Investigating the Impact of Processing Method on Proteins in Nanocomposites

Since there had not been any previous literature reports on the incorporation of protein fibrils into a polymer matrix, it was important to investigate the impact of the polymer-fibril mixing on the protein control and protein fibrils and ensure that the fibrils remained intact. ThT assay, TEM and CR staining with polarising light microscopy were utilised to ascertain if fibrils were retained in the PVOH + fibril films, and whether there was any indication of amyloid formation in the control PVOH + protein control films.

4.5.1 Characterising insulin fibrils in the nanocomposites by ThT and TEM The ThT assay was carried out on PVOH + insulin mixtures to determine the impact of processing on the insulin fibrils and insulin control. Figure 4.5 shows the intensity of ThT fluorescence and suggests that the dialysis and mixing of insulin fibrils with PVOH did not affect the β-sheet content (columns 4 and 5). Similarly the insulin control was not affected by the steps involved in preparing a PVOH-native insulin mixture, since the ThT fluorescence remained negligible (columns 1 and 2), pre and post PVOH mixing.

0 10000 20000 30000 40000 50000 60000 70000 80000 1 2 3 4 5 R el ati ve fl u o re sce n ce i n ten si ty (4 82 n m )

Figure 4.5 ThT assay of casting solutions with insulin control and insulin fibrils, with

and without PVOH. Lane 1: 0.6% insulin control, Lane 2: 2.5% PVOH + 0.6% insulin control, Lane 3: 0.6% insulin fibrils before dialysis, Lane 4: 0.6% insulin fibrils after dialysis in 12 mM HCl, Lane 5: 2.5% PVOH + 0.6% insulin fibrils. All PVOH + protein solutions were made up with dialysed protein solutions at pH 4. Insulin fibrils were prepared in 25 mM HCl (100 mM NaCl, pH 1.6), at 60°C for 24 hr. ThT assay was carried out in situ on the plate reader, in triplicates, with 198.4 μl of protein solution and 1.6 μl of (2.5 mM) ThT in the wells.

The TEM micrographs of the two composite solutions from Figure 4.5 (Lane 2 and 5) prior to casting are shown in Figure 4.6. In the images, observation of theproteins and protein nanofibres is made particularly difficult by the presence (and thickness)of the PVOH, which strongly reduces the image contrast. Figure 4.6 (a) shows that native insulin is present in the form of dark globular aggregates in the films. Figure 4.6 (b) shows dark contrast, which may have a nanofibre network, but the image is not distinct. However, further characterisation of the composite solutions was carried out using CR and examination of films under cross polarised light.

Figure 4.6 TEM analysis of the 2.5% PVOH composite mixture. (a) PVOH + 0.6%

insulin control, (b) PVOH + 0.6% insulin fibrils. Insulin control and fibrils had been dialysed before mixing with PVOH.

4.5.2 Using CR to detect amyloid in PVOH nanocomposites Further attempts were made to investigate the stability of insulin control and

insulin fibrils in PVOH films using CR, which has a structure as shown in Figure 4.7. CR dye is a diazo dye of symmetrical structure (Puchtler et al. 1962) which forms hydrophobic interactions with the β-sheets of the fibrils (Westermark et al.

1999). Light microscopic studies demonstrated that the birefringence of amyloid deposits increases intensely after staining with CR (Puchtler and Sweat 1965).

N N NaO3S +_NH 3 N N H3N+ SO3Na

Figure 4.7 The structure of Congo red.

Insulin (control and fibrils) solutions were stained with CR dye prior to mixing with PVOH and casting. Staining was carried out by adding CR at 1 mg/ml to the solutions used to make the protein control and protein fibrils respectively. The solutions were allowed to equilibrate for 30 min to allow the CR to bind. The protein solutions were dialysed to remove any unbound CR. Figure 4.8 shows the PVOH films with insulin fibrils at varying levels.

(a) (b) (c) (d)

Figure 4.8 2.5% PVOH-insulin fibrils composites with CR in casting dishes, while the

water was evaporated from the solutions. (a) PVOH + no fibril control, (b) PVOH + 0.2% insulin fibrils, (c) PVOH + 0.4 % insulin fibrils, (d) PVOH + 0.6% insulin fibrils.

Films composed of PVOH + no protein control, PVOH + insulin control, PVOH + insulin fibrils with CR were viewed with a light microscope fitted with a cross polarising lens. The CR dye emits a bright yellow-green birefringence under polarised light when bound to amyloid fibrils (Puchtler and Sweat 1965) . Figure 4.9 demonstrates the CR binding on 2.5% PVOH films with and without the insulin fibrils and insulin control. PVOH + no protein control films showed no

yellow-green birefringence observed in the controls when examined under unpolarised light.

The films with insulin fibrils (Figure 4.9 f) showed yellow-green birefringence under the cross polarised lens, confirming the presence of amyloid fibrils in the films. No yellow-green birefringence was observed for PVOH + insulin control films under cross polarised light (Figure 4.9 d). The film manufacture method did not induce fibrillation of the insulin control. Similarly the PVOH + no protein control film showed no amyloid present (Figure 4.9 b).

Unpolarised light Cross polarised light

(a) (b) (c) (d) (e) (f)

Figure 4.9 PVOH films when viewed under unpolarised and cross polarised lens.

Images are at 40x magnification: (a) and (b) PVOH + no protein control + CR, (c) and (d) PVOH + insulin control + CR, (e) and (f) PVOH + insulin fibrils + CR. Fibrils were prepared in HCl (25 mM HCl, 100 mM NaCl). Protein solutions were dialysed and the buffer was diluted at pH 4.

The impact of film manufacture method on crude crystallin protein was also assessed by CR. Figure 4.10 shows that the PVOH + crystallin fibrils (Figure 4.10 d) do not show a yellow-green birefringence similar to the crystallin control films. This can be attributed to the low concentration of fibrils in the crystallins and CR requires a significant level of amyloid fibrils to be able to bind. However, CR served as a diagnostic tool for in vitro detection of amyloid in polymer nanocomposites such as PVOH + insulin fibril film method development work.

Unpolarised light Cross polarised light

(a) (b)

(c) (d)

Figure 4.10 2.5% PVOH films when viewed under unpolarised and cross polarised

lens. Images were taken 10x magnification: (a) and (b) PVOH + crystallin control + CR, (c) and (d) PVOH + crystallin fibrils + CR. Fibrils were prepared in 10% TFE and final concentration in the films were 0.6%. Protein solutions were dialysed and the buffer was diluted at pH 4.

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