Journal: Pharmaceutics

Article Title: Tailored Intranasal Albumin Caged Selegiline-α Synuclein siRNA Liposome with Improved Efficiency in Parkinson’s Model

doi: 10.3390/pharmaceutics17020243

Figure Lengend Snippet: Factors and responses in D-optimal design for Sel-siRNA loaded liposomes.

Article Snippet: 1, 2-distearoyl-snglycero-3-phosphocholine(DSPC),1, 2-dioleoyl-3-trimethylammonium-propane(DOTAP), N-palmitoyl-sphingosine-1-[succinyl (methoxypolyethylene glycol) 2000] (C16-PEG2000 Ceramide) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). α-Synuclein siRNA (siSNCA 2 ) and non-Targeting siRNA Control (siNEG) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). siAtto655 was obtained from Dharmacon (Cambridge, UK).

Techniques: Liposomes, Zeta Potential Analyzer

Journal: Pharmaceutics

Article Title: Tailored Intranasal Albumin Caged Selegiline-α Synuclein siRNA Liposome with Improved Efficiency in Parkinson’s Model

doi: 10.3390/pharmaceutics17020243

Figure Lengend Snippet: The effect of lipid composition on particle size, zeta potential, Sel, and siRNA encapsulation efficiency. Data obtained from a physicochemical characterization of the prepared liposomes were used to generate a predictive DoE model. Lipid interactions defined between CH ( A ), DSPC ( B ) and DOTAP ( C ) when C16-PEG2000 ( D ) was used at 2.5% ( left ), 3.75% ( middle ), 5% ( right ) on particle size (Y1) ( A ), zeta potential (Y2) ( B ), Sel EE% (Y3) ( C ), and siRNA EE% (Y4) ( D ) are presented as contour plots.

Article Snippet: 1, 2-distearoyl-snglycero-3-phosphocholine(DSPC),1, 2-dioleoyl-3-trimethylammonium-propane(DOTAP), N-palmitoyl-sphingosine-1-[succinyl (methoxypolyethylene glycol) 2000] (C16-PEG2000 Ceramide) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). α-Synuclein siRNA (siSNCA 2 ) and non-Targeting siRNA Control (siNEG) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). siAtto655 was obtained from Dharmacon (Cambridge, UK).

Techniques: Zeta Potential Analyzer, Encapsulation, Liposomes

Journal: Pharmaceutics

Article Title: Tailored Intranasal Albumin Caged Selegiline-α Synuclein siRNA Liposome with Improved Efficiency in Parkinson’s Model

doi: 10.3390/pharmaceutics17020243

Figure Lengend Snippet: The effect of lipid albumin coating on physicochemical characters of the optimized Lip Sel-siNEG . Coating Sel-siRNA-loaded liposomes resulted in increased particle size and surface negativity ( A ). The EE% of both Sel and siRNA is inversely proportional to the concentration of HSA used in the coating ( B ). The amount of associated HSA in the optimized liposomes is quantified by Bicinchoninic Acid Protein Assay (BCA) ( C ). Data are represented as the mean ± SD (n = 3). The amount of associated HSA is directly proportional to HSA concentration.

Article Snippet: 1, 2-distearoyl-snglycero-3-phosphocholine(DSPC),1, 2-dioleoyl-3-trimethylammonium-propane(DOTAP), N-palmitoyl-sphingosine-1-[succinyl (methoxypolyethylene glycol) 2000] (C16-PEG2000 Ceramide) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). α-Synuclein siRNA (siSNCA 2 ) and non-Targeting siRNA Control (siNEG) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). siAtto655 was obtained from Dharmacon (Cambridge, UK).

Techniques: Liposomes, Concentration Assay, Bicinchoninic Acid Protein Assay

Journal: Pharmaceutics

Article Title: Tailored Intranasal Albumin Caged Selegiline-α Synuclein siRNA Liposome with Improved Efficiency in Parkinson’s Model

doi: 10.3390/pharmaceutics17020243

Figure Lengend Snippet: Physicochemical characterization of the optimized liposomes and HSA-coated liposomes loaded Sel and siRNA.

Article Snippet: 1, 2-distearoyl-snglycero-3-phosphocholine(DSPC),1, 2-dioleoyl-3-trimethylammonium-propane(DOTAP), N-palmitoyl-sphingosine-1-[succinyl (methoxypolyethylene glycol) 2000] (C16-PEG2000 Ceramide) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). α-Synuclein siRNA (siSNCA 2 ) and non-Targeting siRNA Control (siNEG) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). siAtto655 was obtained from Dharmacon (Cambridge, UK).

Techniques: Liposomes, Zeta Potential Analyzer

Journal: Pharmaceutics

Article Title: Tailored Intranasal Albumin Caged Selegiline-α Synuclein siRNA Liposome with Improved Efficiency in Parkinson’s Model

doi: 10.3390/pharmaceutics17020243

Figure Lengend Snippet: Cytotoxicity, intracellular uptake, and silencing efficiency of C-Lip Sel-siSNCA2 in SH SY5Y cells. SH-SY5Y cells were pretreated with rotenone (10 nM) for 5 days to induce α-synuclein overexpression. Consequently, cells were incubated with C-Lip Sel-siSNCA2 for 48 h at increasing Sel concentrations (0.01–100 μM). Cell viability was determined by MTT assay and data are presented as viable cells as a percentage of non-treated cells (n = 5) ( A ). The biocompatibility of the optimized C-Lip Sel-siSNCA2 was assessed also on Calu-3 cells. The optimized C-Lip Sel-siSNCA2 has an insignificant effect on the cell viability of both cells (ns: nonsignificant). Intracellular delivery of C-Lip Sel-siAtto655 at concentrations 10, 20, and 30 nM after 4 and 24 h was assessed using flow cytometry. Representative flow cytometry histograms obtained at the 4 h time point are shown in ( B ). Quantitative uptake of siRNA expressed as MFI is shown in ( C ). siRNA uptake was higher at increasing concentrations and incubation times (* p < 0.05). To evaluate gene silencing, SH-SY5Y cells pretreated with rotenone were incubated with C-Lip Sel-siSNCA2 at three different siSNCA 2 concentrations (10, 20, and 30 nM) for 48 h. Representative flow cytometry contour plot for 30 nM siRNA at 48 h is shown in ( D ). Gates were drawn based on isotype controls. The knock-down efficiency of α-synuclein is presented as % positive cells ( E ). Data points represent mean and SD (n = 3). Statistical analysis was performed using One-way ANOVA followed by Tukey’s post-test * p < 0.05.

Article Snippet: 1, 2-distearoyl-snglycero-3-phosphocholine(DSPC),1, 2-dioleoyl-3-trimethylammonium-propane(DOTAP), N-palmitoyl-sphingosine-1-[succinyl (methoxypolyethylene glycol) 2000] (C16-PEG2000 Ceramide) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). α-Synuclein siRNA (siSNCA 2 ) and non-Targeting siRNA Control (siNEG) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). siAtto655 was obtained from Dharmacon (Cambridge, UK).

Techniques: Over Expression, Incubation, MTT Assay, Flow Cytometry, Knockdown

Journal: Experimental Neurobiology

Article Title: Death-associated Protein Kinase 1 Phosphorylates α-Synuclein at Ser129 and Exacerbates Rotenone-induced Toxic Aggregation of α-Synuclein in Dopaminergic SH-SY5Y Cells

doi: 10.5607/en20014

Figure Lengend Snippet: DAPK1 exacerbates rotenone-induced toxic synucleinopathy in SH-SY5Y and MEF cells. (A) SH-SY5Y cells were either mock-transfected or transfected for 24 h with a plasmid encoding FLAG-DAPK1-WT, and treated with 100 nM rotenone for additional 24 h. Cell lysates were fractionated into 1% Triton X-100-soluble and -insoluble fractions, followed by immunoblotting with the indicated antibodies. (B) DAPK1 -WT or DAPK1 -KO MEFs were treated with either DMSO (control) or 100 nM rotenone for 24 h. Cells were lysed with detergent-free lysis buffer and the protein samples were analyzed by native PAGE. (C) After DAPK1 -WT or DAPK1 -KO MEFs were mock transfected or transfected for 24 h with a plasmid encoding FLAG-DAPK1-WT, cells were treated with either DMSO (control) or 100 nM rotenone for an additional 24 h. Representative confocal images of immunostaining of endogenous α-synuclein (green) and FLAG-DAPK1 (red) are shown in the upper panel. In the lower panel, the number of cells showing intracellular and visible α-synuclein aggregates were counted among ~100 cells, and this was repeated five times. Data represent the mean±SEM of five independent experiments (**p≤0.01). (D) Where indicated, DAPK1 -WT or DAPK1 -KO MEFs were mock-transfected or transfected for 24 h with a plasmid encoding FLAG-DAPK1-WT, and treated with either DMSO (control) or 100 nM rotenone for additional 24 h. Representative confocal images of immunostaining of pS129-α-synuclein (green) and FLAG-DAPK1 (red) are shown in the upper panel. In the lower panel, the number of cells showing intracellular and visible α-synuclein aggregates was counted in approximately 100 cells, and the counting was repeated five times. The data represent the mean±SEM of five independent experiments (**p≤0.01). (E) Where indicated, SH-SY5Y cells were transfected for 24 h with plasmids encoding FLAG-DAPK1-WT or FLAG-DAPK1-K42A, or α-synuclein ( SNCA ) siRNA alone or in combination. Cells were then treated with either DMSO (control) or 100 nM rotenone for an additional 24 h. Cell viability was measured using Cell Counting Kit-8. Data represent the mean±SEM of five independent experiments (**p≤0.01).

Article Snippet: The α-synuclein (SNCA)-specific small interfering RNAs (siRNAs) were designed and synthesized by Santa Cruz Biotechnology (sc-29619).

Techniques: Transfection, Plasmid Preparation, Western Blot, Lysis, Clear Native PAGE, Immunostaining, Cell Counting