Having successfully optimised styrene RAFT emulsion polymerisations (Section 3.2.1), and implemented a fluorescent labelling strategy (Section 3.2.2), the nanoparticle rigidity study was initiated by synthesising a series of six fluorescently-labelled 100 nm and 50 nm nanoparticles. As discussed in Section 3.2, both of these particle sizes would be generated with either PS, P(t-BA) or P(n-BA) cores and for clarity will be referred to as 100H, 100M, 100S, 50H, 50M, and 50S for 100 nm and 50 nm hard, medium and soft nanoparticles.
Figure 3.9 (A) Preparation of core-shell nanoparticles via RAFT emulsion polymerisation. (B) Graphical representation of hard, medium and soft nanoparticles.
A library of BODIPYA-labelled PS, P(t-BA) and P(n-BA) with different rigidities were prepared using adapted procedures from section 3.2.2.3 (Figure 3.9). Particle size was tuned by controlling either the DPtarget or the initiator type (ACVA or VA-057), as shown
in section 2.2.3.1 (full conditions can be found in Table 3.7), with all polymerisations achieving high monomer conversions (> 98%) within 3 h. Additionally, as some
107 by coloured samples, an analogous series of non-labelled nanoparticles were prepared using the same conditions but omitting the addition of BODIPYA.
DLS and ζ-potential analysis revealed negatively charged (-30 mV), uniform nanoparticles (PDi ~0.05), with diameters close to targeted values for both fluorescent and non-fluorescent analogues (Figure A3.1 and Figure A3.2). Negligible differences in particle diameter or PDi were observed in culture medium Dulbecco’s Modified Eagle
Medium (DMEM) suggesting the suspensions would be colloidally stable during cell uptake experiments (Table 3.3).
Table 3.3. Characterisation data of synthesised nanoparticles.
Hydrodynamic diameter and ζ-potential
Water DMEM
Full title Core Abb. Dh
(nm)a PDi b ZP (mV)c Dh (nm)a PDi b T g/°C d Fluorescent 100 nm Hard PS 100H 107.6 0.054 -31 112.4 0.051 90.0 100 nm Medium P(t-BA) 100M 102.7 0.056 -25 102.8 0.082 37.8 100 nm Soft P(n-BA) 100S 101.5 0.058 -28 104.7 0.062 -57.5 50 nm Hard PS 50H 53.6 0.062 -24 52.7 0.058 90.0 50 nm Medium P(t-BA) 50M 57.9 0.056 -35 49.1 0.065 31.4 50 nm Soft P(n-BA) 50S 54.2 0.047 -30 55.4 0.050 -58.3 Non-fluorescent 100 nm Hard PS 100HNF 100.1 0.054 -29 109.0 0.056 91.0 100 nm Medium P(t-BA) 100MNF 102.3 0.058 -27 108.4 0.064 37.5 100 nm Soft P(n-BA) 100SNF 105.3 0.058 -31 107.1 0.051 -56.5 50 nm Hard PS 50HNF 58.5 0.056 -27 61.5 0.052 82.5 50 nm Medium P(t-BA) 50MNF 52.5 0.040 -31 50.4 0.052 32.9 50 nm Soft P(n-BA) 50SNF 54.0 0.049 -25 58.3 0.058 -57.8 aMean of three measurements using dynamic light scattering (number distribution). bCalculated using
Equation 2.13. cMean of three measurements. dMeasured using differential scanning calorimetry. Subscripts
with ‘NF’ refer to non-fluorescent nanoparticles. All data reported is for nanoparticles after dialysis purification against a 60/40 1,4-dioxane/water (v/v) mixture.
SEC chromatograms of both fluorescent (RI and UV520 nm detection) and non-fluorescent
(RI detection only) nanoparticles indicated successful chain extension from their respective macro-RAFT agents. From UV520 nm traces, fluorescent derivatives displayed
a large proportion of unconsumed BODIPYA. All fluorescently labelled nanoparticles were therefore subjected to the two-step dialysis procedure established in section 3.2.2.3. The complete removal of unconsumed BODIPYA from the polymer latexes was evident from SEC (UV520 nm detection), while RI detection showed a large reduction in the amount
108 (Figure 3.10a), and the polymer signal in the UV520 nm SEC detection (Figure A3.3),
suggested a high amount of fluorescent monomer remained incorporated, with dialysis procedures completely removing any free BODIPYA. Importantly, the particle size distributions showed negligible differences after purification (Figure A3.1). Furthermore, the unconsumed macro-RAFT agent in non-fluorescent analogues (100HNF, 100MNF,
100SNF, 50HNF, 50MNF and 50SNF) was removed by dialysis against pure water, as
confirmed by SEC (RI detection; Figure A3.4). Notable differences in Mn,SEC between
fluorescent and non-fluorescent nanoparticles were observed, which may be ascribed to differences in chain extension efficiency (amount of residual macro-RAFT agent), leading to either smaller or larger chain length, due to the presence of BODIPYA.
Table 3.4. SEC data for dissolved nanoparticles. Non-fluorescent Fluorescent Mn,th (g mol-1)a M n,SEC (g mol-1)b M w,SEC (g mol-1)b Ðb M n,SEC (g mol-1)b M w,SEC (g mol-1)b Ðb 100H 46,500 42,000 47,900 1.14 52,000 64,900 1.23 50H 25,800 28,500 33,000 1.18 40,800 50,600 1.24 100S 26,800 32,800 42,600 1.27 35,300 53,450 1.51 50S 20,400 21,900 26,000 1.19 27,200 37,000 1.36 100M 26,800 31,500 41,500 1.32 24,400 31,300 1.28 50M 21,600 25,900 33,200 1.28 23,100 29,600 1.28
aCalculated using Equation 2.12 and 1H NMR spectroscopy. bDetermined with THF-SEC calibrated with
PMMA standards.
Fluorescence measurements of the fluorescent nanoparticles showed similar emission and excitation profiles to free BODIPYA (Figure 3.4), suggesting that differences in core chemistry did not affect their spectroscopic properties (Figure A3.5). When placed under a UV lamp (λ=365 nm), bright green fluorescence could be observed from the
nanoparticles (Figure 3.10b).
Differential scanning calorimetry was used to evaluate the Tg of all twelve nanoparticles.
Thermograms revealed Tg values of approximately 90°C, 37°C and -55°C for soft,
medium, and hard varieties, which are in close agreement to literature values for PS, P(t- BA) and P(n-BA) respectively (Figure A3.6 and Figure A3.7). Interestingly, traces for all soft nanoparticles displayed a small melting transition at approximately 0°C, which could be attributed to trace water.
109
Figure 3.10. Digital photographs of the BODIPY labelled nanoparticles 100H, 100M, 100S, 50H, 50M and 50S under natural and UV light (λ = 365 nm).
Transmission electron micrographs of the fluorescent nanoparticles displayed similar particle sizes to those obtained with DLS, and confirmed their spherical morphology. Unsurprisingly, the low Tg (P(n-BA)) analogues were not visible under room temperature
dry microscopy, likely due to these nanoparticles flattening as a consequence of flexibility. Cryo-TEM (performed by Dr Saskia Bakker, University of Warwick) was therefore employed to image the P(n-BA) (soft) nanoparticles, clearly showing spherical nanoparticles with similar sizes to those found with DLS.
Figure 3.11. Dry state transmission electron micrographs of 100H, 100M, 50H, 50M (deposited on graphene oxide coated lacey carbon grids) and cryogenic transmission electron micrographs of 100S and 50S (deposited on lacey carbon grids). Cryo-TEM images were obtained by Dr Saskia Bakker, University of Warwick.
A B
110 Solution phase particle size and morphology were further elucidated with a combination of small angle neutron scattering (SANS) and static light scattering (SLS). As described in section 2.2.4, these techniques are complimentary and can give information on different size domains of nanomaterials. For these experiments, only the non-fluorescent 100 nm analogues were investigated as the incident beam wavelength (λ0= 533) in SLS is
exceptionally similar to the excitation maxima for the fluorescent nanoparticles, and as SLS relies on the intensity of scattered light at various angles, any absorption will artificially reduce this value leading to erroneous results. Additionally, due to beam-line limitations only the 100 nm particles were assessed. By combining SLS and SANS data, a wider q range could be observed. The scattering profiles of all three nanoparticles showed similar features, such as a plateau at low q (q < 0.002; Figure 3.12b). With the combined SLS measurements, data fits using hard sphere models with particle radii of 550Å and dispersities between 0.05 and 0.15 showed good agreement with acquired data. Overall, the characterisation data confirmed the synthesised particles were highly similar to those targeted, with negligible structural differences between the different particles (except rigidity), making them suitable for this study.
Figure 3.12. Small angle neutron scattering (blue circles), static light scattering profiles (green circles) and fits (red line) of 100HNF, 100MNF and 100SNF diluted in deuterium oxide.
111
Figure 3.13 Height profiles of a single (A) 100 nm and (B) 50 nm particles with hard (green), medium (blue) and soft (orange) rigidities respectively determined by AFM. Data obtained by Dr Kai Yu and Dr Christopher Hodges at the University of Leeds.
To elucidate rigidity differences, the height profiles of the six fluorescently labelled nanoparticles were studied using AFM after deposition on silanised glass slides. These studies were performed by Dr Kai Yu and Dr Christopher Hodges at the University of Leeds, UK. The heights of both hard derivatives (100H and 50H) matched theoretical values, however the lower Tg (~-59°C) of the soft derivatives (100S and 50S) caused
particle flattening, spreading them by almost two-fold compared to the hard analogues. Interestingly, the medium (Tg = 37°C) nanoparticles for both sizes displayed an
intermediate maximum height suggesting these analogues did indeed have intermediate rigidity (Figure 3.13). AFM images of the nanoparticles can be found in the appendix (Figure A3.8). 0 100 200 300 400 0 20 40 60 80 100 120 H e igh t ( n m) Distance (nm) 50H 50M 50S 0 100 200 300 400 500 600 0 20 40 60 80 100 120 H e igh t ( n m) Distance (nm) 100H 100M 100S
A
B
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