OhioMod2013:Methods/secondary

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Secondary Functionalization of CP Nanoparticles

Now that we have DNA encapsulated within calcium phosphate nanoparticles that are well-dispersed in an aqueous solution, we now have the option of conjugating secondary functional groups on the surface of the particle, by using amide formation on the citrate-coated nanoparticles.

Phenylethylene Glycol

MethoxyPEG-amine

1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

PEG is a non-toxic water-soluble polyether component that is useful for in vivo applications due to the ability of PEG to shield the nanoparticle from the immune system and the reticuloendothelial system. PEG-coated CP nanoparticles have seen many applications including tumour imaging[1] and systemic deliver of siRNA to tumor[2]. This selectiveness towards tumors can be due to the enhanced permeability and retenetion effect that tumor tissue has towards nanoparticles. We find PEG useful because of its potential for creating osmotic pressure as well as its protection of the CP nanoparticle. The PEG can also be synthesized as PEG-charge conversion polymer to induce endosomal destablization at low pH, thus improving endosomal escapement [3].

• Prepare aqueous solution of 20 mg/mL ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDCI) and methoxyPEG-amine. The carbodiimide will activate the carboxyl groups of the citrate for amide formation. The methoxyPEG-amine is functionalized with an amine group at its end.
• Add to 1 mL of the post-HPLC CP nanoparticle suspension. Incubate for 18 hr at 40 °C with continuous stirring.
• Dialzye the particles to remove excess EDCI and unreacted methoxyPEG-amine.

Avidin Conjugation

Much of the protocol comes from Rahul Sharma in Jim Adair's lab [4]. The biotin-avidin interaction is one of the strongest compound-protein binding complexes, and one of the most common used. By conjugating the avidin protein to the surface of the nanoparticles, we can then bind any biotinylated antibody, protein, or molecule. This gives us a wide range of possiblities for functionalization of the nanoparticles.

• Take a 1 mL of the CPNP suspension and dry under argon while covered from light. After the solvent is evaporated, reconstitute the nanoparticles with 1 mL of PBS.
• Prepare 1 mL aqueous solution of 20 mg/mL EDCI and 1 mL of 6 mg/mL avidin. Add solution to the CPNP suspension in PBS.
• Incubate for 24 hours at 35°C in dark with continuous stirring.
• Centrifuge mixture with a 100 kDa filter device to filter out the excess avidin.

Suggested membrane is Ultracel YM-100 cellulose membrane from Millipore.

Do not use polyethersulfone filter membranes due to binding of CPNPs to membrane.

References

1. Altinoğlu EI, Russin TJ, Kaiser JM, Barth BM, Eklund PC, Kester M, and Adair JH. Near-infrared emitting fluorophore-doped calcium phosphate nanoparticles for in vivo imaging of human breast cancer. ACS Nano. 2008 Oct 28;2(10):2075-84. DOI:10.1021/nn800448r |
2. Li J, Chen YC, Tseng YC, Mozumdar S, and Huang L. Biodegradable calcium phosphate nanoparticle with lipid coating for systemic siRNA delivery. J Control Release. 2010 Mar 19;142(3):416-21. DOI:10.1016/j.jconrel.2009.11.008 |
3. Pittella F, Zhang M, Lee Y, Kim HJ, Tockary T, Osada K, Ishii T, Miyata K, Nishiyama N, and Kataoka K. Enhanced endosomal escape of siRNA-incorporating hybrid nanoparticles from calcium phosphate and PEG-block charge-conversional polymer for efficient gene knockdown with negligible cytotoxicity. Biomaterials. 2011 Apr;32(11):3106-14. DOI:10.1016/j.biomaterials.2010.12.057 |
4. Barth BM, Sharma R, Altinoğlu EI, Morgan TT, Shanmugavelandy SS, Kaiser JM, McGovern C, Matters GL, Smith JP, Kester M, and Adair JH. Bioconjugation of calcium phosphosilicate composite nanoparticles for selective targeting of human breast and pancreatic cancers in vivo. ACS Nano. 2010 Mar 23;4(3):1279-87. DOI:10.1021/nn901297q |