Receptor dependent cellular uptake of synthetic low density lipoprotein by mammalian cells in serum-free tissue culture

Abstract

Low density lipoprotein (LDL) is a normal plasma component, which is of interest in a number of research areas such as hypercholesterolaemia, drug targeting in cancer chemotherapy and as a lipid supplement in tissue culture systems. Currently, however, it can only be obtained by extraction from fresh plasma samples, which yields only small quantities. Synthetic LDL (sLDL) has been prepared using readily available lipid components coupled with a synthetic amphiphatic peptide molecule containing the apoprotein B receptor sequence. sLDL was capable of supporting the growth of Chinese Hamster Ovary (CHO) and fibroblast cells in serum-free culture media in a cholesterol-dependent manner that was related to the presence of the receptor peptide molecule. sLDL could be fluorescently labelled with 3,3'-dioctadecyloxalocarbocyanine perchlorate (DiO), and once labelled was assimilated by CHO and fibroblast cells in a time- and temperature-dependent manner that was dependent upon the presence of the receptor peptide. In addition, assimilation was reduced by an excess of unlabelled native LDL. The results indicated that the interaction of sLDL with CHO and fibroblast cells occurred via a receptor dependent system, most likely the LDL cellular receptor. sLDL is therefore a useful, easily obtained substitute for native LDL with potential applications in the areas of drug targeting to cells and serum-free tissue culture systems.

Uptake of synthetic Low Density Lipoprotein by leukemic stem cells — a potential stem cell targeted drug delivery strategy

“…The physicochemical and analysis results for these materials are similar to those already reported in the literature [18]. The larger diameter of DiO containing sLDL with respect to DiO free samples has been previously observed [18,19] and does not affect cellular uptake.…”

“…sLDL preparation sLDL was prepared using a solvent evaporation method previously reported [18,19], a mixture of phosphatidylcholine, triolein, cholesterol and cholesteryl oleate in the molar ratio 3:2:1:1 was dissolved in dichloromethane and the synthetic peptide (see Materials) added at a molar concentration of 0.03 per mole of total cholesterol. The dichloromethane was then added to a solution of sodium oleate in water for injection and homogenised using an icecooled EmusiFlex-C5 microfluidiser (Avestin, Canada) at pressures up to 30,000psi until the desired particle size was obtained.…”

“…In order to avoid these issues, in this study we have employed synthetic LDL (sLDL) prepared using readily available lipid components coupled with a synthetic amphiphatic peptide molecule containing the Apoprotein B (Apo-B) receptor sequence [18,19]. sLDL can be routinely produced [18] that is physicochemically [20] and biologically [21] equivalent to native LDL.…”

Lipoprotein-Related and Apolipoprotein-Mediated Delivery Systems for Drug Targeting and Imaging

“…The limitation here is that apo-B100 is difficult to separate from plasma-LDL and due to its amphipathic nature, enormous size and structural sensitivity, it is even more difficult to restore apoB-100 to its native form on the surface of microemulsion particles. More recent studies have shown the feasibility of synthesizing artificial LDL particles using lipid microemulsions in combination with synthetic peptides, whereas the peptide sequence corresponds to the amino acid sequence of the LDL receptor binding domain [32-34]. These LDL mimetic particles were actively taken up by cells via LDL receptor mediated endocytosis [34].…”

“…More recent studies have shown the feasibility of synthesizing artificial LDL particles using lipid microemulsions in combination with synthetic peptides, whereas the peptide sequence corresponds to the amino acid sequence of the LDL receptor binding domain [32-34]. These LDL mimetic particles were actively taken up by cells via LDL receptor mediated endocytosis [34]. To extend the protocol, Nikanjam et al [35, 36] have used the same peptide sequence but additionally linked to an 18 amino acid amphiphatic alpha helix that avidly binds to the surface of the lipid emulsion.…”