Preparing Chitosan Nano/Microparticles
Chitosan nano/microparticles are useful for intracellular delivery of negatively charged molecules and presumably can be prepared with any negatively charged molecule. Here, DNA is used as an example. There are many types of chitosan with different molecular weights and percent deacetylation; results will vary depending on the chitosan used. Experiment can be scaled to prepare larger or smaller batches. Procedure is based on Aral et al., STP Pharma Sciences, 10: 83-88 (2000) and Berthold et al., J. Cont. Rel., 39:17-25 (1996).
Dissolve 0.25 wt% chitosan (any type) in 1% acetic acid (takes ~1 day)
Prepare solution of negatively charged molecule in water or a dilute buffer. Concentration will depend on the negative charge and solubility of the molecule. For DNA, with 2.8 nmol of negative charge per mg of DNA a concentration of 1 mg/ml (2.8 mM negative charge) in 0.5X TE works well.
Put 2 ml of 0.25 wt% chitosan in a small glass vial with a small stir bar. Stir on a stir plate at a speed where sample is almost vortexing.
Carefully inject 2 ml of solution containing negatively charged molecule into the stirring chitosan through a 25 gauge needle. Speed of addition affects the initial particle size, but unknown if it will affect the final particle size.
Let solution stir for ~3 hours. Stir speed will affect final particle size, but no measurements have been made to characterize the correlation.
Centrifuge to separate nanoparticles from solution- 2000xg for 10 minutes works well with DNA-chitosan nanoparticles.
If desired, supernatant may be saved for assay to determine efficiency of encapsulation.