presentación de powerpoint · rplp1 and rplp2 (rplp 1/2) are essential for flaviviruses to infect...
TRANSCRIPT
![Page 1: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/1.jpg)
![Page 2: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/2.jpg)
![Page 3: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/3.jpg)
![Page 4: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/4.jpg)
TYPES OF VECTORS
VIRAL VECTORS NON VIRAL VECTORS
High transfection efficiency
Low transfection efficiency
Expensive
Possibility of immunogicity
Low immunogicity
Easy to manipulate and cheaper
![Page 5: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/5.jpg)
NON VIRAL VECTORS. NANOPARTICLES
(Suñé-Pou et al, 2017)
![Page 6: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/6.jpg)
SOLID LIPID NANOPARTICLES (SLN)
▪ Nanoparticles based on biocompatible lipids → Low toxicity.
▪ Easy manufacture process.
▪ Can be manufactured avoiding organic solvents.
▪ Can incorporate drugs inside its structure or outside, on the surface.
▪ Protect the drugs from external agents.
▪ SLN can be functionalized.
![Page 7: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/7.jpg)
SOLID LIPID NANOPARTICLES (SLN)
+
+++
+ +
++
siRNA
cSLNs
--
SLNPLEXES
**cSLNs → cationic Solid Lipid Nanoparticles
---
--- -
---
![Page 8: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/8.jpg)
METHODS. HOT MICROEMULSIFICATION METHOD
1. CENTRIFUGATION2. FILTRATION3. LIOFILIZATION
![Page 9: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/9.jpg)
Stearic Acid 500 mg
Octadecylamine 600 mg
Poloxamer 188 100 mg
PREVIOUS FORMULATION
Matricial Lipids
CationicLipid
Surfactant
![Page 10: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/10.jpg)
COMPARISON OF FORMULATIONS WITH CHOLESTERYL OLEATE
(CO)
![Page 11: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/11.jpg)
DEVELOPED AND TESTED FORMULATIONS
Ref. 12 Ref. 13 Ref.14 Ref. 15 Ref. 16
Stearic Acid 400 mg 300 mg 200 mg 100 mg 0 mg
Cholesteryl
Oleate100 mg 200 mg 300 mg 400 mg 500 mg
Octadecylamine 600 mg 600 mg 600 mg 600 mg 600 mg
Poloxamer 188 100 mg 100 mg 100 mg 100 mg 100 mg
+ Cholesteryl Oleate (CO)- Stearic Acid
Matricial Lipids
CationicLipid
Surfactant
![Page 12: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/12.jpg)
PARTICLE SIZE DISTRIBUTION
A → Reference 12B → Reference 13C → Reference 14D → Reference 15E → Reference 16
![Page 13: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/13.jpg)
MORPHOLOGY (TEM)
A → Reference 12B → Reference 13C → Reference 14D → Reference 15E → Reference 16
![Page 14: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/14.jpg)
ZETA POTENTIAL
SLNs Zeta potential (mV)
Ref. 12 38.3
Ref. 13 35.3
Ref. 14 35.7
Ref. 15 29.5
Ref. 16 41.9
![Page 15: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/15.jpg)
CELL VIABILITY (PROPIDIUM IODIDE ASSAY)
▪ Propidium Iodide is a fluorescentintercalating agent that can be used to staincells.
▪ Propidium iodide is used as a DNA stainin flow cytometry to evaluate cell viability.
▪ Propidium Iodide is not membrane-permeable making it useful to differentiatehealthy cells based on membrane integrity.
![Page 16: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/16.jpg)
CELL VIABILITY (PROPIDIUM IODIDE ASSAY)
• All the formulations do notcause cellular death at theconcentrations studied
• CO-SLNs arebiocompatible in all theconcentrations tested
![Page 17: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/17.jpg)
▪ The Luciferase Assay is an extremely sensitive and rapid reagentfor quantitation of firefly luciferase.
▪ Experiments tot transfect plasmid p3xMHC-LUC with the SLNs asnon viral vector were performed.
▪ The more transfection, the more levels of Firefly Luciferase willbe observed,.
▪ Firefly Luciferase is an enzyme that catalazises the followingreaction:
PLASMID TRANSFECTION. LUCIFERASE ASSAY
![Page 18: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/18.jpg)
PLASMID TRANSFECTION. LUCIFERASE ASSAY
![Page 19: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/19.jpg)
siRNA TRANSFECTION. siEGFPCy3
▪ Cy3 is a cyanine, therefore it can be used as a fluorescent dye.▪ Cy3 is excited with λ = 570 nm-1▪ siRNA can be marked with Cy3, so you can know the % of cells
transfected (flow citometry) and track the transfection in cellswith images (confocal microscopy)
![Page 20: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/20.jpg)
siRNA TRANSFECTION. siEGFPCy3
40-50% of transfection
![Page 21: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/21.jpg)
siRNA TRANSFECTION. siEGFPCy3
A → Negative ControlB → Positive ControlC → Reference 14
BLUE → NUCLEUS (DAPI)RED → SLNPLEXES (siRNACy3)
![Page 22: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/22.jpg)
siRNA TRANSFECTION. siEGFPCy3
BLUE → NUCLEUS (DAPI)RED → SLNPLEXES (siRNACy3)
![Page 23: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/23.jpg)
CHARACTERIZATION OF REF. 14
![Page 24: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/24.jpg)
CHARACTERIZATION OF REF. 14
▪ All the galenic and biologic characterization was performed withthe lyophilized resuspended.
▪ The assays performed were:
• Size
• Zeta Potential
• Morphology and shape
• Surface composition
• Excipients Compatibility
• Critical Collapse Temperature
• Stability Studies
• Cell viability (MTT)
• Studies of SLNplexes formation
• Cellular location of siRNAtransfected
![Page 25: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/25.jpg)
SIZE, ZETA POTENTIAL AND MORPHOLOGY (TEM)
Experiments
Particle size
(nm) PdI
Zeta
potential
(mV)1
2
3
4
5
172
224
255
173
210
0,210
0,209
0,224
0,204
0,226
39,8
34,0
30,0
33,1
36,9Mean 207 0,215 34,76
Standard
Deviation35,06 0,0098 3,74
![Page 26: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/26.jpg)
EXCIPIENTS COMPATIBILITY (DSC)
A: Stearic Acid + CO
B: Stearic Acid + Octadecylamine
C: Stearic Acid + Poloxamer 188
D: CO + Octadecylamine
E: CO + Poloxamer 188
F: Octadecylamine + Poloxamer 188
![Page 27: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/27.jpg)
CRITICAL COLLAPSE TEMPERATURE (DSC)
A: Critical collapse temperature (SLNs) → -18 ˚CB: Critical collapse temperature (SLNs + Trehalose) → -28 ˚C
![Page 28: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/28.jpg)
STABILITY STUDIES
![Page 29: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/29.jpg)
CELL VIABILITY (MTT ASSAY)
![Page 30: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/30.jpg)
CELLULAR LOCATION OF siRNA TRANSFECTED WITH REF. 14
BLUE → NUCLEUS (DAPI)GREEN → CYTOSKELETON (α-TUBULIN)RED → SLNPLEXES (siRNACy3)
HeLa Cells
![Page 31: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/31.jpg)
DENGUE INFECTION AFTER THE
TRANSFECTION OF siP2.1 USING SLN AS A
VECTOR
![Page 32: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/32.jpg)
▪ Dengue Virus (DENV) is the cause of Dengue Feber.
▪ DENV is a Flavivirus, like Zika Virus, Yellow Feber Virus, etc.
▪ More than 100 million of cases around the world.
▪ 25.000 people die because of its infection every year.
▪ Currently, there is not any vaccine.
DENGUE VIRUS
![Page 33: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/33.jpg)
▪ RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells,
because they promote the early accumulation of viral proteins (Campos et
al., J Vir, 2017)
▪ If RPLP1 and RPLP2 are knockdown, the DENV infection decreases extremely
(Campos et al., J Vir, 2017)
RPLP1/2
![Page 34: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/34.jpg)
SLNs+NSC → Negative controlSLNs+siP2.1 → Treatment with the SLNs+siRNA
RESULTS
![Page 35: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/35.jpg)
SLNsNSC
SLNssiP2.1
SLNs+NSC → Negative ControlSLNs+siP2.1 → SLN:siRNA
RESULTS
BLUE → NUCLEUS (DAPI)GREEN → Virus Protein
![Page 36: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/36.jpg)
GREEN → Positive (infected cell)RED → Negative (non-infected cell
+ Signal Intensity (more infection)
RESULTS
SLNsNSC
SLNssiP2.1
![Page 37: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/37.jpg)
NEXT STEPS
![Page 38: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/38.jpg)
Carles Suñé Negre, PhD, PICristina Hernández-Munain, PhD, PI
Silvia Prieto Sánchez, PhDJuan Pablo Muñoz-Cobo Belart, PhDCristina Moreno-CastroYounes El YousfiSofía Boyero-Corral
Salvador Guerrero FernándezBlanca Martínez
JM Suñé Negre, PhD, PIEncarna Garcia Montoya, PhD, PIPilar Pérez-Lozano, PhD, PIJR Ticó-Grau, PhD, PIMontserrat Miñarro, PhD, PI
Manel Roig CarrerasAnna Nardi Ricart, PhDFrancesc Gual PujolIsaac Nofrerias RoigDébora Mercadé Frutos, PhDSusana Fuentes SánchezMarta ToroMaria Balcells MestreLluís Domènech GrauRaquel Mulero LópezMarina Saball TorresCarlos Martín Ramos Bosch, PhDMarta Eduviges Bustos ArayaMaria José LimeresDavid Narváez
MA Garcia-Blanco, MD, PhD, PIShelton S. Bradrick, PhD, coPI
Gaddiel Galarza-Muñoz, PhDReddisiva Prasanth Kambham, PhDRuben Soto-Acosta, PhDRafael K Campos, PhDGeraldine O. Schott-Lerner
Steven T. PowellJingru Fang
![Page 39: Presentación de PowerPoint · RPLP1 and RPLP2 (RPLP 1/2) are essential for flaviviruses to infect cells, because they promote the early accumulation of viral proteins (Campos et](https://reader034.vdocuments.us/reader034/viewer/2022050716/5e39110dd15e837feb18874d/html5/thumbnails/39.jpg)