ABSTRACT
Details of intracellular pathways of cytotoxicity remain unclear for doxorubicin conjugates being studied to treat breast cancer tumours. A high molecular weight gelatine-doxorubicin conjugate was investigated with an emphasis on lysosome participation. The conjugate was synthesised and characterised. Cell uptake and cellular localisation in MCF-7 and triple negative breast cancer (TNBC) MDA-MB-231 cells were determined with fluorescence microscopy. Nuclear content of released DOX was determined by UHPLC. Cytotoxicity was determined by the MTT assay. Lysosome membrane permeabilization (LMP) was followed by lysosomal release of fluorescently labelled dextran. After incubation at an equivalent 10 µM DOX, conjugate lysosome accumulation was substantial in both cell lines by 24 h, at which time the conjugate cytotoxic effect was first observed. By 48 h, the conjugate was nearly fourfold more toxic in TNBC than in MCF-7 cells. The MCF-7 nucleus drug content from conjugate released DOX was small but confirmed intra-lysosomal drug release. The conjugate induced LMP in 100% of TNBC cells but LMP was virtually absent in MCF-7 cells. These results suggest that the conjugate induces cytotoxicity by a lysosomal pathway in MDA-MB-231 cells and has potential for treatment of TNBC tumours. Support: NIH/NCI R15CA135421, the Agnes Varis Trust for Women's Health.
Subject(s)
Biological Transport/physiology , Doxorubicin/metabolism , Doxorubicin/pharmacology , Gelatin/metabolism , Gelatin/pharmacology , Triple Negative Breast Neoplasms/drug therapy , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Movement/drug effects , Cell Survival/drug effects , Drug Liberation/drug effects , Female , Humans , Lysosomes/chemistry , MCF-7 Cells , Triple Negative Breast Neoplasms/metabolismABSTRACT
The water-soluble carbodiimide, 1-ethyl-3-(3-(dimethylaminopropyl)-carbodiimide (EDC) is widely used in protein chemistry. We used EDC-induced gelatin cross-linking as a model for amide bond formation to resolve reaction ambiguities with common variables of buffers, gelatin concentration, and pH. Percentage changes in SEC high molecular weight peak areas were used to follow the reactions. Differences in reaction rate and extent were observed with four commonly used buffers, while differences in extent were observed for commonly used concentrations and pH. We also investigated an anhydride mechanism for aqueous EDC-induced amide bond formation that has received little attention since its proposal in 1995. Gelatin carboxyl groups had a synergistic role during the addition of hydrazine to corroborate the anhydride formation between carboxyl groups. EDC-induced degradation of gelatin was investigated using percentage changes in SEC low molecular weight peak areas. The degradation occurred in excess EDC at neutral to alkaline pH and was enhanced substantially when reacting amino groups were not available. A mechanism of EDC-induced gelatin degradation is proposed and designated the extended Khorana mechanism. This EDC side reaction has the potential to occur in peptides and proteins under similar conditions.
Subject(s)
Cross-Linking Reagents/chemistry , Ethyldimethylaminopropyl Carbodiimide/chemistry , Gelatin/chemistry , Animals , Buffers , Chromatography, High Pressure Liquid , Hydrazines/chemistry , Hydrogen-Ion Concentration , Ligands , Models, Chemical , Molecular Weight , Proteolysis , WaterABSTRACT
PURPOSE: To demonstrate the feasibility of a novel macromolecular delivery system for doxorubicin (DOX) which combines pH dependent DOX release with a high molecular weight and biodegradable gelatin carrier. METHODS: DOX was conjugated to gelatin using an acid labile hydrazone bond and a glycylglycine linker. The gelatin-doxorubicin conjugate (G-DOX) was evaluated for hydrazide and DOX content by spectrophotometry, molecular weight by HPLC-SEC, in vitro DOX release at various pH, and cell growth inhibition using EL4 mouse lymphoma and PC3 human prostate cells. RESULTS: G-DOX hydrazide and DOX content was 47% and 5-7%, respectively of theoretical gelatin carboxylic acid sites. During preparation of G-DOX, the molecular weight decreased to 22 kDa. DOX release was 48% in pH 4.8 phosphate buffer, 22% at pH 6.5, but 10% at pH 7.4. The G-DOX IC50 values in EL4 and PC3 cells were 0.26 µM and 0.77 µM, respectively; the latter value 3 times greater than that of free DOX. CONCLUSIONS: A 22 kDa macromolecular DOX conjugate containing 3.4-5.0% w/w DOX has been prepared. The pH dependent drug release in combination with a biodegradable gelatin carrier offer potential therapeutic advantages of enhanced tumor cell localization and reduced systemic toxicities of the drug.
