1. No measureable binding
Chitosan’s Mileu Alters its Interaction with Protein Biologics
Sean Smith1,2, Srinivas Jayanthi3, David Zaharoff1,2
1Department of Biomedical Engineering, University of Arkansas
2Laboratory for Vaccine and Immunotherapy Delivery, University of Arkansas
3Department of Chemistry and Biochemistry, University of Arkansas
Clean up figure 1
Conclusions
Aim
Future Directions
Explore the trends of pH and salt concentration with other proteins such as bovine serum albumin and lysozyme.
Modify the attributes of chitosan such as the molecular weight and degree of deacytlation.
Explore the effect of different solvents.
Perform in vitro loading and release studies.
The binding of proteins to chitosan in dilute solution is primarily due to ionic interactions.
These ionic interactions are dependent on the pH of the solution and the charge of the protein.
Higher net negative charges on the protein lead to tighter binding.
At physiological salt concentrations the ionic interaction of chitosan to proteins is sharply diminished if not eliminated. Thus, ionic interactions are not likely to be the main source of controlled release under physiological conditions.
Use binding thermodynamics to characterize the effect that environmental conditions including pH, ionic strength, solvent, and temperature have upon chitosan binding to therapeutic molecules in solution.
Use this understanding to predict and optimize the release of therapeutic molecules from chitosan solution.
Introduction
Introduction
Materials and Methods
Preparation of chitosan and Ovalbumin. Chitosan (Sigma Aldrich) with an average MW of 96 kD and degree of deacytlation of 75 was weighed out and dissolved in 0.1 M HCl to form a 10 μM solution. Ovalbumin (Sigma) was dissolved in water at a concentration of 500 μM. Both solutions were dialyzed separately against water and then twice together against water that was adjusted to the desired ionic strength by addition of NaCl. Both solutions were removed from dialysis and adjusted to the appropriate pH using HCl and NaOH. The buffer from the last round of dialysis was saved and also adjusted to the appropriate pH. The concentration of chitosan was determined by mass while that of ovalbumin was determined by A280 following the pH adjustment.
Isothermal Titration Calorimetry. MicroCal’s VP-ITC was used to titrate μM Ovalbumin into 200 nM chitosan. The cell volume was 1.4 mL while each injection volume was 10 μL. The cell was maintained at a temperature of 25 oC and the reaction mixture stirred at 310 RPM. Three blank experiments were performed for each data point and subtracted from the raw titration curves before fitting with MicroCal’s Origin.
Table 2: Summary of titrations performed at each data point. Italics indicate blank experiments that account for extraneous heats.
Why Chitosan?
Results
Second most abundant natural polymer
Highly Biocompatible and biodegradable
Soluble in slightly acidic aqueous solutions
Can be formed into hydrogels, nano/micro particles, powders, and solutions for controlled release applications.
Enhances absorption across membranes.
Currently used in our lab as a component of a cancer immunotherapy.
Only pseudonatural cationic polymer
Table 1: Summary of the thermodynamic characterization of ovalbumin and chitosan under varying conditions. pH
[NaCl] (mM) N K
ΔH (kcal/mol)
ΔS (kcal/mol)
ΔG (kcal/mol)
5.5
13.4
1.86E+06
-3.62E+04
-92.6
-8.58E+03 6
13.5
2.43E+06
-4.63E+04
-107
-1.44E+04
6.5
8.69
6.46E+06
-4.65E+04
-125
-9.22E+03 10
8.41
3.34E+05
-3.49E+04
-92
-7.47E+03
100
No measureable binding
Figure 2: Primarily derived from the shells of crustaceans, chitin can be deacytlated to produce chitosan.1
pH 5.5
[NaCl] 0 mM
pH 6.5
pH 6.0
[NaCl] 10 mM
Sustained Release from Polymers
Physical entrapment within a polymer matrix and binding of the molecule with the matrix. Can include covalent or ionic interactions.
Previous experiments have shown that chitosan solution injected subcutaneously provides sustained release of fluorescently labeled proteins. 2
Can ionic interactions be used to optimize the release of proteins from chitosan?
Investigate the interactions of chitosan and proteins with various charge states by selecting proteins with known isoelectric points and modifying the pH.
Cell
Syringe
Ovalbumin
Chitosan
Buffer
Isothermal Titration Calorimetry
Acknowledgements
References
1. Ravi Kumar MNV. A review of chitin and chitosan applications. React Funct Polym 2000;46:1-27.
2. Zaharoff DA, Rogers CJ, Hance KW, Schlom J, Greiner JW. Chitosan solution enhances both humoral and cell-mediated immune responses to subcutaneous vaccination. Vaccine 2007;25:
3. Freyer MW, Lewis EA. Isothermal titration calorimetry: experimental design, data analysis, and probing macromolecule/ligand binding and kinetic interactions. Methods Cell Biol 2008;84:
The authors would like to thank Suresh Kumar and all of the members of his laboratory for their patience and assistance as we learned the in’s and out’s of ITC.
Known volume per injection
Known volume
Provides a full thermodynamic characterization in a single experiment.
ΔH: Enthalpy Change
K: Binding constant
N: Stoichiometry
ΔS: Entropy change
ΔG: Gibbs free energy
Figure 1: Representative isothermal titration calorimetry curves under a variety of conditions. The upper plots represent the raw changes in heat over time while the bottom curves indicate the integration of those heats to give ΔH for each injection as a function of the molar ratio of ovalbumin to chitosan. Curve fitting was performed using the model for a single set of binding sites in Origin.
Figure 3: Diagram showing the design of a typical isothermal titration calorimeter.3