The Study Of PH-responsive Nanoparticles Based On Hyaluronic Acid As Drug Delivery System

Hydrophobically modified hyaluronic acid-histidine (HH) conjugates were prepared with histidine (His) as the hydrophobic and pH-responsive groups. The self-assembled behavior of the amphiphiles in water solution was investigated. Adriamycin (ADR) was chosen as a model drug to fabricate the drug-loaded hyaluronic acid (HA) nanoparticles and the in vitro release of ADR from the nanoparticles was analyzed. Human breast-cancer cell line (MCF-7) was used to image the nanoparticles'responsiveness to the pH value of endosome and ADR's intracellular release from ADR-loaded HH nanoparticles. Cancer cell viability of the ADR-loaded HH nanoparticles was measured by MTT assay.The chemical conjugates of HA and His were synthesized by covalent attachment of His to HA with amide formation though EDC-mediated reaction. Various HH conjugates with three different degrees of substitution (DS) were prepared by changing the free mole ratios of His to HA (HH5, HH9, HH10). The pH-profile of HH9 conjugates was obtained by acid-base titration. The structure and DS of HH conjugates were identified by 1H NMR spectra. The self-assembled HH nanoparticles solution was obtained by sonication and the self-assembling properties were analyzed by the fluorescence probe test. The critical aggregation concentration (CAC) of HH nanoparticles decreased with the increasing of DS and the CAC determined for HH conjugates HH5, HH9 and HH10 were 0.098mg/ml,0.045mg/ml,0.039mg/ml respectively. The hydrophobicity of the core of nanoparticles was increased as the increasing of DS. The formation and pH-responsiveness of HH nanoparticles was characterized by dynamic laser light scattering. When the amphiphilic conjugates were dispersed in phosphate-buffered saline (PBS; pH 7.4) and sonicated, the conjugates formed self-assembled nanoparticles with the smallest mean diameters of 342nm, and the particle size was decreased with DS increased. However, when the pH of the medium was lowered to 6.0, the particle size was about 1 micrometer, out of nano-level, suggesting that these self-assembled nanoparticles dissociate in acidic pH due to loss of hydrophobicity caused by the protonation of imidazole groups. The transmission electron microscopy (TEM) and scanning electron micrograph (SEM) observation demonstrated the near spherical shape of self-aggregates with a integrated form.ADR was used as a model drug to prepare drug-loaded nanoparticles through hydrophobic interaction, and the feed weight ratio of ADR to HH conjugates was 10wt.%. The loading efficiency was 7.02% and the entrapment efficiency was 87.23%. When the drug loading HH9 nanoparticles were dispersed in phosphate-buffered saline (PBS; pH 7.4), the accumulative drug release amount was 27.98% in 48h. However, when the pH of the medium was lowered to 6.0, the accumulative drug release amount was 28.33% in 6h, and up to 50.97% in 48h. This result also showed the pH-responsiveness of the drug loading nanoparticles.The in vitro cytotoxicity of blank HH9 nanoparticles and cytostatic activity of ADR-loaded nanoparticles were analyzed by the MTT mothod. The endocytosis of ADR-HH9, fluorescent HH9 and ADR-HH9 nanoparticles and ADR's intracellular release were visualized by using fluorescence microscopy. The blank nanoparticles exhibited no cytotoxicity for tumor cells and the cell viability was higher than 95% within 3 days. ADR-loaded HH9 nanoparticles showed higher cytotoxicity versus free ADR at the drug concentration range of 1-10μg/ml. Visual evidence of three kinds of (ADR-HH9, FITC-HH9, FITC-ADR-HH9) nanoparticles's uptake by MCF-7cells was obtained with fluorescence microscopy.The internalized process exhibited high dependence of incubation time. ADR can release from ADR-HH9 nanoparticles into cytoplasm within four hours.