Purdue University Graduate School
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PhD Dissertation-Chemistry-Aayush-2023

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posted on 2023-04-26, 00:16 authored by Aayush AayushAayush Aayush


Learning about ‘behavior’ has always been at the heart of my research endeavors. While my undergraduate work in evolution and ecology exposed me to the science behind why a behavior exists, in my graduate work, I intended to explore how to use something’s behavior to widen its applicability. In this thesis, I will present three works that utilize some of the fundamental

behaviors (i.e., properties) of elastin-like polypeptides (ELP) to improve existing protein purification methods or explore their applicability in bladder cancer imaging and immunotherapy. 

Bladder cancer has high recurrence rates (60-70 % annually) that necessitate multiple follow-up therapies making it one of the costliest cancers per patient. In this work, we have attempted to address two leading causes of the recurrence. First is a low sensitivity (62-84 %) and variable specificity (43-95 %) of white light cystoscopy used to diagnose and remove tumors. We aimed to address the heart of this problem, i.e., the non-specific mode of detection using white light. Only the trained eyes can discern abnormal from normal-appearing tissues even then, leaving up to 45% of tumors unresected to colonize and spread. We developed and characterized near infrared dye-peptide-ligand conjugates (NIR-ELP-ligand) that undergo receptor-mediated binding and internalization to human bladder cancer cells in vitro and tissues ex vivo. By using a molecular target-based probe in combination with NIR imaging, we can aid in improving the detection limit via selective binding to the tumor and reduction in background autofluorescence.

Bacillus-Calmette Guérin (BCG) instillation in the bladder is the gold-standard

immunotherapy used after surgical removal of bladder tumors. This was approved as a response to the inefficiency of surgery alone in improving cancer status. It has succeeded by reducing the recurrence rate to 30-50 %. But it comes with the complications of putting a live mycobacterium

in the human body and giving a patient a urinary tract infection right after surgical tumor resection. Thus, we aimed to deliver nucleic acid as immunotherapeutic cargo in a selective manner to elicit robust anti-tumor immune responses while minimizing the side effects due to its carrier. Towards

this goal, we have developed a highly modular and adaptable ELP-ligand fusion protein-based nucleic acid delivery carrier targeted toward bladder cancer. Before developing targeted peptide-based cancer imaging and nucleic acid delivery modalities, we addressed the Achilles heel of peptide-based approaches. The peptide and protein industry suffers

through complex, time-consuming, inconsistent, and low-yielding purification methods. We have developed a scalable, facile, and reproducible protein purification method that delivers ELP and ELP fusion proteins free of host cell proteins and nucleic acids and has low lipopolysaccharide

content in just 3 h starting from a bacterial pellet. Thus, for a coherent narrative, the thesis is structured as follows:

1. Introduction

2. ELP as a protein purification tag: Development of a rapid purification method for ELPs and ELP fusion proteins.

3. ELP as a cancer imaging agent: Development of NIR-ELP-Ligand imaging probe targeting bladder cancer.

4. ELP as a drug delivery agent: Utilizing ELP-ligand fusion protein in the formulation of targeted nucleic acid delivery carrier to bladder cancer.


Degree Type

  • Doctor of Philosophy


  • Chemistry

Campus location

  • West Lafayette

Advisor/Supervisor/Committee Chair

David H. Thompson

Additional Committee Member 2

Graham H. Cooks

Additional Committee Member 3

Timothy Ratliff

Additional Committee Member 4

Chittaranjan Das