不同脂质载体对超氧化物歧化酶(SOD)渗入小白鼠红细胞能力的观察

本文观察了单纯注射超氧化物歧化酶和脂质体及脂肪乳携带超氧化物歧化酶渗入小白鼠红细胞的能力。发现脂质体及脂肪乳携带超氧化物歧化酶渗入小白鼠红细胞的能力比单纯注射超氧化物歧化酶大(P<0.05～0.01)。该结果为解决超氧化物歧化酶渗入细胞发挥治疗作用提供了简易方法和实验根据。     

Expression ofNeisseria meningitidisclass 1 porin as a fusion protein inEscherichia colli: the influence of liposomes and adjuvants on the production of a bactericidal immune reesponse

High level exxpression of meningococcal class 1 protein was achieved inEscherichia coliusing the p-GEMEX-1 vector, in which the protein was expressed in inclusion bodies, (IB), as a fusion with the bacteriophage T7 gene 10 capsid protein. The fusion protein (FP) was engineered with a factor Xa protease site between the gene 10 and class 1 protein, but treatment with the enzyme resulted in cleavage at additional sites within the class 1 protein. Since it was not possible to remove the leader protein, the intact FP provided an alternative antigen for immunization. Antisera raised to FP, solubilized from IB and incorporated into liposomes, generated a subtype-specific response which was weakly bactericidal for meningococci. In order to remove any possible effect ofE. coliLPS present in IB, the FP was further purified by SDS-PAGE and incorporated into liposomes, either alone ofr in combination with the adjuvants monophosphoryl lipin A or muramyl dipeptide. The incorporation of adjuvants in liposomes resulted in stimulation of the overall immune response to FP, but the resulting antisera were not bactericidal. however and effective bactericidal response was obtained with the purest preparation of FP in liposomes, without any additional adjuvants, revealing that attempts to increase further the immunogenicity of such antigens must not be at the expense of interfering with optimal protein folding     

In vivo gene transfer into the adult mammalian central nervous system by continuous injection of plasmid DNA–cationic liposome complex

Previously reported methods of liposome-mediated direct in vivo gene transfer have been inefficient, especially when performed with highly differentiated, quiescent cells of the adult mammalian central nervous system (CNS). We have therefore improved these procedures based upon a novel concept. Following continuous injection of plasmid DNA–cationic liposome complex which contained a reporter gene encoding E. coli β-galactosidase into the striatum of adult rats, the expression of transgene was dramatically elevated without any adverse effects. This new technique may enable a wide application of liposome-mediated gene transfer technology not only to basic analysis of gene functions in the brain but also for clinical treatment of certain CNS disorders.     

Neural retina limits the nonviral gene transfer to retinal pigment epithelium in an in vitro bovine eye model

We investigated the permeation of liposomal and polymeric gene delivery systems through neural retina into retinal pigment epithelium (RPE) and determined the roles of various factors in permeation and subsequent uptake of the delivery systems by RPE. Anterior parts and vitreous of fresh bovine eyes were removed. Retina was left intact or peeled away. Complexes of ethidium monoazide (EMA)-labeled plasmid DNA and cationic carriers (polyethyleneimine, poly-L-lysine, DOTAP liposomes) were pipetted on the retina or RPE. Two hours later the neural retina was removed, if present, and the RPE cells were detached. Contaminants were removed by sucrose centrifugation, and the RPE cells were analyzed for DNA uptake by flow cytometry. Cellular uptake of FITC-dextrans (molecular weight [mw] 20,000, 500,000 and 2,000,000), FITC-poly-L-lysine (mw 20,000), FITC-labeled oligonucleotide (15-mer), and naked EMA-labeled plasmid DNA was determined after pipetting the solutions on the RPE or neural retina. Location of the fluorescent materials in the retina was visualized with fluorescence microscopy. Neural retina decreased the cellular uptake of DNA complexes by an order of magnitude, the uptake of FITC-dextrans slightly, whereas delivery of polycationic FITC-poly-L-lysine to RPE was almost completely inhibited. Neural retina decreased the cellular uptake of FITC-oligonucleotides, while the uptake of uncomplexed plasmid was always negligible. Conclusions from FACS and fluorescence microscopy were similar: delivery of polymeric and liposomal DNA complexes into RPE are limited by the neural retina. This is due to the size and positive charge of the complexes.     

Enhanced transfollicular delivery of adriamycin with a liposome and iontophoresis

To find a better way to deliver drugs into hair follicles, we tried two approaches: single topical application using various liposomes; and iontophoresis combined with topical application of ionic liposome. After delivery of adriamycin (ADR) to wax-depilated rat skin, the transport of the drug was examined under fluorescence microscopy. Most liposomal ADR showed more effective transdermal and transfollicular penetration than free ADR. Among tested liposomes, the non-ionic GDL liposome (GDL/CH/POE-10 = glycerol dilaulate/cholesterol/polyoxyethylene-10) was the most selective to hair follicles against skin, while the cationic liposome (GDL/CH/POE-10/DOTAP, dioleoyl trimethylammonium propane) containing monocationic DOTAP was less selective; however, it was better at improving the delivery amount and penetration of ADR into the follicles and skin. The DMPC/DMPG (7/3) formulation of anionic PC liposome (DMPC/DMPG = dimyristoyl-phosphocholine/-phospoglycerol) showed results similar to the cationic liposome. The DMPC/DMPG (3/7) formulation yielded poor results, however, probably because of its increased viscosity and anionic property. Although ADR delivery was enhanced by liposomal formulations, topical applications had some limitations in delivery capacity and speed. To accelerate delivery, iontophoresis was combined with the cationic liposome at positive 0.2-0.4 mA/cm(2) for 20-30 min. The resulting delivery of ADR through follicular routes was excellent. This combination method diffused ADR 3.0-fold more efficiently, rapidly and deeply than single topical application of cationic liposomal ADR. This system also achieved a 3.5-fold higher diffusive follicular delivery than a free ADR/iontophoresis combination. Furthermore, it was demonstrated that the tetracationic lipid DOSPER and hydrophile spermine could serve as a cationic additive instead of the monocationic DOTAP in the liposome. These results suggest that the combinative system of the topically applied cationic liposome followed by iontophoresis has a significant synergistic effect on the transfollicular delivery of ADR.     

Improving lipoplex-mediated gene transfer into C6 glioma cells and primary neurons

The development of methodologies for gene transfer into the central nervous system is crucial for gene therapy of neurological disorders. In this study, different cationic liposome formulations were used to transfer DNA into C6 glioma cells and primary hippocampal and cortical neurons by varying the nature of the helper lipid (DOPE, Chol) or a mixture of DOPE and cholesterol (Chol) associated to DOTAP. In addition, the effect of the lipid/DNA (+/-) charge ratio, the association of the ligand transferrin to the lipoplexes, and the stage of differentiation of the primary cells on the levels of transfection activity, transfection efficiency, and duration of gene expression were evaluated. Mechanistic studies were also performed to investigate the route of delivery of the complexes into neurons. Our results indicate that DOTAP:Chol (1:1 mol ratio) was the best formulation to transfer a reporter gene into C6 glioma cells, primary hippocampal neurons, and primary cortical neurons. The use of transferrin-associated lipoplexes resulted in a significant enhancement of transfection activity, as compared to plain lipoplexes, which can be partially attributed to the promotion of their internalization mediated by transferrin. While for hippocampal neurons the levels of luciferase gene expression are very low, for primary cortical neurons the levels of transgene expression are high and relatively stable, although only 4% of the cells has been transfected. The stage of cell differentiation revealed to be critical to the levels of gene expression. Consistent with previous findings on the mechanisms of cell internalization, the experiments with inhibitors of the endocytotic pathway clearly indicate that transferrin-associated lipoplexes are internalized into primary neurons by endocytosis. Promising results were obtained in terms of the levels and duration of gene expression, particularly in cortical neurons when transfected with the Tf-associated lipoplexes, this finding suggesting the usefulness of these lipid-based carriers to deliver genes within the CNS.     

Analgesic duration and kinetics of liposomal bupivacaine after subcutaneous injection in mice

1. The objective of the present study was to assess the time-course profile of analgesia and bupivacaine concentrations at the site of injection after subcutaneous administration of a single dose of standard bupivacaine or a novel controlled-release liposomal bupivacaine formulation. 2. Groups of mice were injected subcutaneously with 0.2 mL of 0.5% standard bupivacaine or 0.5, 1 or 2% liposomal bupivacaine. 3. A prolonged duration of analgesia occurred in mice receiving liposomal bupivacaine. In the liposomal groups, the bupivacaine remained at the injection site for more than 96 h, compared with approximately 8 h in groups injected with standard bupivacaine. 4. These results confirm that the prolonged analgesia observed after injection of the liposomal formulation is associated with sustained higher levels of bupivacaine at the site of injection.     

Magainin-Mediated Disruption of Stratum Corneum Lipid Vesicles

Pharmaceutical Research -     

Manganese superoxide dismutase-plasmid/liposome (MnSOD-PL) administration protects mice from esophagitis associated with fractionated radiation

Intraesophageal administration of manganese superoxide dismutase-plasmid/liposome (MnSOD-PL) prior to single fraction radiation has been shown to protect mice from lethal esophagitis. In our study, C3H/HeNsd mice received fractionated radiation in two protocols: (i) 18 Gy daily for four days with MnSOD-PL administration 24 hr prior to the first and third fraction, or (ii) 12 Gy daily for six days with MnSOD-PL 24 hr prior to the first, third, and fifth fraction. Control radiated mice received either no liposomes only or LacZ (bacterial beta-galactosidase gene)-plasmid/liposome (LacZ-PL) by the same schedules. We measured thiol depletion and lipid peroxidation (LP) in whole esophagus and tested the effectiveness of a new plasmid, hemagglutinin (HA) epitope-tagged MnSOD (HA-MnSOD). In fractionation protocols, mice receiving MnSOD-PL, but not LacZ-PL (200 microl of plasmid/liposomes containing 200 microg of plasmid DNA), showed a significant reduction in morbidity, decreased weight loss, and improved survival. Four and seven days after 37 Gy single fraction radiation, the esophagus demonstrated a significant increase in peroxidized lipids and reduction in overall antioxidant levels, reduced thiols, and decreased glutathione (GSH). These reductions were modulated by MnSOD-PL administration. The HA-MnSOD plasmid product was detected in the basal layers of the esophageal epithelium 24 hr after administration and provided significant radiation protection compared to glutathione peroxidase-plasmid/liposome (GPX-PL), or liposomes containing MnSOD protein, vitamin E, co-enzyme Q10, or 21-aminosteroid. Thus, MnSOD-PL administration significantly improved tolerance to fractionated radiation and modulated radiation effects on levels of GSH and lipid peroxidation (LP). These studies provide further support for translation of MnSOD-PL treatment into human esophageal radiation protection.