Characterization and in vitro methotrexate release from methotrexate/gelatin conjugates of opposite conjugate bond polarity.

Purpose: Our laboratory has previously prepared gelatin/ methotrexate (MTX) conjugates containing mixed conjugation sites and by-product crosslinking, both of which may alter conjugate effectiveness. In this study, we prepared and evaluated gelatin/MTX conjugates having specific conjugate bond sites and minimal by-product crosslinking.

Methods: Opposite polarity conjugates were produced by coupling gelatin having blocked amino groups with MTX (G-MTX) and by coupling MTX having blocked amino groups with gelatin (M-GEL) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl. Amino groups were blocked using citraconic anhydride and deblocked under acidic conditions. Gelatin and MTX contents were determined spectrophotometrically. The stability of each conjugate was determined by evaluating their in vitro release of MTX in isotonic buffer at pH 7.4 and 37 degrees C for 7 days.

Results: The G-MTX and M-GEL conjugates contained 21 and 1.2 mole MTX/mole gelatin and released 12 and 17% MTX by 7 days resulting in pseudo-first order release rate constants of 0.76x10(-3) and 1.0x10(-3) hr(-1), respectively. Alternate MTX species (< or =10%) were detected during the release study and were attributed to low molecular weight gelatin/MTX fragments and MTX polymers.

Conclusions: Gelatin/MTX conjugates having opposite conjugate bond polarities and minimal by-product crosslinking have been produced and slowly released MTX by hydrolytic cleavage indicating good stability for future cell culture studies.