OhioMod2013:Results

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Nanoparticle Characterizatione)
Current revision (22:01, 1 July 2013) (view source)
(Nanoparticle Characterizatione)
 
(6 intermediate revisions not shown.)
Line 21: Line 21:
Use inductively coupled mass spectroscopy to determine Ca:P ratio.  
Use inductively coupled mass spectroscopy to determine Ca:P ratio.  
===Zetasizer===
===Zetasizer===
-
Determine size and zeta potential of particles.  
+
Determine size and zeta potential of particles. With a high zeta potential we can infer stability, as it can indicate a high repulsion between nanoparticles, hence avoiding flocculation from the emulsion.
 +
===Fourier-transform Infrared Spectroscopy===
 +
Confirm citrate functionalization
 +
 
===Gas Pycnometry===
===Gas Pycnometry===
Determine density of nanoparticles
Determine density of nanoparticles
===TEM===
===TEM===
-
Take direct measurements of ~100 particles. Determine consistency of size.  
+
Take direct measurements of ~100 particles using ImageJ software. Determine consistency of size.
 +
 
===Dissolution===
===Dissolution===
Perform titration of nanoparticles until dissolution of nanoparticles.  
Perform titration of nanoparticles until dissolution of nanoparticles.  
Line 31: Line 35:
===Endurance===
===Endurance===
Store nanoparticles at 25°C and 4°C. Measure rate of precipitation over week. How long can we store the nanoparticles?
Store nanoparticles at 25°C and 4°C. Measure rate of precipitation over week. How long can we store the nanoparticles?
 +
 +
===Uptake Efficiency===
 +
For liposomal encapsulation efficiency: Use impermeable DNA intercalator dye such as propidium iodide to stain the liposomes before and after ethanol dissolution. Will see an increase in flourescence after ethanol dissolution that correlates with amount of origami in the liposome.
 +
 +
For calcium phosphate encapsulation efficiency: Add DNA intercalator, stain before and after acid dissolution of calcium phosphate. Increase in flourescence correlatees with origami protected by calcium phosphate.
==Serum Assay==
==Serum Assay==
Line 40: Line 49:
==Cytotoxicity==
==Cytotoxicity==
-
Perform MTT assay to measure metabolic activity of culture.
+
Perform MTT assay to measure metabolic activity of culture. Or use brightfield.  
==Cellular Uptake==
==Cellular Uptake==
 +
Use propidium iodine- a cell impermeable DNA intercalating agent.
Use carbazole-based biscyanine fluorophores to measure intact DNA origami intracellularly.  
Use carbazole-based biscyanine fluorophores to measure intact DNA origami intracellularly.  
Also used flourescent tagged DNA origami.  
Also used flourescent tagged DNA origami.  
Line 57: Line 67:
Use histoligical staining by Von Kossa method to measure particle uptake into cells.  
Use histoligical staining by Von Kossa method to measure particle uptake into cells.  
[[Image:VonKossa.png|200px|right|thumb|Von Kossa reaction. Dark cells indicate particle uptake]]
[[Image:VonKossa.png|200px|right|thumb|Von Kossa reaction. Dark cells indicate particle uptake]]
 +
 +
==In vivo==
 +
Use indocyanine green near-infrared imaging to track within body
 +
 +
==Transfection Efficiency==
 +
Beta-galactosidase
 +
GFP-encoding plasmid. (gWiz GFP)
 +
Luciferase.
 +
Can perform floursecent/lucifer assay, western blot of mouse organs.

Current revision

Home        Introduction        Design        Methods        Results        Team        Internal       


Contents

Results

Nanoparticle Characterizatione

HPLC

Take A260 peaks, determine rate of DNA encapsulation vs free DNA.

Ca:P ratio

Use inductively coupled mass spectroscopy to determine Ca:P ratio.

Zetasizer

Determine size and zeta potential of particles. With a high zeta potential we can infer stability, as it can indicate a high repulsion between nanoparticles, hence avoiding flocculation from the emulsion.

Fourier-transform Infrared Spectroscopy

Confirm citrate functionalization

Gas Pycnometry

Determine density of nanoparticles

TEM

Take direct measurements of ~100 particles using ImageJ software. Determine consistency of size.

Dissolution

Perform titration of nanoparticles until dissolution of nanoparticles. Run gel for DNA origami vs. free DNA after dissolution. Is origami intact?

Endurance

Store nanoparticles at 25°C and 4°C. Measure rate of precipitation over week. How long can we store the nanoparticles?

Uptake Efficiency

For liposomal encapsulation efficiency: Use impermeable DNA intercalator dye such as propidium iodide to stain the liposomes before and after ethanol dissolution. Will see an increase in flourescence after ethanol dissolution that correlates with amount of origami in the liposome.

For calcium phosphate encapsulation efficiency: Add DNA intercalator, stain before and after acid dissolution of calcium phosphate. Increase in flourescence correlatees with origami protected by calcium phosphate.

Serum Assay

Apply DNA encapsulated particles vs free DNA origami vs PEGylated particles to bovine serum. Measure intact origami after period of time via agarose gel electrophoresis.

Apply endonucleases/DNase to encapuslated vs free DNA origami. Measure intact orgimai after periods of time via agarose gel elctrophoresis.

Cytotoxicity

Perform MTT assay to measure metabolic activity of culture. Or use brightfield.

Cellular Uptake

Use propidium iodine- a cell impermeable DNA intercalating agent. Use carbazole-based biscyanine fluorophores to measure intact DNA origami intracellularly. Also used flourescent tagged DNA origami. Also use flourescent tagged Calcium Phospahte nanoparticles. potential for confocal microscopy.

Use chloroquinine to inhibit lysosomal fusion/acidification. Measure effect. May also use Cytochalasin D to inhibit endosome travel. Measure effect

Measure transfection of confluent cells vs recently split fast-growing cultures.

If using gWiz GFP encoding, use fluorescent microplate reader to measure transfection.

Use histoligical staining by Von Kossa method to measure particle uptake into cells.

Von Kossa reaction. Dark cells indicate particle uptake
Von Kossa reaction. Dark cells indicate particle uptake

In vivo

Use indocyanine green near-infrared imaging to track within body

Transfection Efficiency

Beta-galactosidase GFP-encoding plasmid. (gWiz GFP) Luciferase. Can perform floursecent/lucifer assay, western blot of mouse organs.

Personal tools