A new strategy for intracellular delivery of enzyme using mesoporous silica nanoparticles: superoxide dismutase.

We developed mesoporous silica nanoparticle (MSN) as a multifunctional vehicle for enzyme delivery. Enhanced transmembrane delivery of a superoxide dismutase (SOD) enzyme embedded in MSN was demonstrated. Conjugation of the cell-penetrating peptide derived from the human immunodeficiency virus 1 (HIV) transactivator protein (TAT) to mesoporous silica nanoparticle is shown to be an effective way to enhance transmembrane delivery of nanoparticles for intracellular and molecular therapy. Cu,Zn-superoxide dismutase (SOD) is a key antioxidant enzyme that detoxifies intracellular reactive oxygen species, ROS, thereby protecting cells from oxidative damage. In this study, we fused a human Cu,Zn-SOD gene with TAT in a bacterial expression vector to produce a genetic in-frame His-tagged TAT-SOD fusion protein. The His-tagged TAT-SOD fusion protein was expressed in E. coli using IPTG induction and purified using FMSN-Ni-NTA. The purified TAT-SOD was conjugated to FITC-MSN forming FMSN-TAT-SOD. The effectiveness of FMSN-TAT-SOD as an agent against ROS was investigated, which included the level of ROS and apoptosis after free radicals induction and functional recovery after ROS damage. Confocal microscopy on live unfixed cells and flow cytometry analysis showed characteristic nonendosomal distribution of FMSN-TAT-SOD. Results suggested that FMSN-TAT-SOD may provide a strategy for the therapeutic delivery of antioxidant enzymes that protect cells from ROS damage.

[1]  Can Li.  Chiral Synthesis on Catalysts Immobilized in Microporous and Mesoporous Materials , 2004 .

[2]  Zongxi Li,et al.  Mesoporous silica nanoparticles in biomedical applications. , 2012, Chemical Society reviews.

[3]  H. Vogel,et al.  Reversible site-selective labeling of membrane proteins in live cells , 2004, Nature Biotechnology.

[4]  T. Yamasoba,et al.  Topical application of the antiapoptotic TAT-FNK protein prevents aminoglycoside-induced ototoxicity , 2011, Gene Therapy.

[5]  M. Fröba,et al.  Periodic mesoporous organosilicas (PMOs): past, present, and future. , 2006, Journal of nanoscience and nanotechnology.

[6]  Y. Sohn,et al.  Transduction of PTEN proteins using the tat domain modulates TGF-β1-mediated signaling pathways and transdifferentiation in subconjunctival fibroblasts. , 2012, Investigative ophthalmology & visual science.

[7]  M. Kiernan,et al.  Pathophysiology of neurodegeneration in familial amyotrophic lateral sclerosis. , 2009, Current molecular medicine.

[8]  Victor S-Y Lin,et al.  A mesoporous silica nanosphere-based carrier system with chemically removable CdS nanoparticle caps for stimuli-responsive controlled release of neurotransmitters and drug molecules. , 2003, Journal of the American Chemical Society.

[9]  Chung-Yuan Mou,et al.  Mesoporous silica nanoparticles as nanocarriers. , 2011, Chemical communications.

[10]  B. Rollins,et al.  Chronic expression of monocyte chemoattractant protein‐1 in the central nervous system causes delayed encephalopathy and impaired microglial function in mice , 2005, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[11]  A. Sun,et al.  Paraquat-Induced Free Radical Reaction in Mouse Brain Microsomes , 2004, Neurochemical Research.

[12]  J Barsoum,et al.  Tat-mediated delivery of heterologous proteins into cells. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[13]  X. Li,et al.  Paraquat induced activation of transcription factor AP-1 and apoptosis in PC12 cells , 1999, Journal of Neural Transmission.

[14]  Su Jin Lee,et al.  Differential regulation of inducible nitric oxide synthase and cyclooxygenase-2 expression by superoxide dismutase in lipopolysaccharide stimulated RAW 264.7 cells , 2009, Experimental & Molecular Medicine.

[15]  K. Griebenow,et al.  Stimulus-responsive controlled release system by covalent immobilization of an enzyme into mesoporous silica nanoparticles. , 2012, Bioconjugate chemistry.

[16]  Steven F Dowdy,et al.  Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis , 2004, Nature Medicine.

[17]  Chung-Yuan Mou,et al.  The effect of surface charge on the uptake and biological function of mesoporous silica nanoparticles in 3T3-L1 cells and human mesenchymal stem cells. , 2007, Biomaterials.

[18]  J. Ribbe,et al.  One-step purification of recombinant proteins with the 6xHis tag and Ni-NTA resin. , 1995, Molecular biotechnology.

[19]  Chung-Yuan Mou,et al.  Well-Ordered Mesoporous Silica Nanoparticles as Cell Markers , 2005 .

[20]  V. Bindokas,et al.  Superoxide production in rat hippocampal neurons: selective imaging with hydroethidine , 1996, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[21]  Magdalena Plebanski,et al.  Methods for nano-particle based vaccine formulation and evaluation of their immunogenicity. , 2006, Methods.

[22]  C. Shih,et al.  Identification of Three Mutations in the Cu,Zn‐Superoxide Dismutase (Cu,Zn‐SOD) Gene with Familial Amyotrophic Lateral Sclerosis: Transduction of Human Cu,Zn‐SOD into PC12 Cells by HIV‐1 TAT Protein Basic Domain , 2005, Annals of the New York Academy of Sciences.

[23]  Brian G. Trewyn,et al.  Mesoporous Silica Nanoparticles for Drug Delivery and Biosensing Applications , 2007 .

[24]  María Vallet-Regí,et al.  Ordered mesoporous materials in the context of drug delivery systems and bone tissue engineering. , 2006, Chemistry.

[25]  D. Luo,et al.  Biologically active core/shell nanoparticles self-assembled from cholesterol-terminated PEG-TAT for drug delivery across the blood-brain barrier. , 2008, Biomaterials.

[26]  Carl O. Pabo,et al.  Cellular uptake of the tat protein from human immunodeficiency virus , 1988, Cell.

[27]  Ta-Liang Chen,et al.  Lipopolysaccharide induces apoptotic insults to human alveolar epithelial A549 cells through reactive oxygen species-mediated activation of an intrinsic mitochondrion-dependent pathway , 2011, Archives of Toxicology.

[28]  S. Marklund,et al.  Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. , 1974, European journal of biochemistry.

[29]  V. S. Lin,et al.  Mesoporous silica nanoparticles deliver DNA and chemicals into plants. , 2007, Nature nanotechnology.

[30]  V. S. Lin,et al.  Luciferase and luciferin co-immobilized mesoporous silica nanoparticle materials for intracellular biocatalysis. , 2011, Journal of the American Chemical Society.

[31]  Chung-Yuan Mou,et al.  Size effect on cell uptake in well-suspended, uniform mesoporous silica nanoparticles. , 2009, Small.

[32]  I. Fridovich,et al.  Superoxide radical and superoxide dismutases. , 1995, Annual review of biochemistry.

[33]  P. Riley,et al.  Thymosin β4 protein therapy for cardiac repair. , 2012, Current pharmaceutical design.

[34]  Victor S-Y Lin,et al.  Mesoporous silica nanoparticles for intracellular delivery of membrane-impermeable proteins. , 2007, Journal of the American Chemical Society.

[35]  S. Gottesman,et al.  Protein quality control: triage by chaperones and proteases. , 1997, Genes & development.

[36]  I. Hwang,et al.  Transduced Tat-DJ-1 protein protects against oxidative stress-induced SH-SY5Y cells death and Parkinson disease mouse model , 2010, 2010 IEEE International Conference on Bioinformatics and Biomedicine Workshops (BIBMW).

[37]  A. Surolia,et al.  Sugar-quantum dot conjugates for a selective and sensitive detection of lectins. , 2007, Bioconjugate chemistry.

[38]  P. Coupland,et al.  Using fluorescent pH-sensitive nanosensors to report their intracellular location after Tat-mediated delivery. , 2009, Integrative biology : quantitative biosciences from nano to macro.

[39]  Soo‐Young Choi,et al.  HIV-1 Tat-mediated protein transduction of Cu,Zn-superoxide dismutase into pancreatic β cells in vitro and in vivo , 2004 .

[40]  S. Emr,et al.  A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast , 1995, The Journal of cell biology.

[41]  J. Serratosa,et al.  Absence of the cell cycle inhibitor p21Cip1 reduces LPS‐induced NO release and activation of the transcription factor NF‐κB in mixed glial cultures , 2005, Glia.

[42]  M. Howarth,et al.  Targeting quantum dots to surface proteins in living cells with biotin ligase. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[43]  Wei-Hsuan Chen,et al.  The FASEB Journal express article 10.1096/fj.05-4288fje. Published online October 17, 2005. , 2022 .

[44]  Yu-Chen Lin,et al.  Specifically and reversibly immobilizing proteins/enzymes to nitriolotriacetic-acid-modified mesoporous silicas through histidine tags for purification or catalysis. , 2011, Chemistry.

[45]  J. Porath,et al.  Metal chelate affinity chromatography, a new approach to protein fractionation , 1975, Nature.

[46]  F. Hartl,et al.  Pharmacologic shifting of a balance between protein refolding and degradation mediated by Hsp90. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[47]  A. Fève.  Current status of tyrosine hydroxylase in management of Parkinson's disease. , 2012, CNS & neurological disorders drug targets.

[48]  Victor S-Y Lin,et al.  A polyamidoamine dendrimer-capped mesoporous silica nanosphere-based gene transfection reagent. , 2004, Journal of the American Chemical Society.

[49]  V. Briner,et al.  Tumor necrosis factor-alpha and lipopolysaccharide induce apoptotic cell death in bovine glomerular endothelial cells. , 1999, Kidney international.