Pure assemblies of peptide form homogenous nanofibrils in solution whereas GAG associated assemblies form a heterogeneous mixture of aggregates.

Somatostatin, substance P and LHRH self-assemblies formed both in the presence and absence of GAGs were characterized by synchrotron small angle X-ray scattering (SAXS). Three types of SAXS intensity profiles were detected as a function of peptide concentration for somatostatin (figure 4.12) and LHRH (figure 4.13). Additionally, figure 4.13 shows SAXS intensity profiles obtained for 5%, 10% and 20% of substance P assemblies.

SAXS intensity profiles given in figure 4.12A were gained from supernatants of 10% (w/w) somatostatin aggregates formed in the presence of sub-equimolar concentrations of GAGs during the lag phase. Guinier analysis was conducted for those SAXS profiles to obtain the radius of gyration (Rg), which is a parameter that provides information about the size of species formed in solution [31, 32](Table 4.4). These Rg values were also compared with previously published Rg values for pure somatostatin monomer and somatostatin-heparin complexes obtained from all atom molecular dynamic (MD) simulations [14]. When comparing with MD simulation data, pure somatostatin forms dimers or small oligomers in solution. This observation is supported by previously reported work for somatostatin derivative lanreotide which states that the dimer is a thermodynamically stable intermediate in their self-assembly pathway [22]. According to the Rg values calculated herein, molecular species present within the supernatants of the GAG-peptide aggregates is almost four times greater than that of the pure somatostatin. In addition, Rg values obtained from MD simulation for the somatostatin- heparin complex are similar to the Rg values calculated in this study. These data support the fact that high binding affinity of negatively charged GAG molecules to positively charged peptide leads to the formation of GAG bound aggregates which is the main species in the supernatants of GAG-somatostatin samples.

118

Figure 4.12: Synchrotron SAXS intensity profiles for Somatostatin assemblies. (A): SAXS plots obtained for

supernatants of 10% w/w peptide-GAG samples with sub-equimolar heparin/chondroitin and during the lag phase of 10% w/w somatostatin. (B) SAXS plots for 1% w/w peptide samples with/without GAGs. (C) SAXS plots for 5% w/w peptide samples with/without GAGs. (D) SAXS plots for 20% w/w samples with/without GAGs.

Table 4.4: Radii of gyration (Rg) obtained from SAXS Guinier plots for somatostatin (Figure 4.12A) and LHRH (Figure 4.13A) and reported from all atoms molecular dynamics for somatostatin [14].

Guinier analysis (Rg, Å) Molecular dynamics (Rg, Å)

Somatostatin 9.75 (90%) 5.5-7.0 (monomer) Somatostatin+chondroitin 22.22 (77%) Somatostatin+heparin 22.11 (77%) 23-32 (aggregates) LHRH 8.53 (87%) LHRH+chondroitin 9.14 (80%) LHRH+heparin 8.38 (93%)

119

Figure 4.13: Synchrotron SAXS intensity profiles for LHRH assemblies. (A): SAXS plots obtained for

supernatants of 10% w/w peptide-GAG samples with sub-equimolar heparin/chondroitin and during the lag phase of 10% w/w LHRH. (B) SAXS plots for 5% w/w LHRH samples with/without heparin. (C) SAXS plots for 20% w/w Substance P with/without heparin.

120

Figure 4.14: Synchrotron SAXS intensity profiles for substance P assemblies formed in the presence and absence of heparin. SAXS intensity profile of 5%w/w substance (A) without (B) with heparin. SAXS intensity

profile of 10%w/w substance (C) without (D) with heparin. SAXS intensity profile of 20%w/w substance (E)

without (F) with heparin.

Surprisingly, in the case of LHRH, we observed similar Rg values for pure LHRH (8.53 Å), LHRH-chondroitin (9.14 Å) and LHRH-heparin (8.38 Å) oligomers (Figure 4.13A). Since LHRH-GAGs aggregates are larger and heavier and insoluble it is possible that they might have fallen to the bottom of the capillary and have been missed by the X-ray beam, while pure LHRH oligomers are found in the top part of the capillary. We might hence not have captured LHRH-GAGs aggregates.

121 The second type of SAXS intensity profiles was obtained for somatostatin (1% and 5%) (Figure 4.12B and 4.12C) and LHRH (5%) (Figure 4.13B) at an equimolar concentration of GAGs to peptide. These SAXS intensity profiles display a slope that leans towards q-x _{(x>0) in q range}

0.01-0.6 Å. These slopes are due to the scattering of nanoscale colloids in solution which provides information on their morphology such as fibrillar, crystals and aggregates. All calculated slopes and corresponding interpretation according to previous reports are tabulated in table 4.5.

Table 4.5: Slope (q−x_{) of the SAXS intensity profiles at low scattering vector values: q in the}

range 0.01–0.02 Å−1 _{corresponding to distance in the range 310–630 Å (31–63 nm).}

Sample q-x _{min q}-x _{max Interpretation [10]}

Somatostatin

1%SST 0.808 1.948 q-1_{: rod and q}-2_{: flat 2D}

5%SST 0.771 0.828 q-1_{: rod}

1%SST+CS 3.602 4.048 q-4_{: unstructured/ spherical aggregates}

5%SST+CS 3.186 4.098 q-2: 2D flat and q-4: unstructured/spherical aggregates

1%SST+hep 3.821 3.632 q-2: 2D flat and q-4: unstructured/spherical aggregates

5%SST+hep 3.394 3.559 q-2: 2D flat and q-4: unstructured/spherical aggregates

LHRH

5%LHRH 0.903 1.140 q-1_{: rod}

5%LHRH+CS 3.897 4.138 q-4_{: unstructured/ spherical aggregates}

5%LHRH+hep 3.340 3.365 q-2_{: 2D flat and q}-4_{: unstructured/spherical}

aggregates

According to the slope interpretation, somatostatin assemblies formed in the presence of GAGs mainly consist of unstructured or spherical aggregates, whereas pure somatostatin assemblies at the same concentration consist of rod-like (nanofibrils) and flat 2D structures (laterally associated fibrils). These results aligned with TEM and AFM data obtained for the same range of concentrations suggest that the presence of heparin leads to the precipitation of somatostatin protofilaments. Similar results were obtained for LHRH assemblies as well (Figure 4.12B). Pure LHRH assemblies consist of rod-like (nanofibrils) structures, while GAGs mediated

122 LHRH assemblies at the same concentration consist of unstructured or spherical aggregates (Table 4.5).

Figure 4.12 (D) shows a third type of SAXS intensity profiles obtained for high concentrations of (20% w/w) somatostatin both in the presence and absence of GAGs. The correlation reflections observed for pure somatostatin are completely absent in the presence of heparin but present to a lesser extent in the case of chondroitin. These correlation peaks (6.3 nm and 12.5 nm) are proposed to arise from inner symmetries within the self-assemblies. Based on fibril diameter obtained for pure somatostatin (1.5 nm) from AFM data, these distances can be interpreted as laterally-associated arrays of fibrils. These results are consistent with images obtained for pure somatostatin by cryo-SEM that showed fibre arrays. Indeed, this observation also supported by parallel β-sheet networks arises due to the lateral association of fibrils as identified by IR spectroscopy. The absence of correlation peaks in heparin-somatostatin assemblies aligns well with the results obtained by AFM and cryo-SEM, which showed disordered aggregates due to the loss of long-range order and loss of parallel beta-networks as detected by IR spectroscopy. The LHRH assemblies formed in the presence of heparin also display loss of reflections by SAXS, which typically arise due to the presence of a hexagonal network within the assembly (Figure 4.13D). LHRH-heparin SAXS intensity profile shows two other peaks corresponding to d-spacing of 27.3 Å and 10.6 Å which could be assigned to inner symmetries within the assembly and distance between β-sheet networks respectively. This result is also in accordance with IR spectroscopic results, which showed a loss of parallel β-sheet network for LHRH-heparin assemblies.

In contrast to somatostatin and LHRH, in the case of substance P, oscillations for nanotubes were observed for both pure and heparin-assisted assemblies for all the concentrations studied (Figure 4.14). Substance P nanotubes formed both in the presence and absence heparin were fitted to a core-shell cylinder with an internal diameter (di), a wall thickness (twall) and a tube

length (l). Table 4.6 displays modelled parameters of substance P nanotubes both in the presence and absence of heparin.

123 Table 4.6: Modelled parameters of substance P nanotubes

SP concentration (w/w) Diameter/di (nm) Wall thickness/wallt (nm) Length/l (nm) 5% 6.54 0.90 100.14 5%+heparin 6.13 0.94 181.88 10% 6.20 1.21 100.14 10%+heparin 6.85 1.47 181.88 20% 6.13 1.21 100.14 20%+heparin 7.02 0.92 120

Both pure substance P nanotubes and heparin assisted nanotubes show approximately similar diameter and wall thickness. However, at all concentrations studied heparin assisted tubes are longer than that of pure substance P nanotubes. The influence of concentration on tube internal diameter, wall thickness and length were not observed for both pure substance P and heparin assisted assemblies. These results suggest that heparin might not have an influence on nanotube growth as evidenced by similar tube diameter and wall thickness in both types of assemblies. However, heparin might influence tube length by making tubes longer.

As previously reported for somatostatin analog lanreotide which is also a positively charge peptide, electrostatic repulsions between peptide nanotubes are important in tube growth [22]. Typically, interactions between positively charged amino acids in peptide structure and negatively charged sulphate groups in heparin molecules might screens repulsive interactions between peptide nanotubes. However, due to the lack of interactions between substance P and heparin, nanotubes formed at both cases show similar tube diameter.

124

4.4 Discussion