Poly(L-Tyrosine) Based β-Sheet Polypeptide Nanoarchitectures for Drug Delivery Applications

Abstract

Synthetic polypeptides continue to be one of the most challenging areas for chemists to mimic the properties of natural proteins to impart artificial intelligence in macromolecular systems for both fundamental understanding and technology development. Natural proteins have balanced architecture control by cleverly choosing the amino acid sequences and lengths to manipulate α-helical and -sheet secondary structures for precise globular self-assembly to carry out biological action under physiological conditions. In the area of synthetic polypeptides, the excellent solubility of the α-helical polypeptides enabled the synthesis of block copolymers to partially mimic the α-helical secondary structures in the aqueous medium for diverse biomedical applications. On the contrary, L-amino acid bioresources that promote -Sheet structures are one of the most difficult systems to be handled by synthetic polymer chemists. This restriction is associated with the uncontrollable-precipitation of the -Sheet polypeptide chains in their synthesis. The heterogenous polymerization was found to be further responsible for the “non-living” nature with dead propagation chains which turned to be inefficient for making block copolymer architectures. The thesis work addresses this important problem in one of the most difficult -Sheet promoting L-amino acid bioresources ʟ-tyrosine. ʟ-Tyrosine has an aromatic unit containing amino acid; thus, it is very difficult to control the ROP process, and the entire literature only documents the synthesis of oligomers (10-12-mers). Further, the poly(ʟ-Tyrosine) is insoluble, and precipitates and non-living during the synthesis. Therefore, the existing synthetic methodology cannot make high molecular weight polypeptides and they could not be expanded to block copolymer macromolecular architectures. As part of the thesis work, an elegant steric hindrance-induced ring opening polymerization (ROP) strategy is introduced to access high molecular weight -sheet poly(ʟ-tyrosine) having more than 250 units, and expand the scope of the methodology to access unexplored poly(ʟ-tyrosine) based higher-order -sheet block copolymers. In this strategy, a tert-butyl benzene unit is employed as a steric handle that imbibed excellent solubility in the propagating polypeptide chains in common organic solvents associated with living-ROP characteristics. The living-ROP process enabled the synthesis of well-defined di-block copolymers either by employing poly(ʟ-tyrosine) living-chain ends as macro-initiators or growing the poly(ʟ-tyrosine) chains on the top of the pre-existing macroinitiator constituted by poly(ʟ-glutamate) or poly(ʟ-lysine). Acid-catalyst post-polymerization deprotection has restored the poly(ʟ-tyrosine) chains in their nascent -sheet conformations, and the di-block copolymers yielded pH-responsive core-shell polyelectrolyte nanoparticles consisting high molecular weight poly(ʟ-tyrosine) -sheet at the middle and anionic or cationic random-coils in the periphery, respectively. Thioflavin-T fluorescence assay established the presence of strong -sheet secondary structures in these poly(ʟ-tyrosine) core-shell polyelectrolytes. The newly developed -sheet core shells were found to be non-toxic to mammalian cell lines and opened new avenues for the unexplored tyrosine-based block copolymers for long-term impact both in material and biomedical applications. The process reported here opens up a new platform for building high molecular weight -sheet polypeptides by reversible conformation-transition strategy and also creates a new avenue to explore diverse block copolymer architectures that have been distant dream so far. The ring-opening polymerization-induced self-assembly (ROPISA) approach is one of the most effective tool to generate the nano-assembly in situ while the polymer is growing itself. Where the block copolymer is grown on the top of the hydrophilic initiator to synthesize the amphiphilic architecture. As part of the thesis work ʟ-Tyrosine amino acid-based ROPISA technology has developed. In this approach, the ʟ-Tyrosine based monomer is employed with amine-terminated PEG as a hydrophilic macroinitiator to polypeptide di-block copolymer self-assembly. Synthesized nano-particles are non-hemolytic in nature. Further self-assembled nanoparticles were tested for in situ encapsulation study with IR-780, Nile red (NR), Rhodamine-B, and 8-Hydroxypyrene-1,3,6-trisulfonic acid tri-sodium salt (HPTS), and showed excellent capsulation ability. Overall, the developed approach is unique in nature to generate in situ self-assembly as well as to encapsulate the cargo while growing polymeric self-assembly. This newly designed approach of in situ dye loading during ROPISA process can open up a new vision for polypeptide-based nano-formulations for biomedical applications.