ENFOQUE DE PROMOCIÓN PROACTIVA DE INVERSIONES

Charge controlled nanoprecipitation of PCL-MA was investigated first via the methodology developed previously for the nanoprecipitation of PCL-diol (section 4.2.1).

pH Average Particle Diameter (nm)

7.4 60 ± 6 8.0 40 ± 4 9.0 22 ± 1 10.0 23 ± 1 11.0 21 ± 2 12.0 26 ± 5 13.0 51 ± 4

Table 4.5 – Effect of pH on particle diameter for PCL-MA. Particle diameter is reported as the intensity distribution. Error represents ± 1 SD, n = 9

Figure 4.10 – Effect of co-solvent pH on the diameter of PCL-MA nanoparticles. Particle diameter is reported as the intensity distribution. Error bars represent ± 1 SD, n = 9.

0 10 20 30 40 50 60 70 7.0 8.0 9.0 10.0 11.0 12.0 13.0 D iam et er ( n m ) pH

163 The pKa of carboxylic acids in water is typically in the range 3 – 4.[308] As such the majority of carboxyl groups are charged (-COO-) across the investigated pH range. The presence of charged groups reduces the hydrophobicity of PCL and promotes electrostatic repulsion which was observed as a reduction in particle size. The diameter of the particles was observed to decrease linearly from 60 ± 6 nm at pH 7.0 to 22 ± 1 nm at pH 9.0. A particle diameter minima was found between pH 9 – 11 with only small variations in particle size observed within this range. The smallest diameter nanoparticles were observed to form at pH 11 (21 ± 2 nm). As such the data suggests complete ionisation of all terminal carboxyl groups at pH 9.0 and above. As the pH was increased from 12 to 13 the particle diameter was observed to increase as well as the variability of the data. No changes in molecular weight were observed when analysing the samples by 1H NMR spectroscopy, in agreement with previous analyses. Increasing the pH to 12 and 13 additionally increased the ionic strength of the buffer solution. As such a higher concentration of counter ions were present in the electric double layer surrounding the carboxyl groups present on the nanoparticles’ surface. Increased shielding of the particles’ charge could increase the hydrophobicity of the particles, promoting aggregation and increasing particle diameter. However, no investigation of the electric double layer was undertaken and as such the exact reason for the increase in particle diameter remains unclear.

Having investigated the effect of pH on the particle size of PCL-MA nanoparticles, the experiments were repeated using previously synthesised PCL-oTHPA. The data for PCL- oTHPA were compared to those gathered for PCL-MA (Figure 4.11).

pH Average Particle Diameter (nm)

7.4 44 ± 10 8.0 26 ± 11 9.0 14 ± 1 10.0 19 ± 5 11.0 16 ± 2 12.0 19 ± 2 13.0 57 ± 7

Table 4.6 – Effect of pH on particle diameter for PCL-oTHPA. Particle diameter is reported as the intensity distribution. Error represents ± 1 SD, n = 9

164 Figure 4.11 – Comparison of nanoparticle diameters produced via charged controlled

nanoprecipitation of PCL-MA, in black, and PCL-oTHPA, in red. Error bars represent ± 1 SD, n = 9.

An almost identical trend in particle diameter was observed for the precipitation of PCL- oTHPA nanoparticles as for PCL-MA nanoparticles. At all pH values, except pH 13, PCL- oTHPA nanoparticles were observed to be ≥ 5 nm smaller than PCL-MA nanoparticles produced via the same methodology. The reduction in particle size is attributed to the presence of the bridging ether moiety which further increases the hydrophilicity of the polymer terminus. Furthermore, the smallest particles of PCL-oTHPA were observed to form at pH 9 (14 ± 1 nm), in contrast to pH 11 for PCL-MA (21 ± 2 nm). Overall, charge controlled nanoprecipitation of carboxyl terminated PCL presents a significant improvement over conventional solvent- displacement methods when attempting to access ultra-low nanoscale materials.

0 10 20 30 40 50 60 70 7.0 8.0 9.0 10.0 11.0 12.0 13.0 D iam et er ( n m ) pH

165

4.3 Chapter Summary

Nanoprecipitation is a facile methodology for the production of polymeric nanoparticles which commonly utilises commercially available materials and simple laboratory equipment. However, the diameter of particles reported in the literature by this method varies significantly. The importance of carefully selected precipitation parameters was highlighted in this work when developing the method for nanoprecipitation of PCL-OH. Particle diameter was shown to vary between 387 ± 43 and 107 ± 11 as influenced by the initial and final polymer concentrations. Furthermore, the data shown here supports the conclusions previously reported by Reisch et al regarding the strong influence of terminal polymer moieties on particle size and utility of the charge controlled nanoprecipitation method. The formation of polymeric nanoparticles with average diameters ˂ 100 nm was achieved via precipitation of carboxyl terminated PCLs in a range of alkaline solutions. The smallest particle diameter recorded was 14 ± 1 nm for PCL-oTHPA precipitated in pH 9 buffer solution. The hydrophilicity of the polymer terminus was also shown to contribute to the final particle diameter by comparison of PCL-MA and PCL-oTHPA nanoparticles formed by this method.

While the data presented in this chapter is preliminary it serves to highlight the facile nature of charge controlled nanoprecipitation to the formation of particles with an average diameter ˂ 100 nm. In the future, the synthesis of polymers with a wider variety of ionisable terminal moieties could provide additional control over the properties of nanoparticles and the sizes achieved. While particles baring an overall positive charge have been observed to exhibit higher cytotoxicity[306], compared with negatively charged particles, polymers with cationic terminal groups could be synthetically interesting. Furthermore, the polymers synthesised in this work bare terminal vinyl moieties which went unexplored as synthetic handles for further modification of the polymer properties. The utility of thiol-ene and thiol-Michael coupling has already been explored in this work and could conceivably be extended to the introduction of additional ionisable groups, for example by thiol-ene coupling with thioglycolic acid. The influence of multiple ionisable groups on the properties of nanoparticles produced by this method is currently unexplored.

Due to the availability of equipment this work only characterised particles by DLS methods. This work would benefit from a more comprehensive analysis including independent

166 verification of particle size by imaging techniques such as transmission electron microscopy (TEM); quantification of the particles’ surface charge, such as zeta potential measurements; determination of long term particle stability, including the influence of changing buffer solution after particle formation and determination of the nanoparticles’ drug loading potential. Such analysis would guide future developments in this area and provide a more comprehensive overview of the factors at work in such systems.

167