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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.

SUBMITTER: Xin X

PROVIDER: S-EPMC6037042 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Drug-delivering-drug platform-mediated potent protein therapeutics <i>via</i> a non-endo-lysosomal route.

Xin Xiaofei X Teng Chao C Du Xiaoqing X Lv Yaiqi Y Xiao Qingqing Q Wu Yubing Y He Wei W Yin Lifang L

Theranostics 20180606 13

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. <b>Methods:</b> The platform was prepared by loading functional protein on pure drug ...[more]

PMID: 30026860

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Project description:Recessive loss-of-function mutations in ATP13A2 (PARK9) are associated with a spectrum of neurodegenerative disorders, including Parkinson's disease (PD). We recently revealed that the late endo-lysosomal transporter ATP13A2 pumps polyamines like spermine into the cytosol, whereas ATP13A2 dysfunction causes lysosomal polyamine accumulation and rupture. Here, we investigate how ATP13A2 provides protection against mitochondrial toxins such as rotenone, an environmental PD risk factor. Rotenone promoted mitochondrial-generated superoxide (MitoROS), which was exacerbated by ATP13A2 deficiency in SH-SY5Y cells and patient-derived fibroblasts, disturbing mitochondrial functionality and inducing toxicity and cell death. Moreover, ATP13A2 knockdown induced an ATF4-CHOP-dependent stress response following rotenone exposure. MitoROS and ATF4-CHOP were blocked by MitoTEMPO, a mitochondrial antioxidant, suggesting that the impact of ATP13A2 on MitoROS may relate to the antioxidant properties of spermine. Pharmacological inhibition of intracellular polyamine synthesis with α-difluoromethylornithine (DFMO) also increased MitoROS and ATF4 when ATP13A2 was deficient. The polyamine transport activity of ATP13A2 was required for lowering rotenone/DFMO-induced MitoROS, whereas exogenous spermine quenched rotenone-induced MitoROS via ATP13A2. Interestingly, fluorescently labeled spermine uptake in the mitochondria dropped as a consequence of ATP13A2 transport deficiency. Our cellular observations were recapitulated in vivo, in a Caenorhabditis elegans strain deficient in the ATP13A2 ortholog catp-6 These animals exhibited a basal elevated MitoROS level, mitochondrial dysfunction, and enhanced stress response regulated by atfs-1, the C. elegans ortholog of ATF4, causing hypersensitivity to rotenone, which was reversible with MitoTEMPO. Together, our study reveals a conserved cell protective pathway that counters mitochondrial oxidative stress via ATP13A2-mediated lysosomal spermine export.

Dataset Information

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

Publications

Drug-delivering-drug platform-mediated potent protein therapeutics <i>via</i> a non-endo-lysosomal route.

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