Properties And Preparation Of N-Lactosam-O-Histamine Acylated Carboxymethyl Chitosan Nanoparticles

Nanocarrier systems have been applied in tumor therapy for improving targeting and bioavailability of drugs, reducing side effects of anti-tumor drugs, etc. Especially, nanocarrier systems can avoid the reticuloendothelial system (RES) because of the small nano-size. The pH-sensitive nanoparticles occur swelling or collapsing upon external pH stimuli, and releasing drug. It will be very promising in cancer treatment to combine the feature of pH-sensitive nanoparticles with the weak acid environment and vascular permeability of cancer.In this paper, the pH-sensitive nanoparticles were prepared by introducing galatose ligand and imidazole group of histamine into O-carboxymethyl chitosan (O-CMCS) in order to respond to the physiological pH of tumor tissue. Thus, N-lactosam-O-histamine acylated carboxymethyl chitosan (LHCS) was synthesized as an amphiphilic polymer, which can form self-assembled nanoparticles in phosphate buffer solution (pH7.4). The nanoparticles can maintain the micelle structure at about pH 7.4, but disassemble and release drugs at about pH 6.5. The main contents and conclusions of study are as follows:(1) N-lactosam-O-carboxymethyl chitosan was synthesized by 0-carboxymethyl chitosan (O-CMCS) and lactose under the catalystis of KBH4 through the reductive amination. Then N-lactosam-O-histamine acylated carboxymethyl chitosan (LHCS) was synthesized by N-lactosam-O-carboxymethyl chitosan and histamine under the catalystis of N-(3-dimethyl amino-propyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccimide (NHS) through the acylation. The five products of different degree of substitution (DS) of histamine were synthesized by controlling the ratio of raw materials. Intermediates and target compounds were characterized by Fourier Transform Infrared Spectroscopy (FTIR) and 1H-Nuclear Magnetic Resonance Spectroscopy (1H-NMR). The DS of galactose ligand was measured by the potentiometric titration method, and the DS of histamine groups was measured by the elemental analysis method. The results showed that it was confirmed by the structure characterization that N-lactosam-O-histamine acylated carboxymethyl chitosan was successfully synthesized. The DS of galactose ligand was 59.44%, and the highest DS of histamine was 8.36. (2) The N-lactosam-O-histamine acylated carboxymethyl chitosan (LHCS) nanoparticles were prepared by the probe ultrasonication method. The critical micelle concentration (CMC) of LHCS was studied by the steady-state fluorescence probe 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 morphology of the nanoparticles was observed under TEM which showed an almost spherical shape at pH 7.4, but cannot be found at pH 6.5 due to disassembling of the nanoparticles. And the diameter of the nanoparticles was between 106.1-173.6 nm. It also indicated that the size of nanoparticles was decreasing with the increasing of the DS of histamine groups.(3) The encapsulation efficiency (EE) and the drug releasing behaviors in vitro of the nanoparticles were studied. The release behaviors of the drug loaded nanoparticles in release medium (pH 6.5,7.4) were studied by the dialyzer method. And the release behaviors of the nanoparticles of different DS of histamine groups of LHCS were compared. The results showed that the nanoparticles can maintain a lower drug releasing rate at physiological conditions in normal tissues (pH 7.4), but showed a significantly accelerated releasing rate at cancer conditions (pH 6.5) and the total cumulative releasing rates were increasing. It also confirmed that the nanoparticles of the higher DS of histamine groups were much more easily releasing drugs at pH 6.5 due to the depolymerization of the hydrophobic core in nanoparticles.Therefore, the N-lactosam-O-histamine acylated carboxymethyl chitosan (LHCS) nanoparticles with the pH-sensitive propery responded to the weak acid environment of cancer, which can be expected as a pH-sensitive nanocarrier for the anti-cancer drugs.