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Abstract

Ionic self-assembling peptides have emerged as promising nano-biomaterials, with direct

applications in the fields of bioengineering and applicative medicine. (RADA)4 is a self-

assembling 16 residue containing peptide, with alternating hydrophobic and hydrophilic residues.

This characteristic amphiphilic nature of the peptide allows it to self-assemble into β-sheets,

forming higher order three-dimensional (3D) structures. The resulting 3D hydrogels are non-

immunogenic, highly hydrated (containing > 99% water), and can respond to physiological

stimuli. The (RADA)4 hydrogels can serve as ECM (extra cellular matrix) scaffolds, assist in

drug delivery, and can achieve rapid hemostasis.

This dissertation consists of two major parts: 1) fundamental study of the self-assembly of

the model ionic complementary peptide (RADA)4, and 2) in-vitro biocompatibility assessment of

the (RADA)4 peptides.

In the fundamental study, the secondary structure of (RADA)4 was examined with

Circular Dichroism (CD), and compared to that of its two variants: (RADA)4K5 and (RADA)4S5.

The effect of peptide concentration and temperature on the secondary structure was also studied.

It was found that all but (RADA)4K5 peptides formed successful β-sheets, consequently forming

nanofibers, whereas (RADA)4K5 resulted in the formation of aggregates rich of primarily random

coil sequences. Further, studies were conducted to determine the critical concentration of

(RADA)4K5 for successful nanofiber formation. The 3D nanostructure of the peptide was affected

by the amino acid sequence as well as by the temperature induced denaturation of the peptide.

Quantitative structural analysis of all the samples were carried out using an online DICHROWEB

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server, by comparing the secondary structure molar ellipticity of the peptides collected with that

of seven reference proteins (data confirmed via X-ray crystallography).

Single-molecule Florescence Correlation Spectroscopy (FCS) was used to confirm the

molecular interaction of the pristine (RADA)4 nanofibers with 25% (RADA)4K5. The effect of

hydration on these self-assembling peptides was investigated via Differential Scanning

Calorimetry (DSC), over a range of temperatures. The Equilibrium Water Content (EWC) in

(RADA)4 was comparable to (RADA)4S5 and (RADA)4K5, even at varying compositions. The

content of non-frozen bound water increased upon appending either Lysine or Serine residues to

the (RADA)4 peptide. Microscopy techniques such as Atomic Force Microscopy (AFM), and

Transmission Electron Microscopy (TEM), were also employed to visually inspect the higher

order structures formed by these peptides.

The second part of this dissertation focuses on the in-vitro biocompatibility of the

(RADA)4 based peptides. The PAC-1, CD62-P, and CD42 markers were used to study platelet

activation (via Flow Cytometry) and a time based clotting analysis was conducted to evaluate the

hemostatic ability of peptides. Complement C3a ELISA assay was conducted with (RADA)4

based peptides to gain more insight into the biocompatibility. The pristine (RADA)4 nanofibers

caused a rapid clot formation, but yielded a low platelet activation and low C3a activation.

Whereas, (RADA)4K5 peptide displayed a significantly higher complement activation, when

compared to both (RADA)4S5, and the (RADA)4 peptide, likely due to the free NH3 groups and

steric hindrance in packing. The overall trend of the platelet activation among the three variants

of the peptides remained consistent: (RADA)4K5 activated the platelets to the highest, whereas

(RADA)4S5, and (RADA)4 showed comparable platelet activation. However, it should be noted

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that both the platelet and complement activation by these peptides were lower than the already

considered biocompatible biopolymers.

Lastly, morphology analysis of the platelets in contact with the hydrogels was conducted

by calculating the Morphology Score (MS) using a Kunicki scoring system, and the platelets

were visualized using Scanning Electron Microscopy (SEM). An overall Kunicki morphology

score of above 385 was achieved for all the peptides, where a score of less than 200 represent

poor retention of morphological characteristics associated with platelets that are active. Overall,

the (RADA)4 based peptides had a lower, or comparable platelet and complement activation,

when compared with the already in-use biomaterials (such as poly(methyl methacrylate), and

dextran), making them a desirable material to further investigate.

The work in this dissertation, not only provides the fundamental knowledge to design

novel biomaterials for direct application in medicine, but also provides the stepping stone for

further in-vitro and in-vivo biocompatibility analysis, required for any further medical

application.