Cholic Acid Conjugated Macromolecular Amphiphiles with Potential Bio-uses

Sahoo, Subhasish (2022) Cholic Acid Conjugated Macromolecular Amphiphiles with Potential Bio-uses. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Subhasish Sahoo (18RS052)) 18RS052.pdf - Submitted Version Restricted to Repository staff only Download (9MB)

Abstract

Chapter 1: General Introduction to Cholic Acid-Based Macromolecules with Biomedical Applications. In recent decades, polymeric materials have been extensively investigated for their widespread therapeutic applications in various biomedical fields. In particular, biomolecule conjugated polymers continue to draw attention due to their distinctive physicochemical and biological properties. By appending biomolecules on polymeric constructs, these synthetic macromolecules can be strategized as multi-motive theragnostic agents. In this chapter, we intend to showcase the biomedical applications of polymeric materials containing cholic acid (CA) as the biomolecular counterpart. CA is the most abundantly produced bile acid during cholesterol metabolism in the human body and is also the least expensive one. The fascinating properties such as inimitable facial amphiphilicity, convenient bioavailability, trivial cytotoxicity and excellent biocompatibility make CA unique to design numerous cholic acid-based polymers (CAPs) in the diverse areas of biomedical research. This chapter primarily focuses to the recent achievements (2011-present) on the obligatory role of the CA moiety in CAPs in different biomedical applications. Especially the therapeutic applications which include drug delivery, gene transfection, antimicrobial activity, bioimaging, diagnosis, wound healing, and other miscellaneous bio-applications are highlighted herein. Chapter 2: Controlling the Membrane Perturbation by Tuning Charge Variable Cholate-Based Macromolecules. Access to the interior of the cell by destabilizing the natural barrier of the cell membrane is rudimentary for drug and therapeutics delivery. Taking unique facial amphiphilicity advantages of cholic acid to facilitate switching over impermeable cellular membranes, we report the synthesis of three different series of charge variable (anionic, cationic and neutral) amphiphilic copolymers with various cholate content and their membrane destabilization behavior. The cholate containing amphiphilic copolymers self-organize into 80-100 nm sized nano-aggregates in aqueous media, where cholate moieties resided in the core of the self-assembled nanoparticle, confirmed by¹1H NMR spectroscopy. The cholate containing polymeric micelles proficiently destabilize the liposomes at pH 5.0 and pH 7.4. Interestingly, the cholate appended neutral polymer exhibited up to 15-fold enhancement in membrane destabilization efficiency in comparison with the respective cholate-based anionic or cationic polymers. Mechanistic studies using fluorescence, dynamic light scattering (DLS) and transmission electron microscopy (TEM) established that the polymer-membrane interaction, subsequent reorganization and insertion of cholate to bilayer plays the crucial role. Model drug (Nile red) encapsulation and release studies suggested the therapeutic potential of the cholate-conjugated polymeric nanoclusters. Chapter 3: Cholate Conjugated Cationic Polymers for Regulation of Actin Dynamics. Cytoskeletal movement is a compulsory necessity for proper cell functioning and is largely controlled by actin filament dynamics. The actin dynamics can be finetuned by various natural and artificial materials including cationic proteins, polymers, liposomes, and lipids, although most of the synthetic substrates have toxicity issues. Herein, we show actin nucleation and stabilization with a synthetic family of CA conjugated cationic macromolecules. Architectural conjugation of CA is designed by attaching it to the polymer chain end, as well as to the side chain of the polymer. The side-chain cholate content is also varied in the copolymer, which results in self-aggregation in aqueous media above a certain CAC. Below the CAC, the in vitro actin dynamics modulation behaviour is studied using pyrene actin fluorescence assay, actin co-sedimentation assay, DLS, and TEM. These polymers are nontoxic to the HeLa cells, and the 2% cholate conjugated cationic copolymer show maximum enhancement of G-actin nucleation, as well as F-actin stabilization. We elucidate the underlying dynamics of the actin polymerization process under the influence of cationic copolymers with cholate pendants. Finally, we proposed macromolecular self-aggregation as a unique tool for modulating actin dynamics, as revealed from the experimental findings. Chapter 4: Multifunctional Alternating “Bitter-Sweet” Macromolecular Architecture. The design and synthesis of a multifunctional macromolecular architecture featuring alternating cholic acid and glucose pendants in a polymer side-chain are reported. The target architecture was prepared by RAFT copolymerization of styrene conjugated cholic acid (St-CA: the bitter monomer) and acetyl protected glucose appended maleimide (MI-GLU: the sweet monomer) using polyethylene glycol (PEG) conjugated chain transfer agent (CTA). Removal of the acetates resulted in amphiphilic “bitter-sweet” alternating copolymers that were self-assembled in aqueous media having CA containing bitter core and sugar-coated sweet shell. DLS measurements in water, FESEM and TEM confirmed the formation of 40 to 75 nm sized micellar nanoscaffolds, depending on the chain-length of the copolymers. The nanoparticles successfully encapsulated hydrophobic molecules as witnessed via fluorescence spectroscopy using Nile red (NR) as an exemplary guest. Interestingly, the alternating copolymer recognized β-cyclodextrin (β-CD) through the formation of inclusion complexes with lateral cholate moieties in the polymer as evident from 2D NMR and nuclear overhauser effect (NOE) experiments. It is worth noting that the polymer and its inclusion complex were found to be capable of recognizing Concanavalin A (Con A), as shown by turbidimetric assay and isothermal titration calorimetry (ITC). Interestingly, the inclusion complex of the alternating copolymer showed significantly higher autofluorescence in the presence of Con A with respect to that of un-complexed one. Thus, the present study offers a simple way to prepare a multifunctional alternating copolymer having hydrophobic molecule encapsulation, inherent fluorescence, inclusion complex formation with β-CD and lectin recognition capabilities. Chapter 5: Polymerization Induced Self-Assembly (PISA) Generated Cholesterol-Based Block Copolymer Nano-Objects in Non-Polar Solvent. A family of nanostructured morphologies made of poly(cholesteroyl methacrylate)-b-poly(styrene) (PCMA-b-PS) copolymers are prepared via reversible addition-fragmentation chain transfer (RAFT) dispersion. The number average degree of polymerization (DPn) of the PS segment was targeted from 15 to 150, to obtain spherical, worm-like, fibrillar, and vesicular nano-objects as evidenced from field emission scanning electron microscopy and transmission electron microscopy, supported by dynamic light scattering experiments. Numerous morphologies were also observed with increasing total solid content (5 to 25 wt%) of the copolymer. Thermo-reversible gel-sol transition was studied with higher wt% of copolymers. The developed strategy accounts for both the graft architecture and structural heterogeneity of the lyophilic block, made it possible to evaluate the morphological transitions in block copolymer solution at different block length ratios, different total solid contents, and temperatures, and to provide an in-depth understanding of the experimental results.

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