OhioMod2013:Methods/CPNP Synthesis

Home        Introduction        Design        Methods        Results        Team        Internal       

Calcium Phosphate Nanoparticle Synthesis with DNA Encapsulation

Double Microemulsion Preparation

A microemulsion is a mixture of oil, water, and surfactant. In our case, we are using reverse microemulsion win which nano-scale water droplets are dispersed in an oil phase. The surfactant allows for the formation of the emulsion of other otherwise immiscible phases of oil and water. The microemulsion provides us a controlled nanoscale environment for synthesis of the microparticles within the nano-scale droplets of water.

Aqueous solution preparation

• Vacuum aspirate for two hours distilled water (and ethanol) to create CO₂ free dionized water solution. This is to prevent the CO₂ from affecting the reaction or from microbubbles from forming with freezing solutions. The gas free dionized water can be stored for up to one weeks and should be used for all aqueous solutions.
• Make solution of 1 x 10^-2 M CaCl₂. Use probe sonicator for at least 10 minutes.
• Make solution of 1 x 10^-3 disodium phosphate with 8 x 10^-4 disodium silicate. Stir for at least 30 minutes. The silicate serves as a nucleation agent for the calcium phosphate [1].

Also, add the DNA origami to the phosphate solution. The DNA and phosphate solution should have little interaction until the calcium is introduced.

• Prepare 1 x 10^-3 sodium citrate solution. This is for coating the CP nanoparticle to allow disperal.

Microemulsion A

• Create 14 mL of 29% Igepal CO-520 in cycloehxane.

Watch out! Igepal CO-520 is cancer-suspecting agent. Use gloves.

• Add 650 μL of 1 x 10^-2 M CaCl₂, and allow to equilibrate for 3 minutes.

Microemulsion B

• Create 14 mL of 29% Igepal CO-520 in cycloehxane.
• Add 650 μL of the phosphate, silicate, origami solution, and allow to equilibrate for 3 min.

Mix A and B

• Use syringe pump to slowly add microemulsion A to B for ~2-10 minutes under constant stirring.
• Quench the precipitation by adding the 1 x 10^-3 sodium citrate solution and allow to react for 10 minutes.

The sodium citrate will serve to disperse the nanoparticles and thus halt the mineralization of the calcium phosphate [2]. It will also present carboxyl functional groups on the surface of the nanoparticle, for later amide conjugation if we so desire. 4 x 10^-2 M aminopropyltriethoxysilane(APTES) can also be used as a dispersant for a longer period of time, if we want to have amine-functionalized nanoparticles.

References

1. Adair, JH et al. Heterogeneous Deposition of Calcium Phosphates at the Silicon(Hydrous) Oxide-Water Interface. Urolithias 2. New York: Plenum Press; 1995 p. 181-187.

2. Leeuwenburgh SC, Ana ID, and Jansen JA. Sodium citrate as an effective dispersant for the synthesis of inorganic-organic composites with a nanodispersed mineral phase. Acta Biomater. 2010 Mar;6(3):836-44. DOI:10.1016/j.actbio.2009.09.005 |