Functional Polydiacetylenes for Colorimetry Sensing and Protein Delivery

Abstract

Polydiacetylene (PDA) is a unique conjugated polymer formed by the photopolymerization of diacetylene monomers. PDA generally has an intense blue color and could undergo a color transition as a signaling response toward the external stimuli encountered. Due to diacetylene's amphiphilic properties, it can be directly dispersed in an aqueous system and yield self-assembled liposome bilayers. However, the structure of the diacetylene-based liposome depends on its dispersion process. Liposomal PDA has great potential, especially in the biological application. However, PDA liposome has intrinsic issues such as coagulation and aggregation during storage in an ambient condition which further affects the system's sensitivity. Therefore, in this dissertation, the designed and tuned self-assembly process of PDA-based vesicular liposome was investigated to be used as a stable and sensitive platform in biological applications. Our first attempt is designing and utilizing the zwitterionic diacetylene for lysophosphatidylcholine (LPC) sensing. We designed and developed a stable and sensitive zwitterionic vesicular liposome (PDZ). PDZ vesicles displayed blue to purple-red color changes and fluorescence turn-on features simultaneously upon interaction with LPC due to the occurring electrostatic and hydrophobic interaction. Furthermore, utilizing a multichannel microfluidic gradients chip, a fluorescence sensing system was applied to measure the LPC concentration simultaneously under optimized flowing conditions within 10 min. Secondly, we designed and tuned non-polymerizable bulk diaminotriazine functionalized diacetylene (PCDA-DAT) to be self-assembled as a responsive liposome. PCDA-DAT was self-assembled with 10,12-pentacosadiynoic acid (PCDA) and various phospholipids. The insertion of phospholipids resulted in notable changes in the stability and sensitivity of PDA liposomes. Among the various phospholipid, the insertion of zwitterionic DMPC showed enhanced stability as measured by the size and zeta potential over the storage time of the liposome. Next, the developed liposome was utilized as a sensitive and selective probe for thymine and thymine-rich ssDNA. The developed liposome undergoes color changes as a consequence of perfectly matched hydrogen bond interaction between the thymine group and diaminotriazine head group of the PCDA-DAT. Furthermore, we also utilized a PCDA-based vesicular liposome to resolve the issues in colorimetric sensing of singlet oxygen (1O2). A photodynamic color sensor for visualizing photoactivated 1O2 using PCDA liposome and a mini singlet oxygen generator (miniSOG) was investigated here. The photoactivated miniSOG yielded 1O2 and subsequently reacted to the PCDA liposome, leading to a rapid color change from colorless monomeric PCDA into blue-colored polydiacetylene (PDA) liposomes (from yellow to green in the miniSOG-mixed solutions) through electron delocalization-mediated polymerization. As a result, a yielded color transition of PCDA to PDA enabled the rapid and straightforward detection of photoactivated 1O2 and the rapid determination of 1O2-scavengers in a microplate. Notably, PCDA liposomes exhibited better sensitivity and stability for photoactivated 1O2 compared to a classical chromogenic reagent. Lastly, despite the widespread use of arginine-rich peptides in the intracellular delivery of cargoes, their translocations through endocytosis often promote endosomal entrapment and limit the cytosolic release in the living cells. Polydiacetylene (PDA) liposomes were developed as protein carriers and visual indicators for cell cytosolic delivery. As a model protein, we adopted a cyclized tetra-arginine (RRRR)-conjugated enhanced green fluorescence protein (Cyc R4-EGFP) that has more liposome-penetrating ability than a linear counterpart (Lin R4-EGFP). Notably, PDA liposomes loaded with Cyc R4-EGFP promoted a rapid cytosolic delivery of Cyc R4-EGFP in living cells as a result of membrane fusion, which was easily detected using red and green fluorescence of PDA and protein, respectively. In contrast, no intracellular delivery was observed for PDA liposome-free proteins. This finding suggests that the developed PDA vesicular liposome is not only a colorimetric probe but also very useful as a promising carrier for cytosolic protein delivery in living cells.