(96d) Designing and Characterizing Chitosan Particles for Drug and Vaccine Delivery Applications

Chitosan is a nontoxic, biodegradable, and biocompatible polysaccharide useful for several biomedical applications. The purpose of this study is: (1) to use electrospraying, a less widely studied technique, for the generation of micro- and nanoparticles; (2) to develop an empirical relationship between the chitosan particle size and shape and the electrospraying process parameters; and (3) to apply this model for improved encapsulation of bovine serum albumin. The generation of chitosan micro- and nanoparticles allows for the use of a noninvasive, controlled method of administering bioactive agents in drug and vaccine delivery applications. By systematically varying experimental parameters, including voltage, flow rate, needle gauge, chitosan molecular weight, concentrations of cosolvents and surfactants, solution viscosity, and surface tension, the condition under which each particle shape forms is determined. Collected particles are quantified using optical microscopy and light scattering techniques. The microscopy images serve as visual representations of the effects each parameter has on the generation of chitosan micro- and nanoparticles. Preliminary results of this study show how the electrospraying parameters are dependent upon one another. Adjusting one parameter changes the appropriate range of operation of other process and system parameters. Using proper choices of process variables, particles with diameters ranging from the nanometer to micrometer range are obtained. This goal of this study is to develop an empirical model for the production of micro- and nanoparticles for drug and vaccine delivery systems, especially for encapsulating protein-based vaccines in polysaccharide carriers.

KEYWORDS electrospraying, polysaccharide, microparticles, drug delivery

1. Introduction

Electrospraying is a technique that utilizes an electric field to reshape the meniscus of a solution at the tip of a tube, causing the droplets of solution released from the controlled continuous dispensing mechanism to be microscopic in size. Preliminary data have been collected for chitosan solutions that were emitted through the electrospray apparatus to form spherical micro- and nanoparticles of chitosan.

The formation of polyelectrolyte complexes (PECs) with chitosan and bovine serum albumin (BSA) has been evaluated, the results of which will be applied to the electrospraying process. PECs are formed between oppositely-charged polymers due to electrostatic interaction of polyions. Chitosan, a polycation in mildly acidic to neutral conditions,^[1,2] aggregates with the model protein BSA due to the anionic nature of the protein under appropriately selected solution pH.

This study focuses on identifying critical process parameters and highlighting important trends in electrospraying so that in the future electrospraying can be a reliable method of creating polymer nanoparticles for drug and vaccine delivery applications.^[3]

2. Methodology

2.1 Electrospraying

Chitosan particles were prepared by electrospraying chitosan dissolved in acetic acid. Chitosan solutions of known concentrations were placed in a 10 mL syringe connected to the emitter electrode through silicone tubing. The solution was sprayed from the emitter using a syringe pump that provided a constant specified rate throughout the experiment. At a sufficiently high voltage, the liquid meniscus took the form of an axisymmetric cone, called the Taylor cone. In this cone-jet mode, as the electrostatic forces exceed the surface tension of the solution, a straight, stable jet, < 100 mm in diameter, was formed at the apex of the cone. The end of the jet broke into droplets while traveling toward the collector electrode. The size of the emitted droplets was smaller than that obtained in the absence of the electric field. Different collection media were used throughout the study. The particles collected were quantified using optical microscopy.

2.2 Microparticle synthesis by polyelectrolyte complexation

Solutions of chitosan and BSA were created and adjusted to the desired pH. These solutions were combined in an equal ratio and diluted to be evaluated with dynamic light scattering and UV-vis spectroscopy.

3. Results

3.1. Chitosan Solution Properties

These physical properties of the chitosan solutions were compared with those of 10% acetic acid that served as the solvent.

Molecular weight and pH. The pH of solutions with chitosan concentrations between 1.0 and 2.5 % (w/v) at low, medium and high molecular weights were measured. The molecular weight of chitosan did not have a noticeable impact on the pH of the solution, but the pH increased with the concentration of chitosan.

Surface Tension. Surface tension was an important factor to be analyzed as the chitosan solution must have surface tension low enough to obtain small droplets, but high enough to penetrate the surface barrier of the liquid collection media (yielding a dispersion of microparticles in the collection medium, without forming a film at the surface). The surface tension of each solution was measured using a bubble pressure tensiometer. Overall, the higher the concentration and molecular weight of chitosan in the solution, the higher the surface tension of the solution. To lower the surface tension, ethanol, or other cosolvents or surfactants, were added to the solution.

Ionic Conductivity. The lower the conductivity of a solution being electrosprayed, the more stable the Taylor cone and stable jet is likely to be. As the chitosan concentration was increased, the conductivity of the solution also increased. Molecular weight of chitosan did not have a significant effect on conductivity. Knowledge of the relationship between surface tension and conductivity will not only allow electrospray stabilization, but also make it possible to electrospray smaller and more spherical chitosan microparticles.

Viscosity. The viscosity of the polymer solution is expected to affect both the shape and size of the microparticles The kinematic viscosity of each solution was measured using an Ubbelohde viscometer. Viscosity was found to increase as the concentration and molecular weight of chitosan in the solution increased. It was also found that more viscous solutions, such as 2.5 % of the high molecular weight chitosan, formed a more stable jet at higher flow rates, while less viscous solutions, including 1.0 % of the low molecular weight chitosan and 1.0 % of the high molecular weight chitosan, led to a more stable jet at lower flow rates.

3.2. Effect of Emitter Diameter

Needles of three different sizes (23, 27, and 30 gauge) were used as emitters. After analysis of their influence on electrospraying, the 27-gauge needle was found to be the optimal size for this study and will be used for all future experiments.

3.3 Chitosan-BSA Polyelectrolyte Complexes

Particle size. Prior to electrospraying of chitosan with BSA, particle formation within solutions was characterized. A pH value in the range of 5–6, higher than the isoelectric point of BSA (~4.7), was found to be suitable for the desired particle size and particle size distribution. The lower molecular weight chitosan resulted in smaller particles than the higher molecular weight chitosan.

Incorporation of BSA in Microparticles. Centrifugation of the dispersion, and UV-vis spectroscopy of the supernatant solution, was used to determine the fraction of BSA complexed with chitosan in the microparticles. BSA absorbance at 280 nm wavelength was used. More BSA was incorporated in the particles as its concentration in the solution increased. The degree of incorporation was also higher at pH 5 than at pH 6. Up to approximately 47 % of the BSA was found to be complexed with chitosan under these conditions.

The effects of cosolvent/surfactant concentration in the chitosan solution, the flow rate, and the applied voltage on cone-jet formation and the particle size and size distribution, are also under investigation. Taylor cone was not formed below 4 kV, while the jet became unstable above 6 kV.

4. Conclusion

Preliminary results indicate that a limited pH range is ideal for chitosan-BSA complexation. Electrospraying experiments have shown that it is not possible to optimize solution and process variables such as conductivity, viscosity, surface tension, flow rate and voltage independently. Therefore, a combination of properties should be considered for tailoring particle size and shape. Utilizing these optimal parameters will lead to a steady jet and Taylor cone, and thus the formation of particles that are suitable candidates for drug and vaccine delivery applications­­.

Acknowledgements

Financial support for TS and AP, from the Clarkson University Honors Program, is greatly appreciated.

References

[1] Pillai, C. K. S., Paul, W., & Sharma, C. P. (2009). Chitin and chitosan polymers: Chemistry, solubility and fiber formation. Progress in Polymer Science, 34(7), 641-678. doi: 10.1016/j.progpolymsci.2009.04.001

[2] Myrick, J. M., Vendra, V. K. & Krishnan, S. (2014). Self-assembled polysaccharide nanostructures for controlled-release applications. Nanotechnology Reviews, 3(4), 319-346. doi: 10.1515/ntrev-2012-0050

[3] Bock, N., Woodruff, M., Hutmacher, D., & Dargaville, T. (2011). Electrospraying, a reproducible method for production of polymeric microspheres for biomedical applications. Polymers, 3, 131-149. doi: 10.3390/polym3010131