE liposomes + LTP I, and ³H-CSA liposomes + LTP I) onto an FPLC column. Our hypothesis is that CSA will bind to the neutral lipid-binding site of LTP I because of its high solubility/interaction with cholesterol and triglycerides. Results. Coincubation of LTP I with ³H-CE liposomes resulted in a significant decrease in the LTP I peak reported at fraction 10 and the appearance of a broad LTP I peak at fractions 30-34 compared to control. Coincubation of LTP I with ³H-CSA liposomes resulted in a significant decrease in the LTP I peak reported at fraction 10 and fraction 30 compared to control. In addition, 30% of the original radioactivity associated with ³H-CSA liposomes was found coeluted with the unbound LTP I peak at fraction 10. Taken together, these findings suggest that CSA does bind to the neutral lipid-binding site of LTP I but may also bind to other regions along the LTP I molecule. Conclusions. We have determined that LTP I mediated transfer of CSA between lipoproteins may be a result of the direct binding of CSA to LTP I at both its neutral binding site and potentially other binding sites along the molecule. These findings provide further evidence that the distribution/redistribution of drugs among plasma lipoproteins facilitated by LTP I may serve as a possible mechanism for determining the ultimate fate of drug compounds     

Interactions of the glycopeptide antitumor antibiotics bleomycin and tallysomycin with deoxyribonucleic acid in vitro

Tallysomycin (TLM), an experimental glycopeptide antitumor antibiotic related to bleomycin (BLM), at a concentration of 4.8 × 10^?5 M sequesters Fe²⁺ and nicks circular DNA to 75–80 per cent in 45 min. The DNA scission reaction which requires oxygen and is suppressed by other divalent ions and by EDTA is pH dependent and shows optima at 9.6 and 11.2. BLM under comparable conditions shows three pH optima at 9.3. 10.6 and 11.2. TLM binds to DNA more strongly than BLM. especially at pH 7.0 to 4.7. The three intermediates, O^?₂, H₂O₂ and OH, are implicated in the DNA scission by tallysomycin as indicated by (1) inhibition by Superoxide dismutase, (2) inhibition by catalase. and (3) e.s.r. detection of the spin-trapped PBN · OH nitroxide radical, respectively. The A,T-specific DNA binding agents, netropsin and distamycin, enhance the TLM-induced cleavage by 7 per cent and 9.5 per cent, while G.C-specinc agents, olivomycin and chromomycin-A₃, enhance the scission by 15 per cent and 16 per cent, respectively. TLM and BLM suppress the extent of psoralen-photo-induced DNA cross-linking by 24 per cent and 53 per cent, respectively. TLM with the spermidine moiety truncated by spermidine oxidase binds to DNA with less efficiency (5.5 per cent) than does the parent antibiotic (19 per cent). However, the modified TLM also sequesters Fe²⁺ and nicks DNA with comparable efficiency to TLM under similar conditions. This suggests (1) that the spermidine moiety is not involved in binding Fe²⁺ at the ‘active site’ of the antibiotic responsible for DNA cleavage, and (2) that in binding to DNA, at least of TLM Fe²⁺, intercalation by the bithiazole moiety is more significant than electrostatic attraction by the spermidine chain. The results are in accord with a mode of action in which TLM sequesters Fe²⁺. binds to DNA and produces OH radicals close to the duplex to cleave the latter. A chemical mechanism is suggested for this process.