Theranostics 2018; 8(13):3474-3489. doi:10.7150/thno.23804

Research Paper

Drug-delivering-drug platform-mediated potent protein therapeutics via a non-endo-lysosomal route

Xiaofei Xin1, Chao Teng1, Xiaoqing Du1, Yaiqi Lv1, Qingqing Xiao1, Yubing Wu1, Wei He1,✉, Lifang Yin1,2,✉

1. Department of Pharmaceutics, School of Pharmacy, China Pharmaceutical University, Nanjing, 210009, PR China.
2. Key Laboratory of Druggability of Biopharmaceutics, China Pharmaceutical University, Nanjing, 210009, PR China.


Protein therapeutics is playing an increasingly critical role in treatment of human diseases. However, current vectors are captured by the digestive endo-lysosomal system, which results in an extremely low fraction (<2%) of protein being released in the cytoplasm. This paper reports a drug-delivering-drug platform (HA-PNPplex, 200 nm) for potent intracellular delivery of protein and combined treatment of cancer.

Methods: The platform was prepared by loading functional protein on pure drug nanoparticles (PNPs) followed by hyaluronic acid coating and was characterized by dynamic light scattering, transmission electron microscopy, and gel electrophoresis. In vitro, cellular uptake, trafficking, and cytotoxicity were evaluated by flow cytometry and confocal laser microscopy. Protein expression was assayed by western blot. In vivo, blood circulation and biodistribution were studied using a fluorescence imaging system, antitumor efficacy was assessed in a caspase 3-deficient tumor model, and biocompatibility was determined by comparison of hemolytic activity and proinflammatory cytokines and tissue histology.

Results: HA-PNPplex delivered the functional protein, caspase 3, to cells via bypassing endo-lysosomes and raised the caspase-3 level 6.5-fold in caspase 3-deficient cells. Promoted tumor accumulation (1.5-fold) and penetration were exhibited, demonstrating a high tumor-targeting ability of HA-PNPplex. HA-PNPplex rendered a 7-fold increase in caspase 3 in tumor and allowed for a 100% tumor growth inhibition and >60% apoptosis, implying significant antitumor activities.

Conclusions: This platform gains cellular entry without entrapment in the endo-lysosomes and enables efficient intracellular protein delivery and resultant profound cancer treatment. This platform, with extremely high drug-loading, is a valuable platform for combined cancer therapy with small-molecule drugs and proteins. More importantly, this work offers a robust and safe approach for protein therapeutics and intracellular delivery of other functional peptides, as well as gene-based therapy.

Keywords: rod-like pure drug particles, caspase 3, caveolae-mediated endocytosis, intracellular protein delivery, endo-lysosomes

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How to cite this article:
Xin X, Teng C, Du X, Lv Y, Xiao Q, Wu Y, He W, Yin L. Drug-delivering-drug platform-mediated potent protein therapeutics via a non-endo-lysosomal route. Theranostics 2018; 8(13):3474-3489. doi:10.7150/thno.23804. Available from