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

Abstract

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.

“Locked” cancer cells are more sensitive to chemotherapy

“…Although PTX‐Ns‐induced apoptosis with higher efficacy compared with free PTX, however, this nanomedicine exhibited lower cytotoxicity at high concentrations. Our previous reports also displayed similar results . Furthermore, combined use with other drugs, such as gene and protein, made this enhancement more profound .…”

“…Our previous reports also displayed similar results . Furthermore, combined use with other drugs, such as gene and protein, made this enhancement more profound . The previous report indicated that internalized PTX‐Ns started to dissolve at 10 hr postinternalization, and therefore demonstrating sustained PTX release over time in cells .…”

“…[54][55][56] Furthermore, combined use with other drugs, such as gene and protein, made this enhancement more profound. 54,55 The previous report indicated that internalized PTX-Ns started to dissolve at 10 hr postinternalization, and therefore demonstrating sustained PTX release over time in cells. 57 Consequently, the sustained release might help the chemotherapeutic agent loaded in nanomedicines kill the cancer cells.…”

“…The drug release from Taxol and PTX‐Ns was tested by a dialysis method under a shaker at 37°C as well. The detailed process and PTX assay were described in our previous report …”

Drug Nanorod-Mediated Intracellular Delivery of microRNA-101 for Self-sensitization via Autophagy Inhibition

“…In our previous reports, we established a drug-deliveringdrug (DDD) platform based on drug nanocrystals [19][20][21]. Via bypassing the digestive endo-lysosomes, the DDD platform maximizes intracellular delivery of biopharmaceuticals and enables the delivery of additional drugs to the target site as well.…”