An improved method for the covalent coupling of proteins to liposomes

Derksen, J.T.P.; Scherphof, Gerrit L.

doi:10.1016/0005-2736(85)90430-4

Cited by 88 publications

(41 citation statements)

References 18 publications

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“…The values for incorporation ratios in parenthesis were calculated by us based on following equation: maleimide lipid molar ratio (of the total lipids in the liposomes)×0.55 (ratio facing outward from the membrane) (Eq. 1 in Chart 1) with the assumption of a 100% reaction yield, which means that all of the anchors contained an attached ligand [33][34][35][36][37][38][39][40][41][42][43][44][45][46][47] . mAb: monoclonal antibody, r.t.: room temperature, n.d.; no data.…”

Section: Methodsmentioning

confidence: 99%

“…33) Most literature reports do not provide these two values or provide sufficiently accurate values to permit the surface topologies to be evaluated. Instead, bioactivities are evaluated with reference to differences in the composition of liposomal lipids [33][34][35][36][37][38][39][40][41][42][43][44][45][46][47] (Table 1). The affinity of a liposome toward binding and its pharmacokinetics are related to its surface topology, 29,30,[48][49][50] which is defined as the ratio of the density of the surface ligand and PEG that are located on the liposomal surface.…”

Section: -9)mentioning

confidence: 99%

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Topology of Surface Ligands on Liposomes: Characterization Based on the Terms, Incorporation Ratio, Surface Anchor Density, and Reaction Yield

Lee

Sato

Hyodo

et al. 2016

Biological & Pharmaceutical Bulletin

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The surface topology of ligands on liposomes is an important factor in active targeting in drug delivery systems. Accurately evaluating the density of anchors and bioactive functional ligands on a liposomal surface is critical for ensuring the efficient delivery of liposomes. For evaluating surface ligand density, it is necessary to clarify that on the ligand-modified liposomal surfaces, some anchors are attached to ligands but some are not. To distinguish between these situations, a key parameter, surface anchor density, was introduced to specify amount of total anchors on the liposomal surface. Second, the parameter reaction yield was introduced to identify the amount of ligand-attached anchors among total anchors, since the conjugation efficiency is not always the same nor 100%. Combining these independent parameters, we derived: incorporation ratio surface anchor density reaction yield. The term incorporation ratio defines the surface ligand density. Since the surface anchor density represents the density of polyethylene glycol (PEG) on the surfaces in most cases, it also determines liposomal function. It is possible to accurately characterize various PEG and ligand densities and to define the surface topologies. In conclusion, this quantitative methodology can standardize the liposome preparation process and qualify the modified liposomal surfaces.Key words active targeting; surface ligand density; incorporation ratio; surface anchor density; reaction yield; surface topology Nanotechnology has the capability to deliver drugs to specific cell types, but it is important to maximize therapeutic effects and minimize unexpected adverse effects, for this technology to be effectively used.1-4) In using nanoparticles for specific drug delivery, they need to have both a stealth function to prevent non-specific recognition by the reticuloendothelial system (RES), 5,6) and an active targeting function to allow them to bind to the specific cell type of interest. 7-9)Polyethylene glycol (PEG) modification is frequently used to confer a stealth function to nanoparticles. 5,6,10) For active targeting, specific ligands for bioactive molecules such as nucleic acids, peptides, proteins, and antibodies are attached to the surface of nanoparticles via a PEG spacer.11) Many attempts have been made to develop various types of specific ligands for use in active targeting, which is critical for targeting non-fenestrated tissues such as the brain, lung and related tissues. [12][13][14] The procedures used to prepare ligands that are used in modifying nanoparticles are relatively complicated because this increases the number of steps in the preparation process that can lead to conditions where ligand molecules can be unstable.15) Therefore, optimal reaction (modification) conditions and methods for quantifying the density of the attached ligand that is attached to the nanoparticles need to be evaluated precisely.Problems are usually encountered in evaluating both the density of the liposomal ligands and the PEG, since a variety of m...

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Section: Methodsmentioning

confidence: 99%

Section: -9)mentioning

confidence: 99%

Topology of Surface Ligands on Liposomes: Characterization Based on the Terms, Incorporation Ratio, Surface Anchor Density, and Reaction Yield

Lee

Sato

Hyodo

et al. 2016

Biological & Pharmaceutical Bulletin

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“…Monoclonal antibodies were coupled to MPB-PE containing liposomes by a sulphhydryl-maleimide coupling method as described previously (Derksen et al, 1985). Briefly, free sulfhydryl groups were introduced in the antibody, using the heterobifunctional reagent SATA.…”

Section: Coupling Of Antibodies To Liposomesmentioning

confidence: 99%

Antiproliferative effect of immunoliposomes containing 5-fluorodeoxyuridine-dipalmitate on colon cancer cells

et al. 1999

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SummaryWe have investigated the antiproliferative action towards CC531 colon adenocarcinoma cells of target cell-specific immunoliposomes containing the amphiphilic dipalmitoyl derivative of 5-fluorodeoxyuridine (FUdR-dP). FUdR-dP incorporated in immunoliposomes caused a 13-fold stronger inhibition of CC531 cell growth in vitro, during a 72-h treatment, than FUdR-dP in liposomes without antibody, demonstrating that the prodrug is efficiently hydrolysed to yield the active drug, FUdR, intracellularly. The intracellular release of active FUdR was confirmed by determining the fate of 3 H-labelled immunoliposomal FUdR-dP. Treatments shorter than 72 h with FUdR-dP in immunoliposomes resulted in anti-tumour activities comparable to, or even higher than, that of free FUdR. The shorter treatments reflect more closely the in vivo situation and illustrate the potential advantage of the use of immunoliposomes over non-targeted liposomal FUdR-dP or free FUdR. Association of tumour cell-specific immunoliposomes with CC531 cells was up to tenfold higher than that of liposomes without antibody or with irrelevant IgG coupled, demonstrating a specific interaction between liposomes and target cells which causes an efficient intracellular delivery of the drug. Since biochemical evidence indicates a lack of internalization or degradation of the liposomes as such, we postulate that entry of the drug most likely involves the direct transfer of the prodrug from the immunoliposome to the cell membrane during its antigen-specific interaction with the cells, followed by hydrolysis of FUdR-dP leading to relatively high intracellular FUdR-levels. In conclusion, we describe a targeted liposomal formulation for the anticancer drug FUdR, which is able to deliver the active drug to colon carcinoma cells with high efficiency, without the need for the cells to internalize the liposomes as such.

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“…The Ab modification with SATA was performed following the protocols as described [16,21] The Ab dissolved in 50 mM phosphate buffer/1 mM EDTA (pH 7 5) at concentrations of 4-10 mg/ml (0 25-1 10 -9 M) were reacted during 60 man at 25 °C under mtrogen and gentle stlrnng with SATA (150 mM) whtch was dissolved in a mlmmal volume of dlmethylformanude The molar ratios of Ab to SATA were 1 24, 1 12 and 1 6 Unreacted SATA was removed by dialysis at 4°C against 1000 volumes of the same buffer over 24 h…”

Section: Modlfzcatton Of Anttbodtes With Sa Tamentioning

confidence: 99%

“…The covalent couphng of Abs, Ab fragments or peptldes to hgands winch are located on the surface of hposomes can be achieved by numerous chemical methods [3][4][5][6][7][8][9][10][11][12][13][14][15][16] Bffunctmnal hgands have become useful tools for the couphng of proteins to hposomes Among a vanety of such molecules, N-succlmmldyl-3-(2-pyndyldlthlo)propinnate (SPDP) is currently one of the most used linkers, although ~t has a number of disadvantages hke its suscept~inhty to traces of reducing agents and the short half-hfe of the reactwe sulfhydryl groups [16] N-Succxnlrmdyl-S-acetyltinoacetate (SATA) is an alternatwe couphng agent [16] In tins work we present the results that were obtained by the direct comparison of Ab couphng methods using SPDP and SATA as hnkers to hposomes which contmned N4-oleylcytosme arabmoslde (NOAC), a hpopinhc denvatwe of arabmosylcytoslne, as cytostatic prodrug [17,18] In particular, the couphng of the two Abs, B8-24 3 specific for the mouse MHC class I H-2k b surface antigen on EL4 mouse T lymphoma cells and IB16-6, specific for a surface antigen on B16 mouse melanoma cells, to small umlamellar drug carrying hposomes was lnvest~gated In addmon, the new highly fluorescent hpopinhc dye N,N'-b~s(1-hexylheptyl)-3,4 9,10-perylenebls(dlcarboxlnude) (BHPD) was used as fluorescence label for the hposomes…”

Section: Introductionmentioning

confidence: 99%

Comparative studies of the preparation of immunoliposomes with the use of two bifunctional coupling agents and investigation of in vitro immunoliposome-target cell binding by cytofluorometry and electron microscopy

Schwendener¹,

Trüb²,

Schott³

et al. 1990

Biochimica et Biophysica Acta (BBA) - Biomembranes

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The two coupling agents SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate) and SATA (N-succinimidyi-Sacetylthioacetate) were compared in their efficiency and feasibility to couple monocional antibodies (Abs) via thioether linkage to liposomes functionalized by various lipophilic maleimide compounds like N-(3-maleimidopropionyl)-N2-palmitoyi-L-lysine methyl ester (MP-PL), N-(3-maleimidopropionyl)phosphatidylethanolamide (MP-PE), N6-(6-maleimidocaproyl)-N2-palmitoyI-L-iysine methyl ester (EMC-PL), and N-(6-maleimidocaproyl)phosphatidylethanolamine (EMC-PE). The composition of the iiposomes was soy phosphatidylcholine (SPC), cholesterol, maleimide compounds and a-tocopherol (1:0.2:0.02:0.01, mol parts), plus N4-oleyicytosine arabinoside (NOAC) as cytostatic prodrug (0.2 mol parts) and a new, lipophilic and highly fluorescent dye N,N'-bis(1-hexylheptyl)-3,4:9,10-perylenebis(dicarboximide) (BHPD, 0.006 mol parts). From the maleimide derivatives MP-PL was the most effective in terms of preservation of the coupling activity in dependence of iiposome storage. The coupling of the monoclonal A B8-24.3 (mouse IgG2b, MHC class I, anti H-gk b) and 11316-6 (rat IgG2a, anti B16 mouse melanoma) to the drug carrying liposomes was more effective and easier to accomplish with SATA as compared to SPDP. Coupling rates of 60-65% were obtained with SATA at molar ratios of 12 SATA:I Ab:40 maleimide spacer groups on the surface of one liposome. The highest coupling rates with SPDP were obtained at the ratio of 24 SPDP: 1 Ab:40 liposomal maleimide groups, with an Ab binding efficiency of only 20-25%. The optimal in vitro binding conditions to specific target cells (EIA for BS-24.3-1iposomes and B16-F10 for IB16-6-1iposomes) were determined by cytofluorometric measurement of the iiposomal BHPD fluorescence with SATA linked Abs. Optimal immunoliposome binding to specific epitopes on the target cells was achieved with 1-2 Ab molecules coupled to one liposome, with immunoliposome concentrations of 20-130 nM and with a small incubation volume of 0.3-0.4 ml. The specificity of the binding of BS-24.3-1iposomes to EIA target cells was visualized by scanning electron microscopy. Antibody mediated endocytic uptake of immunoliposomes could be demonstrated by transmission electron microscopy.

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An improved method for the covalent coupling of proteins to liposomes

Cited by 88 publications

References 18 publications

Topology of Surface Ligands on Liposomes: Characterization Based on the Terms, Incorporation Ratio, Surface Anchor Density, and Reaction Yield

Topology of Surface Ligands on Liposomes: Characterization Based on the Terms, Incorporation Ratio, Surface Anchor Density, and Reaction Yield

Antiproliferative effect of immunoliposomes containing 5-fluorodeoxyuridine-dipalmitate on colon cancer cells

Comparative studies of the preparation of immunoliposomes with the use of two bifunctional coupling agents and investigation of in vitro immunoliposome-target cell binding by cytofluorometry and electron microscopy

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