Association of CpG ODN with Emulsion Formulations
The association of CpG with the oil (squalene) phase is thought to be weak because of the unfavorable energetics of hydrophobic
interactions. The partitioning of oligonucleotides is dominated by strong solvation in the aqueous phase, though surface or
interfacial effects may be occurring through mediation of surfactants. However, even weak association with particles may have
an effect on biological activity. Because of the small difference in density of the two phases, it is necessary to increase
the density gradient by adding a high MW sucrose polymer such as Ficoll PM400 (GE Healthcare). Ficoll can be dissolved in
water to create a solution that has a significantly higher density than water because of its high molecular weight and excellent
water solubility. This allows separation of the phases in an emulsion using a standard microcentrifuge. Alternatively, an
ultracentrifuge may be used to separate the phases without adding the sucrose polymer.3
Table 2. Association of CpG ODN with oil-in-water emulsion
|
To determine the extent of CpG ODN associated with the oil particulates in the SE formulation, a 30% (w/v) solution of Ficoll
in water was mixed at a 1:1 ratio with the SE formulation (10% oil v/v and 1.25 mg/mL CpG ODN) in a microcentrifuge tube and
vortexed briefly. The sample was centrifuged for 30 min at 16,000g. After centrifugation, the sample was carefully removed and the tube bottom carefully punctured with a 27g half-inch needle
to extract an aliquot of the lower aqueous phase. The absorbance at 260 nm was then measured and compared to a CpG ODN standard.
The difference in absorption represents the amount of CpG ODN associated with the oil phase (Table 2). In this example, all
of the CpG ODN was in the aqueous phase, as was the case for a CpG-MF59 formulation that underwent ultracentrifugation.3 This probably is because these emulsions use nonionic surfactants or neutral phospholipids as emulsifiers. Emulsions made
with charged emulsifiers show higher CpG ODN association with the oil phase and somewhat better immune activity; however,
strong oil association is not necessary for synergistic adjuvant effects demonstrated with neutral emulsion-CpG ODN formulations.3
Because CpG ODNs are efficiently ionized at physiological pH, a change in electrophoretic mobility (zeta potential) of the
emulsion can be observed if the CpG ODN molecules associate preferentially into the interface between the oil and water phases.
Differences in the composition and local chemical potentials at the interface can adjust to balance the electrostatic–hydrophobic
interactions that shift as the activity of CpG grows with increasing solution concentration. To measure the change in emulsion
interfacial charge caused by CpG ODN interactions, a titration of zeta potential versus CpG ODN concentration was performed.
The emulsion used here was the MF59-like emulsion described above. The emulsion was diluted 10-fold in water to permit light
beam transmission of the scattering solution, as well as lowering the ionic strength to decrease the conductivity and increase
the Debye length, permitting a more accurate measurement.
Figure 4. Zeta potential measurement of the titration of CpG 1826 in an MF59-like emulsion. Error bars represent triplicate
measurements from a single experiment.
|
The diluted emulsion was titrated against 4.0 mg/mL CpG ODN and changes in emulsion surface charge and curvature were measured
by measuring both zeta potential and particle size as described above. In this example, the effects were subtle, with a gradual
downward slope in zeta potential from 0 to ~0.1 mg/mL CpG ODN, a gradual upward slope from ~0.1 to 0.25 mg/mL (Figure 4),
and an abrupt shift of ~2 mV at ~0.25 mg/mL CpG ODN, possibly caused by a reorganization of the molecules in the interfacial
Stern layer to accommodate the changes in the aqueous phase as solvation of the added CpG becomes more extensive. At higher
CpG ODN concentrations up to 0.9 mg/mL, the zeta potential is relatively constant.
Conclusion
Appropriate formulation of CpG ODN can significantly improve vaccine adjuvant activity. Rational design of CpG ODN formulations
is enabled by thorough characterization of fundamental formulation parameters. In addition, changes in vaccine excipient composition
should be monitored to determine effects on CpG ODN formulation. In this work, simple but informative physicochemical analytical
techniques have been effectively used to determine the extent of CpG ODN association with particulate formulations, namely
aluminum hydroxide and oil-in-water emulsion formulations.
|
