Developing Bio-Inspired Supramolecular Self-Assembly as Anticancer Theranostics


Student thesis: Doctoral Thesis

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Award date5 Sep 2019


Molecular self-assembly is a common process occurring in nature, and has been used as a core approach of bottom-up method to construct nanostructures and nanoobjects for advanced applications. In this thesis, we seek to manipulate the bioinspired self-assembly in living system spatially under the guide of nature’s principles and without complicated synthesis steps. In addition, we also try to explore new candidate of small molecular self-assembly for anticancer theranostics.

In biological systems, locally formed components are responsible for the temporal and spatial regulation of different cellular functions. Therefore, it demands more precise control over the involved molecular interactions within living organisms when developing new therapeutics. Inspired by nature, researchers began to use enzymes that are ubiquitous in cells to trigger self-assembly of small molecules due to their abnormal enzymatic activity to suppress the growth of cancer cells. However, the rational synthesis of self-assembled nanostructures in mitochondria remains unexplored. Here, we propose a new strategy to create spatially confined nanostructures in mitochondria by taking advantage of a unique enzyme trigger Sirt5, a mitochondria-localized enzyme that displays efficient lysine de-succinylase activity. Through the selective bio-catalysis of Sirt5, a soluble non-assembling peptide precursor was successfully transformed into self-assembling building block, leading to the formation of three-dimensional supramolecular network in living cells. We demonstrated that the formation of nanofibers was spatially restricted to the mitochondrial region of cells. Moreover, we have shown that such nanosized fibers are non-toxic, making them highly suitable for serving as cancer diagnostics that indicate the activity of enzymes over-expressed in cancer cells. We envision that the principle revealed here will be applicable to the rational development of stimuli-responsive biological materials and nanodevices.

Other than the diagnostic applications demonstrated in the first part, much work described before has also investigated the use of artificial small molecule modules as biomedicine to control cancer cell fate. However, the mimicry of natural biosystems and corresponding developments are still in the middle of the basic research phase and the replicating and translating of multi-hierarchical self-assembly in living system into practical application is hindered by poor understanding and control of the architecture and processing, especially for pharmaceutical and medical applications. In the second part, we present a 4-(1,2,2-triphenylvinyl) benzoic acid (TPE-COOH,an aggregation induced emission (AIE) probe) conjugated peptide containing 3,4-dihydroxyphenylalanine (DOPA) residues, which could self-assemble into hydrogel for selective inhibition of cancer cell through selective adhesion and long-term release of cytotoxic nanofibres. The retained AIE property allowed directly imaging of the in situ formed peptide hrydrogel thin film and the DOPA residues acted as biological motifs for cell adhesion. Self-assembly of peptide in water produced loose and thin nanofiber network but produced stable and rough nanofiber in phosphate-buffered saline (PBS). Owing to such difference, controlled degradation of hydrogel by protease was achieved in vitro, which allowed these two kinds of film to show different degree of cell growth inhibition of U87MG (human glioblastoma astrocytoma) cells. The hydrogel formed in water also exhibited selective inhibition of cell growth between U87MG and MCF-7 (human breast adenocarcinoma cell line) cancer cells, and the possible explanations for this result may be the different integrin expression levels between U87MG and MCF-7. These results suggested that the strategy of controlled inhibition of cancer cell by DOPA-containing peptide self-assembly may indeed be a promising treatment of cancers through selective adhesion. Our works expand the scope of application of DOPA-functionalized biomaterials inspired by mussel which usually are developed as multifunctional coating materials.

The diversity of natural components and their highly accurate interactions with each other determine that high quality bio-inspired materials should contain diverse supramolecular nanostructures and afford both diagnostic and therapeutic capabilities. Among the abundant endogenous building blocks, peptide is the most explored motif for enzyme-triggered self-assembly as selective cancer therapy, but the short half-life, poor metabolic stability and difficulty in large-scale production of peptide self-assemblies have limited their practical applications. Enzymatically mediated self-assemblies of other motifs like nucleic acids or carbohydrate amphiphiles have emerged in recent years. In the third part of this thesis, inspired by lipid self-assembly, we have developed a simple amphiphile-like lipid which initially self-assemble into nanoparticles in solution and then self-assemble into nanofibers after enzymatic dephosphorylation. Such self-assembly can be instructed by alkaline phosphatase (ALP) in solution condition or by ALP from HepG2 (human hepatocellular carcinoma) cell line in situ. The conversion from nanoparticles formed by the phosphorylated lipid amphiphile to the membrane-localized nanofibers formed the by the dephosphorylation product induced HepG2 cell death. Results demonstrated that this process was positive correlated with the phosphatase concentration, and therefore cells with lower ALP activity were selectively unaffected compared to HepG2 cells. Owing to the facile synthesis process, the prevention of proteolytic degradation in living system and the low risk of inducing drug resistance, this designed simple lipid derivative may have great potential for anticancer theranostics. Therefore, beyond the peptide-based systems that we investigated both in the first and the second part, this research offers new opportunities for small molecular as amphiphiles self-assembly system for enzyme-mediated cancer treatment with higher yield and stability. Most importantly, our work provides new insight for establishing bio-inspired platforms in the future containing multi-components that simultaneously satisfy the demand of both diagnostic and therapeutic capabilities, for example, ahybrid hydrogel system consists of peptides and lipid moieties that responsive to different stimulus independently.