OhioMod2013:Methods/CPNP Synthesis

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*Create 14 mL of 29% (volume) nonionic surfactant poly(oxyethylene)-nonylphenyl ether ([http://www.sigmaaldrich.com/catalog/product/aldrich/238643?lang=en&region=US Igepal CO-520]) in cyclohexane.
*Create 14 mL of 29% (volume) nonionic surfactant poly(oxyethylene)-nonylphenyl ether ([http://www.sigmaaldrich.com/catalog/product/aldrich/238643?lang=en&region=US Igepal CO-520]) in cyclohexane.
:<span style="color:red">Watch out!</span> Igepal CO-520 is cancer-suspecting agent. Use gloves.
:<span style="color:red">Watch out!</span> Igepal CO-520 is cancer-suspecting agent. Use gloves.
-
*Add 650 μL of 1 x 10<sup>-2</sup> M CaCl<sub>2</sub>, and allow to equilibrate for 3 minutes.
+
*Add 650 μL of 1 x 10<sup>-2</sup> M CaCl<sub>2</sub>, and allow to equilibrate for one hour.
===Microemulsion B===
===Microemulsion B===
*Create 14 mL of 29% (volume)[http://www.sigmaaldrich.com/catalog/product/aldrich/238643?lang=en&region=US Igepal CO-520] in cycloehxane.
*Create 14 mL of 29% (volume)[http://www.sigmaaldrich.com/catalog/product/aldrich/238643?lang=en&region=US Igepal CO-520] in cycloehxane.
-
*Add 650 μL of the phosphate, silicate, origami solution, and allow to equilibrate for 3 min.
+
*Add 650 μL of the phosphate, silicate, origami solution, and allow to equilibrate for one hour.
===Mix A and B===
===Mix A and B===
[[Image:Disodium_citrate.png|right|200px|Disodium Citrate|thumb]]
[[Image:Disodium_citrate.png|right|200px|Disodium Citrate|thumb]]
*Use syringe pump to slowly add microemulsion A to B for ~2-10 minutes under constant stirring.  
*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<sup>-3</sup> sodium citrate solution and allow to react for 10 minutes.
+
*Quench the precipitation by adding 225 μL aliquot of the 1 x 10<sup>-3</sup> sodium citrate solution and allow to react for 10-15 minutes.
:The sodium citrate will serve to disperse the nanoparticles and thus halt the mineralization of the calcium phosphate <cite>citrate</cite>. It will also present carboxyl functional groups on the surface of the nanoparticle, for later amide conjugation if we so desire. 4 x 10<sup>-2</sup> M aminopropyltriethoxysilane([http://en.wikipedia.org/wiki/%283-Aminopropyl%29triethoxysilane APTES]) can also be used as a dispersant for a longer period of time, if we want to have amine-functionalized nanoparticles.
:The sodium citrate will serve to disperse the nanoparticles and thus halt the mineralization of the calcium phosphate <cite>citrate</cite>. It will also present carboxyl functional groups on the surface of the nanoparticle, for later amide conjugation if we so desire. 4 x 10<sup>-2</sup> M aminopropyltriethoxysilane([http://en.wikipedia.org/wiki/%283-Aminopropyl%29triethoxysilane APTES]) can also be used as a dispersant for a longer period of time, if we want to have amine-functionalized nanoparticles.
-
*'Dissolve the emulsions with 50 mL of ethanol (pH 7).' This will dissolve the micelles.
+
*'Dissolve the emulsions with 50 mL of ethanol (pH 7, adjust with 1 M KOH).' This will dissolve the micelles.
==Alternatives==
==Alternatives==
Use Aeorsol OT or TritonX-100 from Sigma as surfactants. Try stirring the microemulsions at 4°C for 12 hrs if not optically clear, and add B to A at 4 ml/hr for 12 h in 4°C cold room. See Mozumdar's protocol <cite>Mozumdar</cite>
Use Aeorsol OT or TritonX-100 from Sigma as surfactants. Try stirring the microemulsions at 4°C for 12 hrs if not optically clear, and add B to A at 4 ml/hr for 12 h in 4°C cold room. See Mozumdar's protocol <cite>Mozumdar</cite>
 +
We may also want to dope with metasilicate.
 +
See also PAA-b-PEG to skip the citrate quenching.
==High Performance Liquid Chromatography==
==High Performance Liquid Chromatography==

Revision as of 16:08, 4 May 2013

°C

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Contents

Calcium Phosphate Nanoparticle Synthesis with DNA Encapsulation

Much of the protocol is adapted from Thomas Morgan's report on calcium phosphate encapsulation[1].

Double Microemulsion Preparation

Fig. 1 Reverse micelles in a microemulsion
Fig. 1 Reverse micelles in a microemulsion

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 [2].
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% (volume) nonionic surfactant poly(oxyethylene)-nonylphenyl ether (Igepal CO-520) in cyclohexane.
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 one hour.

Microemulsion B

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

Mix A and B

Disodium Citrate
Disodium Citrate
  • Use syringe pump to slowly add microemulsion A to B for ~2-10 minutes under constant stirring.
  • Quench the precipitation by adding 225 μL aliquot of the 1 x 10-3 sodium citrate solution and allow to react for 10-15 minutes.
The sodium citrate will serve to disperse the nanoparticles and thus halt the mineralization of the calcium phosphate [3]. 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.
  • 'Dissolve the emulsions with 50 mL of ethanol (pH 7, adjust with 1 M KOH).' This will dissolve the micelles.

Alternatives

Use Aeorsol OT or TritonX-100 from Sigma as surfactants. Try stirring the microemulsions at 4°C for 12 hrs if not optically clear, and add B to A at 4 ml/hr for 12 h in 4°C cold room. See Mozumdar's protocol [4] We may also want to dope with metasilicate. See also PAA-b-PEG to skip the citrate quenching.

High Performance Liquid Chromatography

Modern HPLC
Modern HPLC

We will use HPLC with van der Waals interaction to seperate the nanoparticles from the leftover reagents, DNA, and cyclohexane. The nanoparticles will then be eluted into a high-concentration well-dispersed solution. Much is adapted from Wang et al[5].

  • Load the stationary phase with 20 μm silica microspheres. Use the APS-treated negative-charged microspheres, which will bind the carboxyl-terminated nanoparticles.
  • The terminal end of the colum should be connected to a spectrometer with the wavelength set an absorbance that is suited to whichever marker or dye is used.
  • Load the unwashed nanoparticle suspension into the column, followed by ethanol.
  • Elute the column, with the flow rate fixed at 2mL/min.
The silica stationary particles will bind the nanoparticles due to van der Waals attraction when the mobile phase is nonionic. The ethanol will thus wash away the surfactants/cyclohexane.
  • Continue washing until the free DNA content is below limit of UV detection in the fractions. Usually only one pass of the eluent is enough.
  • Once washing is complete, elute the nanoparticles using 70:30 ethanol-water solution (with dionized degassed water of 5 x 10-4 M NaCl pH 7).
The water provides enough ionic attraction to overwhelm the van der Waals interaction of silica and nanoparticles. The nanoparticles will become mobile and will exit the column into a concentration solution.
  • Collect the fractions, and use the fraction with the first major peak in absorbance observed.



Other

  • The nanoparticles can now be conjugate with a variety of compounds to provide it with a secondary functionalization. See secondary functionalization protocol.
  • The nanoparticles were also taken under images, see microscopy protocol.

References

  1. Morgan TT, Muddana HS, Altinoglu EI, Rouse SM, Tabaković A, Tabouillot T, Russin TJ, Shanmugavelandy SS, Butler PJ, Eklund PC, Yun JK, Kester M, and Adair JH. . pmid:19367837. PubMed HubMed [Morgan]
  2. 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. [Adair]
  3. Leeuwenburgh SC, Ana ID, and Jansen JA. . pmid:19751849. PubMed HubMed [citrate]
  4. Singh R, Saxena A, and Subho Mozumdar. Calcium Phosphate-DNA Nanocomposites: Morphological Studies and Their Bile Duct Infusion for Liver-Directed Gene Therapy. Int. J. Appl. Ceramics Technology 5[1]; (2008) p. 1-10. [Mozumdar]
  5. Wang J, White WB, and Adair JH. . pmid:16526702. PubMed HubMed [Wang]
All Medline abstracts: PubMed HubMed
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