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: 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: Stability results of particle size, PDI, and zeta potential. The selected C-Lip Sel-siNEG was incubated with 0%, 10%, and 50% v / v FBS for 4, 24, and 48 h then particle size ( A ), PDI ( B ), and Zeta potential ( C ) were measured using DLS as described. Data points represent mean and SD (n = 3). Statistical analysis was carried out using one-way ANOVA followed by Tukey HSD test and p < 0.05 was considered significant, ns: non-significant. Serum protein had a non-significant effect on albumin-coated liposome particle size, PDI, or Zeta potential.

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, Incubation

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: In vitro characterization of the optimized C-Lip Sel-siNEG. In vitro release profile of Sel from the optimized C-Lip Sel-siNEG in simulated nasal fluids at 35 °C ( A ). Drug release from liposomes is measured by dialyzing C-Lip Sel-siNEG against simulated nasal fluids (pH 7.4). Drug concentration in the dialysate is assessed by HPLC. Datapoint represents mean and SD (n = 3). Morphological characterization of the optimized C-Lip Sel-siNEG by Transmission Electron Micrography ( B ). C-Lip Sel-siNEG appeared as spherical non-aggregate nanostructure.

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: In Vitro, Liposomes, Concentration Assay, Transmission 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: 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: 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: Figure 5. Cytotoxicity, intracellular uptake, and silencing efficiency of C-LipSel-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-LipSel-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-LipSel-siSNCA2 was assessed also on Calu-3 cells. The optimized C-LipSel-siSNCA2 has an insignificant effect on the cell viability of both cells (ns: nonsignificant). Intracellular delivery of C-LipSel-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-LipSel-siSNCA2 at three different siSNCA2 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-3trimethylammonium-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 (siSNCA2) 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: 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: Figure 6. Pharmacokinetics Sel profile and biochemical markers following administration of IN C-LipSel-siSNCA2 and intravenous Sel solution. Sel concentrations in rats’ plasma (A) and brain (B) after administration of various formulations. Animals received Sel at a dose of 1 mg/kg either via IV injection through the tail vein or IN instillation of 10 µL of C-LipSel-siSNCA2 containing 0.75 mg/kg siSNCA2 in each nostril. At each time point, 6 animals were sacrificed from each group and the concentration of Sel in plasma and brain was quantified using LCMS/MS. A significantly higher brain Sel concentration was observed at all time points following IN administration of C-LipSel-siSNCA2 compared to IV solution. Datapoints represent the mean ± SE (n = 6). PD was simulated in rats by IP injection of rotenone (3 mg/kg/day) for 11 days. On days 12 and 20 post-starting rotenone injection, rats received either IN C-LipSel-siSNCA2 or IV Sel solution. On day 30 of the experiment, rats were sacrificed and brain tissues were collected. Brain tissues were homogenized and the concentration of dopamine in the brain homogenate was assessed by HPLC-equipped with a fluorescence detector set at excitation/emission wavelengths of 280/315 nm (C). Catalase activity in the brain tissue homogenate was measured by a colorimetric method at 570 nm (D). MAO-B level was quantified in brain tissue using ELISA (E). Data points represent mean and SE (n = 6). Statistical analysis was performed using Two-way ANOVA followed by Tukey’s post-test, ns: non-significant, * p < 0.05, ** p < 0.01, *** p < 0.001. IN C-LipSel-siSNCA2 administration successfully restored dopamine levels, while catalase activity significantly reduced MAO-B levels in Parkinson’s model rats’ brains.

Article Snippet: 1, 2-distearoyl-snglycero-3-phosphocholine(DSPC),1, 2-dioleoyl-3trimethylammonium-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 (siSNCA2) and non-Targeting siRNA Control (siNEG) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). siAtto655 was obtained from Dharmacon (Cambridge, UK).

Techniques: Drug discovery, Clinical Proteomics, IV Injection, Concentration Assay, Injection, Fluorescence, Activity Assay, Enzyme-linked Immunosorbent 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: Figure 7. Pharmacodynamic assessment of C-LipSel-siSNCA2 Parkinson’s model rats. PD was simulated in rats by IP injection of rotenone (3 mg/kg/day) for 11 days. On day 12 and 20 post starting rotenone injection, rats received either IN C-LipSel-siSNCA2 or IV Sel solution. On day 30 of the experiment, stride length in left and right Forelimb and Hind limbs (A,B), Paw placement (C), and coverage (D) were measured and compared to control healthy rats and rotenone-treated rats. The open field test was carried out over 60 min and the distance crossed by rats was recorded (E). Catalepsy score was measured using bar method by measuring the time required by the rat to remove its forepaws from the rod (F). The rats were scored using a 5-point scale; 0 (0–5 s), 1 (6–15 s), 2 (16–25 s), 3 (26–35 s), 4 (36–60 s), and 5 (>60 s). Data points represent mean and SE (n = 6). Statistical analysis was performed using Two-way ANOVA followed by Tukey’s post-test, ns: non-significant, * p < 0.05, ** p < 0.01, *** p < 0.001. IN C-LipSel-siSNCA2 administration successfully restored locomotor activity in Parkinson’s model rats.

Article Snippet: 1, 2-distearoyl-snglycero-3-phosphocholine(DSPC),1, 2-dioleoyl-3trimethylammonium-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 (siSNCA2) and non-Targeting siRNA Control (siNEG) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). siAtto655 was obtained from Dharmacon (Cambridge, UK).

Techniques: Injection, Control, Activity Assay

Journal: Experimental eye research

Article Title: α-Synuclein modulates fibronectin expression in the trabecular meshwork independent of TGFβ2

doi: 10.1016/j.exer.2022.109351

Figure Lengend Snippet: (A) Primary human TM cells were treated with scrambled (Scr) sequence or siRNA for α-Syn. Probing of lysates for α-Syn shows a single band migrating at 14 kDa in samples treated with scrambled sequence, and significant down-regulation in samples exposed to α-Syn siRNA (lanes 1 & 2). (B) Immunoblot analysis of the neuroretina, CB, and TM isolated from human cadaveric eye shows expression of monomeric α-Syn in all three samples (lanes 1–3). A similar analysis of AH and VH shows monomeric and oligomeric α-Syn (lanes 4 & 5). All membranes were re-probed for β-actin to control for loading. (C) Immunoblotting of protein lysates from bovine CB, TM, and retina shows expression of α-Syn in all three tissues, with significantly higher expression in the retina (lanes 1–7).

Article Snippet: Dexamethasone (D1756) was obtained from Sigma Aldrich, USA. siRNA stocks for α-Syn (sc 29619) and scrambled sequences (sc37007) were obtained from Santa Cruz Biotechnology, USA.

Techniques: Sequencing, Western Blot, Isolation, Expressing, Control

Journal: Experimental eye research

Article Title: α-Synuclein modulates fibronectin expression in the trabecular meshwork independent of TGFβ2

doi: 10.1016/j.exer.2022.109351

Figure Lengend Snippet: (A) Primary human TM cells were treated with scrambled sequence (Scr) or siRNA specific to α-Syn for 48 h, followed by transfection with AdEmpty or AdTGFβ2. After additional 48 h, lysates were analyzed by immunoblotting. Probing for FN, ROCK-1, and α-SMA shows increased expression in cells over-expressing TGFβ2 relative to vector controls as expected (lane 1 & 2). However, this effect is abolished by silencing of α-Syn in the absence (lane 3) or presence (lane 4) of TGFβ2 (lanes 3 & 4 vs. 1 & 2). (B) Quantification by densitometry after normalization with β-actin and α-tubulin shows significant upregu-lation of FN, α-SMA, and ROCK-1 by 2.3 fold, 1.8 fold, and 2.8 fold respectively by TGFβ2 in the presence of endogenous α-Syn (**p < .01, ***p < .001), a significant decrease of 0.4–0.5 fold in all three proteins in cells where α-Syn had been silenced (### p < .001) regardless of active TGFβ2 (ns), n represents the number of biological replicates. (C) Lysates of primary human TM cells treated with scrambled (Scr) sequence or α-Syn specific siRNA were fractionated on three separate SDS-PAGE gels, and processed for immunoblotting. Probing of the first membrane for FN shows significant downregulation by silencing of α-Syn relative to controls (lanes 1 & 2). Probing of the second membrane for ROCK-1 also shows down-regulation by silencing of α-Syn, as is evident (lanes 1 & 2). The third membrane was probed for α-SMA, which also shows less expression in cells where α-Syn had been silenced (lanes 4–6 vs. 1–3). β-actin and α-tubulin were used as a loading control. (D) Densitometry after normalization with β-actin or α-tubulin shows significant downregulation of FN and α-SMA by ~0.7 fold, and ROCK-1 by 0.4 fold in cells where α-Syn had been silenced relative to respective controls. n represents the number of biological replicates. ***p < .001.

Article Snippet: Dexamethasone (D1756) was obtained from Sigma Aldrich, USA. siRNA stocks for α-Syn (sc 29619) and scrambled sequences (sc37007) were obtained from Santa Cruz Biotechnology, USA.

Techniques: Sequencing, Transfection, Western Blot, Expressing, Plasmid Preparation, SDS Page, Membrane, Control

Journal: Experimental eye research

Article Title: α-Synuclein modulates fibronectin expression in the trabecular meshwork independent of TGFβ2

doi: 10.1016/j.exer.2022.109351

Figure Lengend Snippet: (A) Lysates from primary human TM cells treated with scrambled (Scr) sequence or α-Syn specific siRNA were processed for immunoblotting. Probing for TGFβ2 shows downregulation in the absence of α-Syn relative to respective controls (lanes 1 & 2). Re probing of membrane for β-actin was performed as a loading control. (B) Densitometry after normalization to β-actin shows significant downregulation of active TGFβ2 by ~0.75 fold on silencing of α-Syn relative to controls. n represents the number of biological replicates. ***p < .001.

Article Snippet: Dexamethasone (D1756) was obtained from Sigma Aldrich, USA. siRNA stocks for α-Syn (sc 29619) and scrambled sequences (sc37007) were obtained from Santa Cruz Biotechnology, USA.

Techniques: Sequencing, Western Blot, Membrane, Control