Regular ArticleDelivery of Plasmid DNA to Glial Cells Using pH-Sensitive Immunoliposomes
Abstract
Immunoliposomes were constructed with an antibody specific to glial cells. They were used to examine the specificity and efficacy of cell type plasmid transfection. Liposomes contained a β-galactosidase gene under control of an SV-40 promotor. Two different monoclonal antibodies of a different subclass, IgM and IgG, were examined for their targeting ability using immunoliposomes. Cultured C6 glioma (specific target cell type) and NIH 3T3 (control cell type, fibroblast) cells were transfected using these immunoliposomes. Results indicate a three-fold increase in transfection by the glial specific immunoliposomes, "gliasomes", in glial cell culture over control liposomes. Gliasomes were exposed to NIH 3T3 cells and showed no enhanced transfection over control liposomes. Gliasomes were tested for their specificity by the addition of excess antibody to the cell culture in order to saturate specific receptors on C6 glioma cells. Results indicate a reduced transfection, nearly three-fold, in cells that were saturated with excess antibody prior to exposure to the immunoliposomes.
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A horseradish peroxidase-light and electron microscopic study of immunoliposomes utilized for intracellular delivery to the rat striatum
2002, Neuroscience LettersLiposomes can deliver plasmid DNA, viruses, antisense oligonucleotides, and pharmacological agents to the central nervous system. Conjugation of antibodies to liposomes increases delivery specificity. Immunoliposomes created with Thy 1.1 antibody have previously been shown to be effective for neuronal delivery. The intracellular delivery of these immunoliposomes is evaluated by light and electron microscopy. Thy 1.1 conjugated liposomes were loaded with horseradish peroxidase and stereotactically injected into rat striatum. On light microscopy, immunoliposomes were concentrated within 0.2 mm of the injection site 8 h following delivery but, 24 h post-operatively, had diffused more than 0.5 mm from the injection site. With transmission electron microscopy, immunoliposomes were observed entering numerous neurons and some astrocytes in a process distinct from the clathrin-coated pit mechanism. These findings suggest that Thy 1.1 immunoliposomes are effective for intracellular delivery in vivo and their endocytosis occurs independently of a coated pit process. The research has helped to elucidate alternative mechanisms for immunoliposomal delivery. A more fundamental understanding of these attributes is needed to achieve the therapeutic potential of immunoliposomes.
Tracing transgene expression in cancer gene therapy: A requirement for rational progress in the field
2002, Molecular Imaging and BiologyThis review summarizes the status of gene therapy in medicine and the role of molecular imaging in its development. In gene therapy, genetic material is introduced into cells in order to generate a specific biological effect. Natural (viruses) or artificial molecular constructs, named gene therapy vectors, are used to achieve efficient cell transduction. This new form of therapy can be used for treating a broad variety of conditions including hereditary diseases, infections, degenerative disorders and cancer. Monitoring transgene expression using noninvasive imaging techniques is a necessary complement for the development of clinical gene therapy. Recent developments in magnetic resonance imaging afford the possibility of detecting gene transfer in vivo, but the most promising results have been obtained with positron emission tomography (PET). PET allows imaging gene therapy products by administration of a labeled substrate when the transgene codes for an enzyme or by administration of a labeled ligand when the transgene codes for a receptor. In the latter strategy, a membrane molecule (somatostatin or dopamine receptors) is used to detect the selective trapping of its radiolabeled ligand in the transduced cells. One of the approaches for the genetic treatment of cancer consists in transferring the “suicide genes” into tumor cells, the most common being the thymidine kinase (tk) of herpes viruses. Different nucleoside analogs can be labeled for its use as PET reporter probes in order to visualize tk expression. The results of pre-clinical studies are extremely encouraging. Reliable methods for the in vivo tracing of transgene expression in humans have to be developed in order for the field of gene therapy to mature. PET has emerged as a powerful tool to assist in achieving this goal. (Mol Imag Biol 2002;4:27–33)
A simple method for preparation of immuno-magnetic liposomes
2001, International Journal of PharmaceuticsA simple and readily manoeuverable method for preparing immuno-magnetic liposomes that indigenously contain binding sites for attaching other molecules like antibodies on their exterior surface is described. In this method magnetic unilamellar vesicles are prepared from a mixture of phosphatidylcholine, cholesterol, small amounts of a linear chain aldehyde and colloidal particles of magnetic iron oxide, using a reverse phase evaporation technique. The aldehyde (dedecanal) molecules align themselves among the lipid molecules in the bilayer with their aldehyde groups exposed to the aqueous phase, allowing straight attachment of antibody molecules (human-antimouse IgG-FITC in this case) in one single step. The success of this approach is confirmed by fluorescence microscopy as well as binding of the resulting immuno-magnetic liposomes to their corresponding target cells.
Site-directed lipid modification of IgG-binding protein by intracellular bacterial lipoprotein process
1999, Journal of BiotechnologyIgG-binding protein was genetically expressed and lipid-modified in a site-directed manner in Escherichia coli. The DNA sequence encoding the signal peptide and the nine N-terminal amino acid residues of the major lipoprotein of E. coli (lpp) was fused to the sequence of B-domain which was one of the IgG binding domains of Staphylococcal Protein A (SpA). The N-terminal cysteine residue of the resulting protein was enzymatically linked with lipids in the bacterial membrane. The lipid-modified protein was translocated at the bacterial membrane in a manner similar to native bacterial lipoprotein, and it was purified with IgG-Sepharose by affinity chromatography. The lipid modified proteins (lppB1 and lppB5) showed a similar IgG binding activity to unmodified proteins, which was estimated by competitive ELISA. Proteoliposomes of lipid modified proteins were prepared in an elegant fashion so that the IgG binding site should be properly oriented on the surface of an individual liposome by anchoring the lipid-tail into the hydrophobic layer of the liposome membrane. As compared with the unmodified one, the lipid modified protein incorporated into the proteoliposome exhibited higher IgG binding activity.
Stability and pH sensitivity of sulfatide-containing phosphatidylethanolamine small unilamellar vesicles
1996, Biochimica et Biophysica Acta - BiomembranesThe bilayer stabilization effect of sulfatide and the pH sensitivity of sulfatide-containing 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) small unilamellar vesicles were examined by light scattering and the release of entrapped calcein. At 30 mol% sulfatide, stable DOPE/sulfatide vesicles were formed at the physiological pH and their stability was preserved in the presence of human plasma. These vesicles were found to be pH-sensitive and became leaky at pH 6.0 or when there was a pH-gradient across the membrane bilayer. Under such conditions, the amount of calcein released after 24 h incubation at 37°C was increased by one-fold compared to that found at pH 7.4. Our results suggest that the hydration and partial dehydration of the headgroup of sulfatide upon changing pH play an essential role in determining the pH sensitivity of DOPE/sulfatide vesicles, while the importance of the condensing effect of the glycolipid on membrane bilayer is less significant.
Transfecting neurons and glia in the rat using pH-sensitive immunoliposomes
1995, Neuroscience LettersImmunoliposomes were constructed using antibody 5–113 (directed to an antigen on the external surface rat glial cells), the antibody Thy 1.1, and a non-immune antibody. The antibodies were conjugated to N-gluytaryl-phosphatidylethanolamine. Liposomes were constructed with these conjugated antibodies, other lipids and a β-galactosidase plasmid under the control of the cytomegalovirus promoter. When immunoliposomes decorated with one of three different antibodies were injected into the brain or spinal cord of adult rats, the X-gal reaction product was observed in neurons, astrocytes and vascular elements. There was an increase in neuronal labeling when animals were injected with Thy 1.1 conjugated liposomes and there was an increase in glial labeling in animals injected with 5–113 liposomes. In spinal cords, the immunoliposomes appear to penetrate a substantial distance, transfecting neurons several centimeters from the site of delivery. These data suggest that immunoliposomes may provide an effective transfection system for gene delivery in the CNS.