Chao-Sheng Chen1, Clyde M Ofner. 1. Department of Pharmaceutical Sciences, Philadelphia College of Pharmacy, University of the Sciences in Philadelphia, 600 South 43rd Street, Philadelphia, Pennsylvania 19104, USA.
Abstract
PURPOSE: Gelatin-methotrexate conjugates (G-MTX) with known molecular weight (MW), drug load, and charge were prepared and evaluated for growth inhibition on leukemia cells. METHODS: Gelatin (34 to 171 kDa) was reacted with a carbodiimide to prepare G-MTX with high (G-MTX-H) and low (G-MTX-L) drug loads. Cationic conjugates were prepared by ethylenediamine modification. MTX:gelatin molar ratios were determined spectrophotometrically. Isoelectric focusing electrophoresis (IEF) and turbidity were used to measure isoelectric points (IEP). Growth inhibition profiles and IC50 values were determined on HL-60 cells using a modified MTT assay. RESULTS: IC50 values of anionic G-MTX-L (drug loads 0.5:1 to 2.2:1) increased linearly from 46 to 180 nM with MW. But, IC50 values for anionic G-MTX-H (drug loads 7.4:1 to 25:1) showed little, if any, MW dependence and were about two times higher. IC50 values for cationic G-MTX-L ranged from 770 to 2,900 nM and the relationship with MW was non-linear. CONCLUSIONS: The growth inhibition ranking was MTX>anionic G-MTX-L>anionic G-MTX-H>cationic G-MTX-L. High drug load may hinder lysosomal enzyme degradation and drug release and contribute to suppression of the MW effect observed with G-MTX-L. A mechanism change is suggested as the cationic conjugates increase to the highest MW.
PURPOSE: Gelatin-methotrexate conjugates (G-MTX) with known molecular weight (MW), drug load, and charge were prepared and evaluated for growth inhibition on leukemia cells. METHODS: Gelatin (34 to 171 kDa) was reacted with a carbodiimide to prepare G-MTX with high (G-MTX-H) and low (G-MTX-L) drug loads. Cationic conjugates were prepared by ethylenediamine modification. MTX:gelatin molar ratios were determined spectrophotometrically. Isoelectric focusing electrophoresis (IEF) and turbidity were used to measure isoelectric points (IEP). Growth inhibition profiles and IC50 values were determined on HL-60 cells using a modified MTT assay. RESULTS: IC50 values of anionic G-MTX-L (drug loads 0.5:1 to 2.2:1) increased linearly from 46 to 180 nM with MW. But, IC50 values for anionic G-MTX-H (drug loads 7.4:1 to 25:1) showed little, if any, MW dependence and were about two times higher. IC50 values for cationic G-MTX-L ranged from 770 to 2,900 nM and the relationship with MW was non-linear. CONCLUSIONS: The growth inhibition ranking was MTX>anionic G-MTX-L>anionic G-MTX-H>cationic G-MTX-L. High drug load may hinder lysosomal enzyme degradation and drug release and contribute to suppression of the MW effect observed with G-MTX-L. A mechanism change is suggested as the cationic conjugates increase to the highest MW.