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The size and morphology of the self-assembled polymers in water was investigated by light scattering and microscopy analysis. Initially, DLS measurements were performed at angles from 80° to 140° for a sample of PTEGA-b-PLA2 micelles at

1mg·mL-1 _{to evaluate the angular dependence of the apparent diffusion coefficient} Dapp, see Figure 2.14. From the slope of the plot τ-1 vs. q² (Figure 2.14) Dapp was

calculated and found to be 6.13⨯10-15 _m²ms-1_{, a value that according to the Stokes-}

Einstein equation (section 2.5.1) corresponds to a hard sphere with a hydrodynamic radius of 26 nm.

Figure 2.14. Light scattering results from the self-assembly of PTEGA-b-PLA2 in

56 Figure 2.15. Light scattering results from the self-assembly of PTEGA-b-PLA2 in

water at 1 mg·mL-1_{; Plot of τ}-1_q-2 _{vs. q².}

The same sample was then subjected to SLS measurements to further characterise the self-assembled polymer. The plot of Kc/Rθ vs. q² (Figure 2.15), showed an angular

independence of Kc/Rθ within the 10% error applied to laser light scattering

measurements.31 _{A slight slope could be observed which was attributed to the fact}

that the size of the particles is close to the detection limits of the setup: for spherical particles with a radius smaller than λ/20 (i.e. 30 nm) there is a negligible phase difference between light emitted from various scattering centres within the particle. Thus, the scattering intensity is independent of the angle of observation, and is only dependent on the mass of the particle; in this case, a reliable radius of gyration could not be determined.

The spherical morphology of the micelles was further analysed via synchrotron small angle X-ray scattering (SAXS) experiments.‡ _{Analysis of the SAXS curves were fitted}

to a Guinier-Porod plot (Figure 2.16) confirmed the spherical morphology with an Rg value of 10.2nm, such a difference with light scattering results could be explained

57 by the hydration of the PTEGA shell, and thus the radius visible by SAXS would correspond to the radius of the core and a partial layer of the shell, the one which is denser and near the core.

Figure 2.16. SAXS profile and Guinier-Porod fit of the solution of micelles in water at 1mg·mL-1_.

Transmission electron microscopy (TEM) was also employed to determine the morphology of the assembled polymers. To achieve this, a droplet of the solution used for light scattering measurements was allowed to dry on a graphene oxide TEM grid, which owing to its low thickness allows stain-free observation of the sample (Figure 2.17).32

58 Figure 2.17. Representative TEM image of the self-assembled polymer PTEGA-b-PLA2on GO grids (scalebar=50 nm) and size distribution of the

observed particles.§

The average diameter of the observed particles (ca. 26 nm) was smaller than the hydrodynamic diameter determined from dynamic light scattering (ca. 52nm). This could be attributed to fact that in dry conditions, the volume of the hydrophilic layer is reduced by the absence of water, which in DLS analysis contributes to the size of the self-assembly.

Reactions of the dithiomaleimide-containing polymers with

thiophenol

Optimisation of the reaction

Depending on the nature of the thiol conjugated to the maleimide, the resulting DTM can be highly emissive or not, e.g. the fluorescence emission is drastically decreased when maleimides are directly conjugated to an aromatic ring.19 _{In order}

to induce a dual response through one stimulus for the polymer self-assemblies, thiophenol was chosen as the chemical stimulus, as upon thiol exchange with the thio-poly(lactide) hydrophobic segment, the fluorescence was expected to be

59 reduced and the amphiphilic character of the polymer changed. The reaction conditions were first optimised utilising an analogous small molecule model system: di(thioethanol)maleimide in lieu of the polymer. The first conditions tested for this reaction were inspired from the DTM syntheses presented by Muus et al.22 _and

Robin et al. 21 _{The first conditions utilised were 1:2:2 (DTM, thiophenol, imidazole)}

in THF but no conversion was observed over the 15 hours of monitoring by TLC, which could be attributed to the lack of excess of reagents. Then, the ratio were increased (1:5:5, 1:10:10) to push the reaction toward the product, but even with 1:10:10, no conversion was observed. Since the polymer was self-assembled in water, the reaction was also tested in water with a 1:10:10 ratio, some product was observed by HPLC but the conversion was too low to be isolated. The same reaction was tested without the imidazole catalyst to confirm its effect and the product was obtained with the same conversion, so the following attempts were performed without the imidazole catalyst. The ratios were further increased to 1:20 and 1:50 (DTM: thiophenol), and a drastic increase in conversion was observed.

The conclusions of these optimisation experiments were that the reaction can occur in the absence of imidazole, water seems to be a good solvent for the reaction and the optimum number of molar equivalents of thiophenol is 20 for 1 equivalent of DTM. Moreover, the reaction was monitored by HPLC and a complete consumption of the starting material (1) was observed 10 min after the addition. The different peaks were identified utilising purified solution of each compound, the starting material was also detectable by the HPLC fluorescence detector.

60 Figure 2.18. HPLC traces of the reaction mixture before the reaction and 10 min

later; 1 is the starting material, 2 is thiophenol, 3 is the mono-substituted product, and 4 is the di-substituted product.†

Reaction of PTEGA-b-PLA2 micelles with thiophenol

Based on the results obtained from the reaction of thiophenol with the small DTM model compound it was hypothesised that the optimised conditions, i.e. water, 20 equivalents of thiophenol, were suitable for the reaction of thiophenol with the DTM derivative of the micelles. The PTEGA-b-PLA2 micelle solution was treated with 20

equivalents of thiophenol, and then purified by exhaustive dialysis (MWCO = 1 kDa) against water. It was anticipated that the addition of thiophenol to the micelle solution would result in the addition of thiophenol to the maleimide and synchronous elimination of two thiol terminated PLAs.

61 Scheme 2.4. Reaction of PTEGA-b-PLA2 copolymer in water at 1 mg·mL-1 with

thiophenol.

When the reaction was performed in water, a precipitate was observed; it was assumed this precipitate is the water-insoluble PLA. Despite the results observed with the model reaction (section 2.3.2.1), a small amount of organic solvent (dioxane, THF) was added in an attempt to improve the solubility of the detached PLA in the reaction mixture. The percentage of organic solvent in water was increased up to 20 vol%, but in all cases the PLA-SH residue was found to be insoluble in the reaction mixture. Further increase of the organic solvent ratio was found to interfere with the formation of micelles as a consequence of the increased solubility of the two blocks in the medium, thus preventing segregation and subsequent self-assembly. Therefore, the reaction was performed in water and the resultant heterogeneous solution was centrifuged to remove the precipitate which was confirmed by 1_{H NMR spectroscopy to be mainly composed of PLA (Figure 2.19).}

62 Figure 2.19. 1_{HNMR (400 MHz, CDCl}

3) spectrum of the PLA-SH, obtained after

separation following the addition-elimination reaction compared to 1_{H NMR}

spectrum of the starting block copolymer PTEGA-b-PLA2. The characteristic

signal of the PLA protons is highlighted at 5.2 ppm while the absence of the characteristic TEGA proton signals at 4.2 ppm is also indicated.

In order to further confirm the successful scission of the block copolymer into the corresponding homopolymers, SEC characterisation was employed to compare the chromatograms of the parent copolymer with the two isolated polymers, i.e. the dissolved polymer and the precipitate.

63 Figure 2.20. Molecular weight distributions of PLA2, PTEGA-b-PLA2, PTEGA-

SPh, and PLA-SH. SEC in THF, PS calibration.

Sample Mn,SEC (kg·mol-1)

PLA2 8.1

PTEGA-b-PLA2 19.5

PTEGA-SPh 10

The SEC chromatograms showed a clear decrease of the molecular weight upon exposure to thiophenol (PTEGA-b-PLA2 vs. PTEGA-SPh). The SEC chromatogram of

the PTEGA-SPh exhibiting a tailing could correspond to residual cleaved PLA-SH. The trace of the isolated PLA precipitate showed a double peak, corresponding to half of the PLA2 molecular weight and also an equivalent molecular weight of PLA2,

these could be attributed to the cleaved PLA-SH and possibly a disulfide bridging between two PLA-SH chains.

64 It was shown that the reaction of thiophenol with the DTM moiety present at the interface between the PTEGA block and the PLA2 block was successful and the

majority of the PLA residue could be removed from the reaction mixture.

2.3.2.2.1.Characterisation of the fluorescence emission upon exposure to thiophenol