Properties And Preparation Of Gsh-sensitive Polymeric Prodrug: 6-mercaptopurine-carboxymethyl Chitosan

Amphiphilic polymeric prodrugs have been applied in tumor therapy for improving targeting and bioavailability of drugs, reducing side effects of anti-tumor drugs, etc. Especially, these prodrugs consisting of hydrophilic and hydrophobic segments can form self-assemblies which can avoid the reticuloendothelial system (RES) because of the small nano-size. Covalently bonded disulfides can be formed spontaneously by autoxidation of sulfhydryls, primarily via oxidation upon exposure to air, which can reversibly be cleaved in the presence of reducing agents such as glutathione (GSH,2-10 mM) in the cells. It will be very promising in cancer treatment to combine the feature of disulfide bonds and amphiphilic polymeric prodrugs.In this research, we prepared 6-mercaptopurine-carboxymethyl chitosans (6-MP-CMC) with different content of 6-MP with the aid of a heterobifunctional cross-linker N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP). Disulfide bonds which were introduced into 6-MP-CMC in order to respond to the intracellular higher GSH levels. 6-MP-CMC was stable in the media containing micromolar GSH (simulated plasma conditions) but could fast release 6-MP at millimolar GSH levels (simulated intracellular conditions). Also,6-MP-CMC can form self-assembled nanoparticles in phosphate buffer solution (pH7.4). The nanoparticles can maintain the structure in the media containing micromolar GSH, but disassemble at millimolar GSH levels. The main contents and conclusions of study are as follows:(1) Carboxymethyl chitosan (CMC) was prepared by chitosan. 6-mercaptopurine-carboxymethyl chitosan was synthesized in follow:CMC was firstly modified with SPDP to obtain 3-(2-pyridyldithio) propionic acid-modified carboxymethyl chitosan (PDP-CMC). Then PDP-CMC was linked with 6-MP by a thiol-disulfide exchange reaction to form 6-mercaptopurine-carboxymethyl chitosan. The three products of different content of 6-MP were synthesized by controlling the ratio of raw materials. Intermediates and target compounds were characterized by Fourier Transform Infrared Spectroscopy (FTIR) and'H-Nuclear Magnetic Resonance Spectroscopy (1H-NMR). The degree of substitution (DS) of PDP and the content of 6-MP were measured by the elemental analysis method and ultraviolet spectrophotometry. The results showed that it was confirmed by the structure characterization that 6-mercaptopurine-carboxymethyl chitosan was successfully synthesized. The highest DS of PDP was 0.39, and the highest content of 6-MP was 19.0%.(2) The drug releasing behaviors in vitro of the amphiphilic polymeric prodrugs were studied. The release behaviors of the prodrugs in release media of various GSH concentrations (10 mM,2 mM,100μM or 2μM) and different pH (pH 7.4,5.0) were studied by the dialyzer method. And the release behaviors of 6-MP-CMC of different content of 6-MP were compared. The results showed that 6-MP release from 6-MP-CMC was not observed at 2μM GSH concentration and only less than 10% drug was released even at 100μM GSH concentration. By comparison, the obvious 6-MP release from 6-MP-CMC could be observed within an hour in the media containing 2 mM and 10 mM GSH. And the maximum cumulative release rate was 65.1% and 74.4% respectively. The buffer pH and the 6-MP content in 6-MP-CMC had obvious influences on the 6-MP release. At 10 mM GSH, the maximum cumulative release rate at pH 5 was higher than that at pH 7.4.(3) The 6-mercaptopurine-carboxymethyl chitosan (6-MP-CMC) nanoparticles were prepared by the probe ultrasonication method. The morphology of the nanoparticles was observed by transmission electron microscopy (TEM). The particle size of the nanoparticles was measured by dynamic light scattering (DLS). The results showed that the diameter of the nanoparticles determined by dynamic light scattering (DLS) was between 134.1-210.8 nm. The particles observed by Transmission electron microscopy (TEM) were spherical in shape and had the size about 100 nm. Also, the nanoparticles can maintain the structure in the media containing micromolar GSH, but disassemble at millimolar GSH levels. And the disassembled rate was increasing with the increasing of the GSH level.Therefore, the 6-mercaptopurine-carboxymethyl chitosan (6-MP-CMC) with the GSH-sensitive propery responded to the intracellular higher GSH levels, which can be expected as a GSH-sensitive nanoprodrug for cancer therapy.