Abstract
Stimuli-responsive polymers have received considerable attention recently owing to their potential applications in the biomedicine and biotechnology. In this study, a pH-responsive polymer, N-palmitoyl chitosan (NPCS), was prepared by conjugating a hydrophobic palmitoyl group onto the free amine groups of chitosan. The synthesized NPCS was proposed as in-situ forming hydrogels. In-situ forming hydrogels triggered by environmental stimuli have emerged as a promising injectable strategy for various biomedical applications. However, several drawbacks associated with temperature-stimulated hydrogels have been reported. By using a NPCS, this study developed a pH-triggered hydrogel system which showed a rapid nanostructure transformation within a narrow pH range of 6.5 to 7.0. NPCS in an aqueous environment was found to be a shear-thinning fluid and exhibited an instant recovery of its elastic properties after shear thinning, thereby making it an injectable material. Additionally, aqueous NPCS, an associating polyelectrolyte, can be transformed rapidly into hydrogel triggered simply by its environmental pH through a proper balance between charge repulsion and hydrophobic interaction. This in-situ hydrogel system was shown to be nontoxic. Subcutaneous injection of aqueous NPCS (pH 6.5) into a rat model resulted in a rapid formation of a massive hydrogel at the location of the injection. The implanted hydrogel was found to be degradable and was associated with an initial macrophage response, which decreased with time as the degradation proceeded. These results suggest the feasibility of using the developed NPCS hydrogel as an injectable drug/cell delivery system. This study also explored the use of NPCS to develop a nanoprobe. Stimuli-responsive nanodevices, particularly those capable of probing environmental changes, have been extensively studied for detecting biological phenomena intracellularly or extracellularly. In the study, a dual-emission nanoprobe that can sense changes of the environmental pH is reported, based on the concept of conformational changes of pH-responsive nanoparticles (NPs) with Förster resonance energy transfer (FRET) donor (Cy3) and acceptor (Cy5) attached to the free amine groups of NPCS. No apparent cytotoxicity was observed for the synthesized NPCS polymers with different degrees of substitution (DS). Cellular uptake of NPs was significantly enhanced when increasing the DS of NPCS used. Although the macropinocytosis and caveolae-mediated routes were involved in the internalization of NPCS NPs, the caveolae-mediated pathway played a more significant role. Experimental results indicated that NPCS NPs entered cells via caveolae and transiently localized to caveosomes before trafficking to the endosomal pathway. When Cy3-/Cy5-NPCS NP suspensions were excited, the fluorescent intensity of Cy3 band gradually decreased while that of the Cy5 band increased with a decreasing pH, i.e. an indication of FRET. The enhanced fluorescence found at low pH environments in the intracellular fluorescence ratiometric imaging indicated that FRET from Cy3 to Cy5 occurred. These results suggest that the developed Cy3-/Cy5-labeled NPCS NPs have a high FRET efficiency and can be used as a nanoprobe for detecting acidic organelles (lysosomes).