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.
Subject(s)
Antineoplastic Agents/pharmacokinetics , Biological Products/pharmacokinetics , Breast Neoplasms/drug therapy , Caspase 3/pharmacokinetics , Drug Delivery Systems , Nanoparticles/metabolism , Animals , Antineoplastic Agents/administration & dosage , Biological Availability , Biological Products/administration & dosage , Blotting, Western , Caspase 3/administration & dosage , Cell Line, Tumor , Cell Survival/drug effects , Disease Models, Animal , Flow Cytometry , Humans , Mice, Inbred BALB C , Microscopy, Confocal , Models, Biological , Optical Imaging , Rats , Treatment OutcomeSubject(s)
Antibodies, Monoclonal/pharmacokinetics , Caspase 3/pharmacokinetics , Chitosan/pharmacokinetics , Drug Delivery Systems/methods , Nanoparticles/therapeutic use , Receptors, Transferrin/immunology , Animals , Antibodies, Monoclonal/metabolism , Apoptosis/drug effects , Apoptosis/physiology , Blood-Brain Barrier/drug effects , Blood-Brain Barrier/metabolism , Caspase 3/metabolism , Cerebral Arteries/drug effects , Cerebral Arteries/metabolism , Chitosan/metabolism , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Delivery Systems/trends , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Humans , Hypoxia-Ischemia, Brain/drug therapy , Hypoxia-Ischemia, Brain/physiopathology , Mice , Nanoparticles/chemistry , Nerve Degeneration/drug therapy , Nerve Degeneration/physiopathology , Nerve Degeneration/prevention & control , Treatment OutcomeABSTRACT
Target proteins can be functionally encapsulated using a cocoon-like polymeric nanocapsule formed by interfacial polymerization. The nanocapsule is cross-linked by peptides that can be proteolyzed by proteases upon which the protein cargo is released. The protease-mediated degradation process can be controlled in a spatiotemporal fashion through modification of the peptide cross-linker with photolabile moieties. We demonstrate the utility of this approach through the cytoplasmic delivery of the apoptosis inducing caspase-3 to cancer cells.
