Autophagy promotes degradation of polyethyleneimine–alginate nanoparticles in endothelial progenitor cells

Polyethyleneimine (PEI)–alginate (Alg) nanoparticle (NP) is a safe and effective vector for delivery of siRNA or DNA. Recent studies suggest that autophagy is related to cytotoxicity of PEI NPs. However, contribution of autophagy to degradation of PEI–Alg NPs remains unknown. CD34+VEGFR-3+ endothelial progenitor cells isolated from rat bone marrow were treated with 25 kDa branched PEI modified by Alg. After treatment with the NPs, morphological changes and distribution of the NPs in the cells were examined with scanning and transmission electron microscopies. Cytotoxicity of the NPs was analyzed by reactive oxygen species (ROS) production, lactate dehydrogenase leakage and induction of apoptosis. The level of autophagy was assessed with expression of Beclin-1 and LC3 and formation of autophagic structures and amphisomes. Colocalization of LC3-positive puncta and the NPs was determined by LC3–GFP tracing. Cytotoxicity of PEI NPs was reduced greatly after modification with Alg. PEI–Alg NPs were distributed in mitochondria, rough endoplasmic reticula and nuclei as well as cytoplasm. After phagocytosis of the NPs, expression of Beclin-1 mRNA and LC3 protein was upregulated, and the number of LC3-positive puncta, autophagic structures and amphisomes increased significantly. The number of lysosomes also increased obviously. There were LC3-positive puncta in nuclei, and some puncta were colocalized with the NPs. These results demonstrate that the activated autophagy promotes degradation of PEI–Alg NPs via multiple pathways.

[1]  A. L. Le Faou,et al.  Drug delivery by polymeric nanoparticles induces autophagy in macrophages. , 2012, International journal of pharmaceutics.

[2]  Sangeeta Khare,et al.  Guidelines for the use and interpretation of assays formonitoring autophagy (3rd edition) , 2016 .

[3]  Lin-Ping Wu,et al.  High resolution respirometry analysis of polyethylenimine-mediated mitochondrial energy crisis and cellular stress: Mitochondrial proton leak and inhibition of the electron transport system. , 2013, Biochimica et biophysica acta.

[4]  K. Braeckmans,et al.  Lysosomal capturing of cytoplasmic injected nanoparticles by autophagy: an additional barrier to non viral gene delivery. , 2014, Journal of controlled release : official journal of the Controlled Release Society.

[5]  M. Jan,et al.  Autophagy-related gene expression analysis of wild-type and atg5 gene knockout mouse embryonic fibroblast cells treated with polyethylenimine. , 2014, Molecular pharmaceutics.

[6]  Yu-zhen Tan,et al.  Autophagy in endothelial progenitor cells is cytoprotective in hypoxic conditions. , 2013, American journal of physiology. Cell physiology.

[7]  Wolfgang Kreyling,et al.  Polyethylenimines for RNAi-mediated gene targeting in vivo and siRNA delivery to the lung. , 2011, European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V.

[8]  S. Berger,et al.  Autophagy mediates degradation of nuclear lamina , 2015, Nature.

[9]  Robert Clarke,et al.  Guidelines for the use and interpretation of assays for monitoring autophagy , 2012 .

[10]  J. Lippincott-Schwartz,et al.  Deacetylation of nuclear LC3 drives autophagy initiation under starvation. , 2015, Molecular cell.

[11]  R. Langer,et al.  Exploring polyethylenimine‐mediated DNA transfection and the proton sponge hypothesis , 2005, The journal of gene medicine.

[12]  George A. Calin,et al.  RNA interference in the clinic: challenges and future directions , 2011, Nature Reviews Cancer.

[13]  S. Ryter,et al.  Autophagy in human health and disease. , 2013, The New England journal of medicine.

[14]  John J. Rossi,et al.  Strategies for silencing human disease using RNA interference , 2007, Nature Reviews Genetics.

[15]  M. Lagunoff,et al.  Isolation and characterization of circulating lymphatic endothelial colony forming cells. , 2016, Experimental cell research.

[16]  S. Nimesh.  Polyethylenimine as a promising vector for targeted siRNA delivery. , 2012, Current clinical pharmacology.

[17]  Kristen N. Duthie,et al.  Wide varieties of cationic nanoparticles induce defects in supported lipid bilayers. , 2008, Nano letters.

[18]  L. Hsu,et al.  Protective role of autophagy in branched polyethylenimine (25K)- and poly(L-lysine) (30-70K)-induced cell death. , 2012, European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences.

[19]  D. Rubinsztein,et al.  Cytoprotective roles for autophagy , 2010, Current opinion in cell biology.

[20]  D. Green,et al.  Molecular characterization of LC3-associated phagocytosis reveals distinct roles for Rubicon, NOX2 and autophagy proteins , 2015, Nature Cell Biology.

[21]  K. Jensen,et al.  In vitro and ex vivo strategies for intracellular delivery , 2016, Nature.

[22]  D. Dykxhoorn,et al.  Breaking down the barriers: siRNA delivery and endosome escape , 2010, Journal of Cell Science.

[23]  Smadar Cohen,et al.  Alginate biomaterial for the treatment of myocardial infarction: Progress, translational strategies, and clinical outlook: From ocean algae to patient bedside. , 2016, Advanced drug delivery reviews.

[24]  U. Himmelreich,et al.  Standard Cellular Testing Conditions Generate an Exaggerated Nanoparticle Cytotoxicity Profile. , 2017, Chemical research in toxicology.

[25]  Guo-Dong Wang,et al.  Inhibition of Lymphangiogenesis of Endothelial Progenitor Cells with VEGFR-3 siRNA Delivered with PEI-alginate Nanoparticles , 2014, International journal of biological sciences.

[26]  I. Nonaka,et al.  Autophagic degradation of nuclear components in mammalian cells , 2009, Autophagy.

[27]  Seungpyo Hong,et al.  Interaction of polycationic polymers with supported lipid bilayers and cells: nanoscale hole formation and enhanced membrane permeability. , 2006, Bioconjugate chemistry.

[28]  Takeshi Noda,et al.  LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing , 2000, The EMBO journal.

[29]  Yu-zhen Tan,et al.  CD34+VEGFR-3+ progenitor cells have a potential to differentiate towards lymphatic endothelial cells , 2014, Journal of cellular and molecular medicine.

[30]  Ying Yu,et al.  Cytoprotective effect of autophagy on phagocytosis of apoptotic cells by macrophages. , 2016, Experimental cell research.

[31]  Richard Hoogenboom,et al.  Cytotoxicity of polycations: Relationship of molecular weight and the hydrolytic theory of the mechanism of toxicity. , 2017, International journal of pharmaceutics.

[32]  J. Carpenter,et al.  Nuclear LC3-positive puncta in stressed cells do not represent autophagosomes. , 2014, BioTechniques.

[33]  H. Hibshoosh,et al.  Induction of autophagy and inhibition of tumorigenesis by beclin 1 , 1999, Nature.

[34]  S Moein Moghimi,et al.  A two-stage poly(ethylenimine)-mediated cytotoxicity: implications for gene transfer/therapy. , 2005, Molecular therapy : the journal of the American Society of Gene Therapy.

[35]  Kevin Braeckmans,et al.  Exploiting intrinsic nanoparticle toxicity: the pros and cons of nanoparticle-induced autophagy in biomedical research. , 2014, Chemical reviews.

[36]  T. Andresen,et al.  Polycation cytotoxicity: a delicate matter for nucleic acid therapy-focus on polyethylenimine , 2010 .

[37]  A. Pathak,et al.  PEI-alginate nanocomposites: efficient non-viral vectors for nucleic acids. , 2010, International journal of pharmaceutics.

[38]  A. Kenworthy,et al.  Nucleocytoplasmic Distribution and Dynamics of the Autophagosome Marker EGFP-LC3 , 2010, PloS one.