Development of Enzyme-Directed Nanotransformers and Peptide-Based Hydrogels for Biomedical Applications

Abstract

Chapter 1 presents a brief introduction of the research background. It revolves around the development of enzyme-instructed self-assembly (EISA) and its applications in biomedical engineering, including imaging, cancer therapy, engineering, proteins sequestration and trafficking. In addition, various enzyme triggered peptide-based self-assembly, and their functions were also summarized and discussed.

Photodynamic therapy (PDT) is a widely used method for treating various malignant cancers due to its non-invasive feature. Significant progress has been made in the development of highly efficient photosensitizers, particularly the porphyrin family and its derivatives, because of their high quantum yield in ¹O₂ generation. However, the application of porphyrins in PDT is often limited by poor water solubility, low bioavailability, strong dark cytotoxicity, and off-target accumulation. Combining EISA with photosensitizer/peptide co-assembly could provide an efficient approach to overcome the aforementioned limitations of photosensitizers. In addition, it circumvents the need for tedious chemical synthesis and enables the nano-system to accumulate in the tumor site, making it possible to design stimuli-responsive nanomaterials with improved anticancer efficiency. Despite significant progress in EISA, no reports have shown the transformation of co-assembled peptides and photosensitizers into nano-systems within cancer cells, particularly in sub-organelles, to enhance PDT efficacy. Employing responsive nanoplatforms as carriers for photosensitizers represents an effective strategy to overcome the challenges associated with photodynamic therapy (PDT), including poor solubility, low bioavailability, and high systemic toxicity. Drawing inspiration from the morphology transitions in biological systems, a general approach to enhance PDT that utilizes enzyme-responsive nanoplatforms was developed. We first demonstrated the transformation of phosphopeptide/photosensitizer co-assembled nanoparticles into nanofibers when exposed to cytoplasmic enzyme alkaline phosphatase in chapter 2. This transition is primarily driven by alkaline phosphatase-induced changes of the nanoparticles in the hydrophilic and hydrophobic balance, and intermolecular electrostatic interactions within the nanoparticles. The resulting nanofibers exhibit improved ability of generating ROS, intracellular accumulation, and retention in cancer cells.

In chapter 3, we expanded our enzyme-responsive nanoplatform to selectively target mitochondria by mitochondria-specific enzyme sirtuin 5 (SIRT5). SIRT5 as a sub-organelle trigger was employed for transforming the co-assembled nanoparticles into nanofibers, specifically in the mitochondria. Under the catalysis of SIRT5, the succinylated peptide/photosensitizer co-assembled nanoparticles can be transformed into nanofibers specifically within the mitochondria. The resulting nanofibers exhibit excellent capability of modulating mitochondrial activity, enhanced ROS formation, and significant anticancer efficacy via PDT. Consequently, the enzyme-instructed in-situ fibrillar transformation of peptide/photosensitizers co-assembled NPs provides an efficient pathway to address the challenges associated with photosensitizers. We envisage this approach will further expand the toolbox for enzyme-responsive biomaterials for cancer therapy.

Although some small molecule-based inhibitors of proteins and genetic knockout tactics have been developed and were successful in decreasing cellular cancer-related protein levels, the research for alternative ways to selectively control protein levels in the tumor microenvironment continues unabated. EISA utilizes overexpressed enzymes in cancers to produce nanomedicines in situ, which specific interactions will be able to flesh out self-assembling molecules, resulting in selectively triggering the generation of supramolecular nanomaterials in situ. The local enrichment of nanomaterials in targeting sites is a great boon for not only drug delivery but also protein degradation. However, study combined the degradation of protein with EISA has been reported rarely. In chapter 4, inspired by the EISA and protein degradation, we have put forward a creative protein degradation method that combined EISA with degradation to selectively degrade the cancer-related membrane EGFR protein. An ALP-responsive peptide sequence with EGFR targeting ability was designed and synthesized, and its efficacy in the enzyme-instructed self-assembly and in the regulation of EGFR in cancer cells were investigated. As expected, the overexpressed extracellular ALP in cancer cells can efficiently catalyze the dephosphorylation of peptide 1 and lead to the self-assembly to form nanostructures. And the overexpressed membrane EGFR in cancer cells provides plenty of robust binding sites for the self-assembled nanostructures. The self-assembled nanofibers binding simulates the partial denaturation state of EGFR and results in the protein degradation. The cell viability, immunofluorescence analysis, and western bolting results strongly proved the hypothesis. Furthermore, the EGFR degradation pathways were studied by western bolting, suggesting the EGFR was mainly knockout by the proteasomal degradation pathway as well as autophagy access. Our research made use of combining EISA and self-assembly induced protein degradation, developed an efficient and targeted degradation of membrane protein EGFR in cancer cells.

Finally, Chapter 5 provides a summary of all the research projects performed in the PhD study. The coming works and the directions based on ELSA for biological applications were also discussed. We will further utilize the enzyme-instructed self-assembly system for degradation of some proteins of interest. For instance, we next will explore enzyme instructed self-assembly for the protein degradation of some immune-relative proteins, like CD47, CD206. And we also expect to construct the degradation system with synergistic effect for dual-proteins degradation or multi-proteins degradation.

Date of Award	26 Aug 2024
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Hongyan SUN (Supervisor)