Conclusions

Chitosan-TPP and chitosan-carrageenan nanoparticles were generated through ionotropic gelation using flush mixing under a variety of conditions to evaluate the effects of chitosan concentration, chitosan to anion mass ratio and solution pH on the nanoparticle characteristics. The resulting chitosan-TPP nanoparticles had particle diameters ranging between 100 and 400nm, surface charges between 30 and 50mV, and particle size distributions ranging between 0.2 and 0.4. While, the chitosan-carrageenan nanoparticles displayed particle diameters between 200 and 1000nm, surfaces charges between 40 and 55mV, and particle size distributions between 0.2 and 0.35. Both types of chitosan nanoparticles grew in size with an increase in chitosan concentration; however, raising the chitosan to anion mass ratio caused the chitosan-TPP nanoparticles to increase in diameter, while causing the chitosan-carrageenan nanoparticles to decrease in diameter. Direct trends were identified between the chitosan concentration and chitosan to anion mass ratio to zeta potential for chitosan-TPP nanoparticles, while chitosan- carrageenan nanoparticles were shown to have a peak zeta potential at a chitosan concentration of 0.5mg/mL, but demonstrated no other trends. The other important factor studied in the screening process, polydispersity, was determined to increase with an increase in chitosan to anion mass ratios for both types of chitosan nanoparticles. Investigation into the particle morphology revealed that both chitosan-TPP and chitosan-carrageenan nanoparticles had no prominent shape, such as a spherical shape. The chitosan-carrageenan morphology did reveal that a significant amount of these nanoparticles had a hexagonal structure that could indicate semi-crystal structure and growth after ionic gelation induced nucleation. The study on the effect of pH on chitosan nanoparticle formation found that the zeta potential decreased with increasing pH for both types of nanoparticles, as well as determined that particle diameter and

polydispersity for chitosan-TPP nanoparticles could be minimized at a solution pH of 5.5. The exploration into the effect of chitosan MW on nanoparticle formation established that the larger

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the MW the larger the particle diameter; however, nanoparticles produced using medium MW chitosan yielded both higher zeta potential and polydispersity.

From this screening process a more narrow range of conditions were selected to be further investigated using the response surface methodology in hopes of creating a more accurate method of controlling the nanoparticle characteristics. Accurate quadratic RSMs were generated for the chitosan-TPP nanoparticles and their three target characteristics that could be used to minimize particle size to as little as 98nm and polydispersity to as low as 0.16, and maximize zeta potential to ~45mV. RSMs were also successfully generated for the chitosan-carrageenan nanoparticle characteristics of particle diameter, zeta potential and polydispersity by linear, quadratic and 2FI RSMs, respectively. Through manipulation of the process conditions using these RSMs the chitosan-carrageenan nanoparticle diameter and polydispersity could be

minimized to ~260nm and ~0.16, while producing particle zeta potential as high as ~52.5mV is possible. Optimized nanoparticles were produced by balancing the different particle

characteristics’ targets, with slightly more emphasis applied to surface charge. The RSMs yielded a chitosan concentration of 0.6mg/mL, chitosan to TPP mass ratio of 4:1 and a pH level of 5.2 as the optimal conditions for chitosan-TPP nanoparticle production. While, the optimized process conditions determined using the RSMs for chitosan-carrageenan nanoparticles were a chitosan concentration of 0.5mg/mL, a chitosan to carrageenan mass ratio of 3:1 and a pH of 5.

The optimized chitosan-TPP nanoparticles resulted in an encapsulation efficiency of 43.45±0.84% and released ~30% of the encapsulated rHu-EPO in 24 hours and ~68% within a two week period. While, the optimized chitosan-carrageenan nanoparticles yielded an

encapsulation efficiency of 47.97±4.10% and released ~32% of the encapsulated rHu-EPO in 48 hours and ~50% within a two week period. Both of the encapsulation results, for the two

different chitosan nanoparticles, are an improvement over the previously reported encapsulation efficiency of 34.5% [91]. The RSMs were also used to determine the effect of surface charge on rHu-EPO encapsulation and release rate, and demonstrated that increasing the surface charge increases the encapsulation efficiency and produces a more stable drug release, which were trends seen in both types of chitosan nanoparticles. The surface charge study also showed that through manipulation of the zeta potential nanoparticles that release a therapeutic agent within a

80

certain desired timeframe can potentially be produced. The effect of chitosan molecular weight on encapsulation and controlled release was determined through experimentation using low, medium and high MW commercial grade chitosan and established that increasing the MW improved the encapsulation efficiency and decreased the rHu-EPO rate of release from both chitosan nanoparticles. The results of this research demonstrate the potential for chitosan nanoparticles as a drug delivery system for rHu-EPO and other therapeutic agents, and the importance of manipulating process conditions to achieve optimum results.

8.2 Recommendations

When comparing the chitosan-TPP and chitosan-carrageenan nanoparticles the results of this work have shown that chitosan-TPP nanoparticles can be produced that have half the particle diameter of that of the smallest chitosan-carrageenan nanoparticles generated. The difference in particle size can be attributed to the difference in polymer length between TPP and the longer carrageenan polymer. The chitosan-carrageenan nanoparticles over all had a greater zeta potential than that of the chitosan-TPP nanoparticles, which later manifested itself in the encapsulation and controlled release of rHu-EPO where the chitosan-carrageenan nanoparticles outperformed the chitosan-TPP nanoparticles in both the encapsulation and controlled release. Based on the results the chitosan-carrageenan nanoparticles appear to be a better drug delivery mechanism for the encapsulation and controlled release rHu-EPO than chitosan-TPP

nanoparticles. However, the application of rHu-EPO must be taken into account since more importance on particle diameter may mean that chitosan-TPP nanoparticles are more suited for certain applications.

One of the more novel applications of rHu-EPO and potential use for rHu-EPO loaded chitosan nanoparticles pertains to the compound’s tissue protective properties and their ability to help patients recover after acute myocardial infarction (MI) [12, 97]. When developing a drug delivery system for the application of rHu-EPO in treatment of MI the nanoparticle system could be inserted onto the tissue at the site of treatment rather than intravenous injection. For such an application the importance of particle size would be diminished, since the size constraints associated with particles in circulation would be removed. Chitosan-carrageenan particles would be the more ideal of the two types of chitosan nanoparticles created in this study for application

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in MI treatment, due its increased encapsulation and prolonged release of rHu-EPO, as well as the removal of size limitations. The use of high MW chitosan would also increase encapsulation and provide a longer more sustained release.

The chitosan-carrageenan nanoparticles did demonstrate a marked improved in encapsulation and controlled release of rHu-EPO; however, the initial drug burst was still witnessed and is an obstacle to maintaining a consistent and prolonged release. In order to get a more consistent release mechanism the use of a cross-linker may be investigated, but as

discussed in Chapter 3 this is less than ideal due to the potentially harmful production conditions. Chitosan-TPP nanoparticles coated with alginate were reported to have a significantly reduced burst effect when releasing BSA [98]; thus, adding an outer layer, whether alginate or another polyanion, could provide the extra resistance needed to prevent the initial rHu-EPO burst.

Further studies into the effect of nanoparticle structure on the biological activity of rHu-EPO and in vivo release studies need to be performed in order determine the true potential of chitosan nanoparticles for rHu-EPO encapsulation and controlled release.

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