Nanoconjugates are a new class of therapeutic compounds, which may lead to a new therapeutic quality due to the combination of drugs already used in therapy, as well as new innovative substances with high molecular carriers. Synthesis of drug-carrier hybrid systems is one of the ways to implement the “magic bullet” concept propounded at the beginning of the 20th century by Paul Ehrlich, and which was aimed at achieving the selective deposition and release of therapeutic substances in target tissues.

As a model anticancer drug, we chose methotrexate (MTX) – one of the oldest antifolate drugs widely used in the treatment of cancer, rheumatoid arthritis and other diseases. In the present contribution the convenient method regarding quantification of MTX release from selected drug-carrier systems in human plasma are presented. The drug release study was investigated at 37°C in human plasma (which contains enzymes like amylases) and mineral buffers (without enzymes). The method was based on size exclusion chromatography and UV-VIS detection at the wavelength of 372 nm. Conjugates were dissolved in final solutions to a final concentration of 0.8026 mM (MTX equiv.). At selected time intervals, each reaction solution was analyzed for unbound MTX. The observed release half-time was calculated for MTX using the pseudo-first-order kinetics equation. For the method basic parameters such as linearity, range of the method, limit of detection (LOD) and limit of quantification (LOQ), reproducibility and  recovery were validated.

Presented method had a linear character in the range of 2.006 – 802.6µM. LOD of the method was 0.4414 µM, and LOQ was 1.471 µM. Our drug release studies suggest that MTX may be cleaved from the conjugates both via chemical and enzymatic hydrolysis. Stability of the conjugates (half life time) in plasma is much short as the half life time in phosphate buffer at an identical pH. The release results of ester-linked conjugates indicate that a specific base-catalysis was involved in the hydrolysis of the ester conjugate, which is usually observed in weak alkaline solution. The conjugates differing in terms of structure demonstrate significantly different drug release characteristics.

This project was supported by the National Science Centre, Poland (N N302 098434)

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