Chitosan-Carrageenan – Encapsulation and Controlled Release

The optimized process conditions determined using the RSMs were a chitosan concentration of 0.5mg/mL, a chitosan to carrageenan mass ratio of 3:1 and a pH of 5. RSM predicted under these conditions a nanoparticle diameter of ~310nm, zeta potential of ~50mV and a polydispersity of ~0.235 could be achieved and was confirmed by DLS and LDE (Table 7- 4). Similar chitosan-TPP nanoparticles were investigated as an encapsulation and controlled release mechanism for rHu-EPO and yielded an encapsulation efficiency of 34.5% [92], which is considerably lower than the 47.97±4.10% obtained by this study’s chitosan-carrageenan

nanoparticles.

Table 7-3: rHu-EPO encapsulation results within chitosan-carrageenan.

Trial

Encapsulation

Mass Encapsulated (µg) Encapsulation Efficiency (%)

Low MW CS 13.65±0.32 44.36±3.40

Medium MW CSi 14.76±0.58 47.97±4.10

High MW CS 15.58±0.20 50.13±2.11

~45mV - CS-CG NPs 13.77±0.05 44.74±0.51

~40mV - CS-CG NPs 13.01±0.21 42.28±2.17

i _{These chitosan-carrageenan nanoparticles doubled as the ~50mV nanoparticles.}

Under the RSM optimized conditions, the particle diameter and zeta potential of chitosan-carrageenan nanoparticles were tested for both blank and rHu-EPO loaded

nanoparticles. The validity of the RSMs developed was furthered during this study since the blank nanoparticle measurements demonstrated consistency between the actual particle characteristics and those predicted by the RSMs. The average difference between the particle diameter of blank and rHu-EPO loaded nanoparticles was ~19.5nm and the average difference in zeta potential was ~7.5mV. Overall the addition of rHu-EPO was revealed to increase the

particle diameter and decrease the particle zeta potential (Table 7-4). Similar to the results for chitosan-TPP nanoparticles, as the chitosan MW increased so did the drop in zeta potential when rHu-EPO was added. Furthering the theory that the effect may be associated with encapsulation efficiency since, again for chitosan-carrageenan nanoparticles, increasing chitosan MW led to greater rHu-EPO encapsulation. Unlike the chitosan-TPP nanoparticles, the results did not imply

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that the lower the zeta potential without rHu-EPO incorporated the larger the drop in zeta potential when rHu-EPO is encapsulated. The chitosan-carrageenan nanoparticles displayed a trend of increasing difference in particle size between blank and rHu-EPO loaded nanoparticles with increased chitosan MW and decreased initial particle zeta potential. These trends could be explained using the same theories as before where the increase in rHu-EPO encapsulation, thus the amount of rHu-EPO within the structure and on the particle surface, resulted in a greater particle diameter and the decrease in initial particle zeta potential meant a more rapid destabilization of the nanoparticles when rHu-EPO was included.

Table 7-4: Comparison of particle diameter and zeta potential between blank and EPO loaded chitosan-carrageenan nanoparticles.

Trial

Particle Diameter (nm) Zeta Potential (mV)

RSM Prediction Blank NPs EPO- NPs RSM Prediction Blank NPs EPO- NPs LMW CS ~ 221.8±2.7 235.4±1.3 ~ 48.44±1.33 43.86±1.51 MMW CSi 311.0 305.9±2.8 322.2±4.3 50 48.89±0.94 42.22±2.38 HMW CS ~ 307.1±3.3 334.7±3.7 ~ 47.37±0.62 37.43±0.96 ~45mV CS-CG NPs 309.1 316.1±2.4 334.2±3.6 45 44.74±0.73 35.60±2.84 ~40mV CS-CG NPs 316.2 318.8±3.1 340.0±4.1 40 40.82±0.67 33.75±1.43

i _{These chitosan-carrageenan nanoparticles doubled as the ~50mV nanoparticles.}

The effect of chitosan MW on the encapsulation and controlled release of rHu-EPO was studied using three commercially available grades of chitosan (low, medium and high MW). During this comparison all conditions were maintained at the optimum settings determined by the RSMs and only the grade of chitosan was altered. It was found that as the MW of chitosan was increased the encapsulation efficiency rose (Table 7-3), potentially due to an increase in the nanoparticle porosity and changes in the sterical arrangement of the polymer chain, which may allow for a greater number of hydrogen bonding sites between chitosan and the therapeutic agent [96]. Another comparison was the effect of surface charge on encapsulation and controlled release, which utilized the RSMs developed to manipulate the nanoparticle zeta potential. As the

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zeta potential increased from 40mV to 50mV the encapsulation efficiency of the chitosan- carrageenan nanoparticles also increased.

The optimized chitosan-carrageenan nanoparticles (medium MW chitosan) in the first 48 hours released ~32% percent of the encapsulated rHu-EPO, in the first week ~42% and in a two week time-span ~50%. When compared to other nano/microparticle rHu-EPO delivery systems this rate of release is within the 30-40% range reported for the first 48 hours and is less than the 60-80% rate of release obtained during a period of two weeks [9, 92].

Figure 7-3: Comparison of rHu-EPO percent release over time for chitosan-carrageenan nanoparticles produced using low, medium and high MW chitosan. [CS] = 0.5mg/mL, CS:CG = 3:1, 5.0 pH and T= 20±1°C.

For all of the trials conducted, the bulk of the drug release occurred within the initial 24 hours and is most likely due to a large amount of rHu-EPO adsorbed to the particle surface [5]. Altering the chitosan MW from low to high decreased the amount of rHu-EPO released, with the main difference in release rate being within the first 12 hours (Fig. 7-3). The decrease in rHu- EPO release rate, as mentioned earlier, may be due to an increase in the number of bonding sites,

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which could lead to multiple bonds between chitosan and rHu-EPO reducing the rate of release. Also, mentioned previously, the nanoparticle swelling decreases with an increase in chitosan MW, which could contribute to a decreased release rate. The manipulation of nanoparticle zeta potential between 50mV and 40mV demonstrated that decreasing the surface charge leads to increased rHu-EPO release, evidence that manipulation of the nanoparticle surface charge can allow for control over the drug release rate (Fig. 7-4).

Figure 7-4: Comparison of rHu-EPO percent release over time for chitosan-carrageenan nanoparticles with surface charges of ~40mV, ~45mV and ~50mV. Medium MW chitosan, T= 20±1°C.

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Chapter 8