Abstract: In this study a reduction-responsive nanoparticles (NPs) modified with hyaluronic acid (HA) was prepared for the co-delivery of doxorubicin (DOX) and siRNA and then evaluated as a lung cancer targeting delivery system in vitro. The amphiphilic polymer of poly-L-lysine-lipoic acid (PLA) based on poly-L-lysine (PLL) with lipoic acid (LA) was synthesized via amidation reaction and characterized by ¹H NMR. The DOX loaded PLA NPs were prepared via dialysis method, and siRNA was loaded via electrostatic attraction to prepare the co-delivery NPs system (PLA/DOX-siRNA-NPs). Then PLA/DOX-siRNA-NPs were coated with HA to obtain HA-PLA/DOX-siRNA-NPs. The tumor microenvironment-responsive properties under different pH or reduction condition of HA-PLA/DOX-siRNA-NPs were evaluated by investigating the particle size and zeta potential. Cellular uptake of HA-PLA/DOX-siRNA^FAM-NPs by A549 cells and endosomal escape of siRNA were studied using confocal laser scanning microscope (CLSM). ¹H NMR spectrum demonstrated that PLA was successfully synthesized with LA grafting rate of 25.1%. The encapsulation efficiency (EE) and drug loading (DL) of HA-PLA/DOX-NPs was (86.93±8.91)% and (4.17±0.68)%, respectively, and siRNA was loaded at an N/P of 6:1 in carrier. HA-PLA/DOX-siRNA-NPs exhibited a suitable size of (167.3±9.9) nm and negative charge of (-15.5±1.4) mV with the optimal ratio of PLA and HA of 1:3. Additionally, the zeta potential of HA-PLA/DOX-siRNA-NPs significantly increased with charge reversal from negative to positive after the treatment with HAase, and the particle size of HA-PLA/DOX-siRNA-NPs changed significantly under the condition of 10 mmol·L^-1 glutathione (GSH). The release profiles in vitro demonstrated that HA-PLA/DOX-NPs exhibited a maintained release behavior at pH 7.4 and the adding of GSH (10 mmol·L^-1) led to rapid release of DOX from NPs. In vitro cellular uptake and subcellular distribution study demonstrated that themodification of HA enhanced the affinity of NPs to A549 cells and targeting ability, and the cellular uptake of HA-PLA/DOX-siRNA^FAM-NPs significantly increased after the treatment with HAase. It was observed that HA-PLA/DOX-siRNA^FAM-NPs could escape from endo-lysosomes followed by sharp payloads release to their relative targets. All these results demonstrated that the co-loaded NPs have a high entrapment efficiency of DOX and siRNA. And they also exhibited an active tumor targeting efficiency and tumor microenvironment-responsive properties, which were beneficial to cellular uptake and intracellular release of DOX and siRNA. In conclusion, these reduction-responsive NPs modified with HA have great potential as co-delivery systems for antitumor agents and siRNA.