Design,Characterization And Acting Mechanism Studies Of Self-assembling Anticancer Peptides

Cancer is one of the greatest threats to human health, which is difficult to cure and has a high mortality. The most commonly used anticancer drugs are small molecules, but these drugs always have poor solubility and cell selectivity, they are also toxic to normal tissues and can induce multi-drug resistance, which makes cancer more difficult to be cured. Therefore,researchers are looking forward to developing a new kind of anticancer drugs which could effectively overcome the difficulties mentioned above.Anticancer peptides are a kind of peptides which could inhibit the growth of or even kill cancer cells, and most of them are derived from antimicrobial peptides. A majority of anticancer peptides are lytic peptides, which target on cell membrane and can induce cell death without getting into cells, hence they rarely cause drug resistance. Besides, as they are peptides, they also have advantages like great bioactivity, high biocompatibility et al. Therefore,anticancer peptides are considered as ideal drug candidates for cancer treatment. However, anticancer peptides also have many disadvantages, their cytotoxicity needs to be enhanced, their acting mechanism are unclear, and they always have poor in vivo stability. We need to optimize them to before their clinic application.Molecule self-assembly is a phenomenon exists universally, and peptide is an important kind of building blocks that are capable of self-assembling.The process of peptides self-assembling is governed by several non-covalent interactions and is responsive to various environmental conditions, thus peptides are considered as "smart" biomaterial, which shows great application potential in cell culture, drug delivery and tissue engineering. Self-assembling could also affect the property and function of anticancer peptides by changing their structure and charge distribution, which finally alter their environmental responsiveness, cell selectivity and stability. As a consequence, the introduction of self-assembling is considered an effective approach for the reconstruction and optimization of anticancer peptides.We tried to overcome the problems which limit the application of anticancer peptides from two aspects. On one hand, we mutated the sequence of an anticancer peptide for anticancer ability improvement, and investigated the acting mechanism of anti cancer peptides in depth. The analysis and conclusion we made on the structure-function relationship of anticancer peptides could be beneficial for the design of novel anticancer peptides and peptide-based therapeutics. On the other hand, by inducing peptide self-assembling through delicate design, we attempted to enhance the serum stability of anticancer peptides. The understanding of the relationship between peptides nanostructure, bioactivity and stability could be applicable to a wide range of functional peptides, not limited to anticancer peptides, for the development of new types of peptide therapeutics. Finally, we utilized the principles of peptide self-assembly for construction of environment responsive peptide hydrogel, which greatly broaden the application of peptide self-assembly.In this dissertation, we used methods of chemistry, biology and cell biology to synthesize and characterize anticancer peptides, analyzed their acting mechanism in depth, and used the principles of self-assembling for the design of anticancer peptides and the construction of peptide-based material with environment responsiveness. The main progress lists as follows:1. Design, optimization and acting mechanism investigation of novel anticancer peptides. An anticancer peptide ZXR-1 (FKIGGFIKKLWRSKLA)derived from a known anticancer peptide mauriporin was developed, and a mutant ZXR-2 (FKIGGFIKKLWRSLLA) with only one residue difference at the 14th position (Lys?Leu) was also engineered. Results from MTT showed that replacement of the lysine with leucine made ZXR-2 more potent than ZXR-1 in general. While results from confocal, western blot and flow cytometer showed that even with only one residue mutation, the two peptides displayed distinct anticancer modes of action. ZXR-1 could translocate into cells, target on the mitochondria and induce cell apoptosis, while ZXR-2 directly targeted on the cell membranes and caused membrane lysis. The variance in their acting mechanisms might be due to the different amphipathicity and positive charge distribution. In addition, the two Ile-Leu pairs (3-10 and 7-14) in ZXR-2 might also play a role in improving its cytotoxicity and changing its acting mode. Further study on the structure-function relationship of the two peptides may be beneficial for the design of novel anticancer peptides and peptide based therapeutics.2. Construction, characterization of self-assembling anticancer peptides and investigation on their structure-function relationship. Through appending a pair of glutamic acid and asparagine to either the N-terminus or the C-terminus of a lytic peptide PTP-7(FLGALFKALSKLL), two self-assembling anticancer peptides EN (EN FLGALFKALSKLL) and NE(FLGALFKALSKLLNE) were designed. This simple, yet rational sequence modification was made to change the amphiphilic pattern and secondary structural content of the parent peptide, thereby modulated its self-assembly process. It was found that the N-terminus modified peptide favors the formation of nanofibrils and the peptide with C-terminal modification formed micelles. Although both nanostructures showed prolonged action profiles and improved serum stability compared to the parent peptide, the morphology of the nanostructures is highly critical to manipulate the release profile of the free peptide from the assembly and regulate their bioactivity. Construction of different amphiphilic pattern could effectively manipulate the time-action profile and stability, we believe the self-assembly approach demonstrated in this study can be applied to a variety of therapeutic peptide drugs to improve their stability and therapeutic activity for the development of carrier-free drug delivery system.3. Construction of environment responsive peptide hydrogel through self-assembly. A pH and calcium dual-responsive peptide amphiphile (PA) was designed from the RTX(GGXGXDXUX) motif, and its hydrogelation could be induced through either lowering environment pH or addition of calcium.Characterization of secondary structure, morphology and hydrogelation behavior were conducted, and it was found that the formation of ?-sheet structure and nanofibers network are highly crucial for PAs hydrogelation.Besides, through residue mutation, the aspartic acid within the PAs was found responsible for the pH and calcium responsiveness. Our understanding on the relationship between peptides secondary structure, morphology and hygelation behavior could be beneficial for further construction of peptide-based environment responsive hydrogel, and broaden the application of peptide self-assembly.