## 4 Discussion  
Chitosan's quick gelling ability on contact with polyanions relies on the creation of inter- and intramolecular cross-linkages mediated by polyanions. This process was shown to be easy to modulate through variation of intrinsic or extrinsic variables, leading to the production of particles with different sizes and charges. Although most previous works focused on the modulation of reaction intrinsic factors (such as chitosan and TPP concentration, chitosan MW or chitosan-to-TPP ratio) [22; 23; 33; 34], this work focused on four reaction extrinsic factors. Of the studied factors, the best described in the literature is environmental pH value, as it was described as being fundamental to ionic gelation, with high pH values leading to chitosan deprotonation. This, in turn, will not be able to interact ionically with TPP's tripolyphosphoric ions. In fact, [PERSON] and [PERSON] [34] showed that at pH values below 4.5, it was difficult to obtain a unimodal NP population, whereas at pH values above 5.2, microparticle formation occurred. Similarly, [PERSON] and [PERSON] [22] stated that at higher pH values, NPs with large size are produced, as deprotonation leads to lower electrostatic repulsion and thereby increases the probability of particle aggregation. [PERSON], [PERSON] [33] showed that when the pH value is three, there is a scarcity of NPs produced due to the TPP's decreasing charge density. This behaviour is in line with the results reported in this work, as NPs were only stably produced at pH 5. Interestingly, the influence of the pH value on the NPs' ZP is quite contentious, as some authors have stated that the environmental pH has no effect on NP ZP as long as it stays within the range where stable NPs are produced [33], whereas others have described NP ZP as being highly sensitive to environmental pH value variation [22]. When considering the other assayed factors, the existing body of work is almost non-existent. [PERSON], [PERSON] [35] reported rotation speed as being a significant factor in NP formation, and [PERSON], [PERSON] [34] reported that an increase in rotation speed from 200 to 800 rpm led to a narrowing in particle size distribution, which is probably due an acceleration in TPP dispersion and a shear force increase which, in turn, improves monodispersity. Similarly, [PERSON], [PERSON] [36] reported that an increase from 200 to 600 rpm led to a reduction of particle size, whereas further increases in rotation speed had no effects. This falls in line with the results obtained at pH 5, as the increase of rotation speed created smaller NPs. Furthermore, the existence of a spot of activity at 500 rpm is similar to the pattern observed by [PERSON], [PERSON] [36], as in their work, they also reported an optimum rotation speed for their test conditions. On the other hand, for addition and reaction time, our results contradict previous research, as these were found to be non-influential in NP formation [35; 36]. The FTIR data obtained showed that chitosan and TPP interacted at a molecular level, an observation which is in line with previous works, and the spectra acquired showed the previously reported chitosan and TPP electrostatic interaction and subsequent complex formation [27; 30; 37]. Freeze-drying is one of the most common techniques used to ensure long term stability of polymeric NPs, as storage in aqueous suspension leads to solubilisation and/or degradation of the polymers, and dried NPs are more easily redispersed. However, freeze-drying has some drawbacks, as aggregation or fusion of NPs may occur, and the formation of ice crystals may lead to their destabilization [23]. These drawbacks were evident in this work, as NPs' size and PDI levels rose after freeze-drying, which is a possible consequence of aggregation due to strong inter- and intramolecular hydrogen bonding. This was also reported by [PERSON], [PERSON] [38] and [PERSON], [PERSON] [39], but was contrary to the findings of [PERSON], [PERSON] [36], who reported that freeze-drying led to a decrease in particle size.