Modulation of integrin affinity by changing the RGD-motif

After the UV-Vis-nIR characterization we investigated how different ssDNA-peptide/SWCNT hybrids affect RGD binding to integrins. For this purpose, we have developed an enzyme-linked immunosorbent binding assay (ELISA). This ELISA quantifies the binding between fibrinogen and soluble αIIbβ3 integrin. After adding RGD-containing polymer/SWCNTs into the system, they will compete with fibrinogen for the binding to αIIbβ3 integrin. Thus, we can quantify the binding affinity of various RGD-containing polymer/SWCNTs to the integrin. As mentioned in the theory section 2.2.4,αIIbβ3 integrin is prevalent on platelets and binds to the RGD-motif [179]. Figure 35 illustrates the schematics of the assay.

Figure 35: Inhibition of integrin by ssDNA-RGD/SWCNTs. Schematic of a binding assay developed to quantify the affinity between RGD-containing polymer/SWCNTs and solubleαIIbβ3integrin. Without RGD-motifs, a bright color can be observed. The binding chain goes from fibrinogen to integrin, then to two antibodies, and ends with horseradish peroxidase (HRP) as the final block. After addition of a substrate, HRP forms yellow product that can be detected. Upon introduction of RGD-containing polymer/SWCNTs into the system, the binding chain is disrupted and the observed intensity decreases.

The sequence of this ELISA is as following: first, we coat the wells with fibrinogen that is known to bind integrin [177]. A sequence of integrin and two antibodies follows, with horseradish peroxidase (HRP) as the final block. After addition of a substrate, HRP forms yellow product that can be detected at 492 nm. Upon introduction of RGD-containing polymer/SWCNTs into the system, it competes with fibrinogen for the freeα_IIbβ₃ integrin.

When ssDNA-RGD/SWCNT-integrin is washed out of the system, the binding chain is

disrupted and the observed intensity decreases. For a detailed description of the ELISA procedure we refer to section 3.2.4.

To account for possible non-specific binding between ssDNA-peptide/SWCNTs and integrin, we used a second peptide sequence: Arg-Gly-Glu (RGE). The difference between RGD and RGE is just a slight change in the last amino acid: from aspartic acid (D) to glutamic acid (E). Glutamic acid has one carbon-carbon bond more and is therefore slightly longer than the aspartic acid. However, for its binding to integrin the change is essential. The RGE peptide sequence displays no binding affinity to the integrin binding pocket [188]. Therefore, ssDNA-RGE/SWCNTs hybrids as a control analyte help to exclude any unspecific bindings.

Typical binding curves for ssDNA-RGD- and ssDNA-RGE/SWCNTs are shown in Fig. 36.

Figure 36: Typical inhibition curves for ssDNA-peptide/SWCNT hybrids. (a) Inhibi-tion curves for (GT)15-RGD/SWCNTs (black squares) and (GT)15-RGE/SWCNTs (red circles). Intensities are normalized to the maximal value without ssDNA-peptide/SWCNTs (100% intensity) and the background signal of well-plates with-out integrin (0% intensity). (b) A comparison of inhibition curves for linear (C)₂₀ -RGD/SWCNTs (black squares) and bridged (C)20-RGD-(C)20/SWCNTs (red circles).

Four parameter logistic function fits are shown as the red lines. Dashed orange line marks the 50% drop in the initial intensity (50% inhibition, IC50). Error bars are standard errors (n = 2).

IC50 values were calculated using a 4-parameter logistic function to fit the experimental data:

y=d+ a−d

1 + ^x_cb , (31)

witha as the minimum value that can be obtained, d as the maximum value, cas the point

of inflection andbas Hill’s slope of the curve (steepness). The rearranged equation allows to calculate the concentration of x at the point ofy = 50 %, thus determining IC50 values for ssDNA-peptide/SWCNTs hybrids. Figure 37 demonstrates color-coded IC50 values, with black as the lowest and white as the highest IC50 value.

Figure 37: IC50 values of ssDNA-peptide/SWCNTs competitively binding to α_IIbβ3

integrin. The values were calculated by logistic function fits from the ELISA assay, with black as the lowest and white as the highest IC50 value.

When comparing IC50 values for various sequences, there might be a tendency for hybrids containing poly-(GT) (sequences of repeating guanine and thymine nucleotides) to display higher affinity compared to poly(cytosine) containing hybrids. On the one hand, the IC50 val-ues might be affected by varying affinity between different ssDNA sequences and SWCNTs.

This binding affinity was measured in an AFM study by pulling various ssDNA sequences from the SWCNTs surface [138]. Based on the applied force, Iliafar et al. established follow-ing rankfollow-ing for bindfollow-ing strength of different homopolymeres: poly(T) >poly(A) >poly(G)

≥ poly(C), with values ranging from 11.3 to 7.58 k_BT per nucleotide. On the other hand, Jena et al. recorded fluorescence response of SWCNTs upon a oligonucleotide displacement and so investigated the affinity of poly(GT) to SWCNTs [262]. Their findings showed no cor-relation between the stability of a ssDNA/SWCNT complex and ssDNA length or SWCNT chirality. Instead they proposed that individual SWCNT chiralities have an enhanced affin-ity for a specific ssDNA sequence, e.g. (GT)₆ was identified as the recognition sequence with the highest affinity for (8,6) SWCNTs.

In Fig. 37 no direct correlation of IC50 values with either ssDNA length or sequence is apparent. The lowest IC50 value was observed for bridged (C)₂₀-RGD-(C)₂₀/SWCNT (20 nM), while its linear equivalent showed the highest IC50 value (309 nM). In contrast, bridged (GT)₁₅-RGD-(GT)₁₅/SWCNT (110 nM) had higher IC50 value than its linear counterpart (29 nM). That suggests that all three parameters, ssDNA sequence, length and geometry (linear vs. bridged), modulate the affinity of ssDNA-peptide/SWCNTs to α_IIbβ₃ integrin.