L- Amino Acid Based Polyester Nanocarriers For Drug Delivery and Bioimaging

Abstract

This thesis work is aimed to explore new classes of amphiphilic and biodegradable polyester and their fluorescent nano-assemblies based on natural L-aspartic acid resources for accomplishing anticancer drug delivery and intracellular bio-imaging in cancer cells. L-aspartic acid resource was converted into multi-functional monomer and it was polymerized under melt conditions in the presence of hydrophilic and hydrophobic diols to yield amphiphilic copolymers. These polymers were designed in order to possess inbuilt pH responsive BOC urethane hanging groups and esterase-responsive ester backbone making them dual stimuli containing amphiphilic polyesters. The BOC urethanes underwent cleavage and transformation into cationic moieties NH3+ in the highly acidic endosomal compartments of the intracellular regions (trigger 1). The ester containing backbone was susceptible to cleave under the presence of esterase enzyme present in the hydrolytic enzyme rich lysosomes which acts as the (trigger-2). Three different therapeutically active drugs curcumin (CUR), doxorubicin (DOX), and topotecan (TPT) were loaded into the polymer scaffolds and their cytotoxicity were studied in breast cancer (MCF 7) and cervical (HeLa) cancer cell lines. The uptake of these drugs was monitored using confocal microscopy. Further, L-amino acid based amphiphilic luminescent polyester was developed using π-conjugate luminescent materials and their FRET (fluorescence resonance energy transfer) probes for colour-tunable intracellular imaging were demonstrated. Highly luminescent oligo-phenylenevinylene (OPV) with π-conjugated cores were tailor made into melt polymerisable diols and were subjected to thermo-selective melt trans-esterification polymerization with modified multi-functional L-aspartic acid monomers along with hydrophilic tri-ethylene glycols and hydrophobic dodecanediol to yield OPV-labelled luminescent amphiphilic polyesters. Comprehensive photo-physical analyses exhibited that the OPV and NR (a hydrophobic encapsulated dye) were constricted within close proximity and aligned at apt geometry within the nano-domains of polymer micelles for highly efficient excited energy transfer from OPV excited state to NR ground state. The OPV tagged nascent polymer nanoparticles and OPV-NR FRET probes were tested for their cytotoxicity in normal cell lines (wild-type MEF, WT-MEF), and cancer cells {breast cancer (MCF 7), cervical cancer (HeLa) cells} and were found to be biocompatible. The cellular uptake studies of these nanoparticles revealed that luminescent polymers and the FRET probes could be transported efficiently across the cell membrane and these could accumulate within the cytoplasm of the cells. Live cell imaging experiments manifested the co-localisation of donor and acceptor molecules in the lysosomal compartments of the cells which was supported by lyso-trackers, which provided direct evidence for endocytotic pathway as the mode of entry of these nanoparticles. The enzymes found in lysosomes of the cells such as esterase, chymotrypsin, trypsin, and GSH reducing agents were employed to study the bio-degradability of the polymeric micelles and were found to efficient in degrading the polymers. Aggregation induced emission (AIE) chromophore tagged L-amino acid based biodegradable polyester have been designed and their fluorescent nano-scaffolds were explored as multi-colour tunable FRET nano-probe for bio-imaging in cancer cells. AIE capable hydroxyl functionalized tetraphenylethyelene (TPE) diol was tailor-made through multi-step reaction and it was subjected to melt transesterification with L-aspartic acid monomer to yield new blue-luminescent amphiphilic polyesters. These AIE polyesters self assembled into compact spherical nanoparticles and they were found to be capable of encapsulating water insoluble red-fluorophore Nile red (NR) and green-fluorophore drug Curcumin (CUR). Two FRET probes TPE-NR and TPE-CUR were constructed in the TPE conjugated polymer nano-system as donor and CUR and NR as acceptors. Third FRET probe was build between CUR-NR using non-TPE amphiphilic polyester scaffolds. Confocal and live cell imaging experiments threw light on the accumulation of these nanoparticles in the cytoplasm of the cells and demonstration of FRET within the cell lines showed the stability of the nanoparticles during the efficient uptake into the cells. The cleavage of these nanoparticles in the presence of lysosomal enzymes were demonstrated by using various enzymes and the FRET as probe was used to study the bio-degradation of these nanoparticles in in vitro experiments. The melt polycondensation methodology was further extended to enzyme-responsive hydroxyl and carboxylic acid dual functional polyesters synthesized from natural L-amino acid residues. L-Aspartic acid was converted into a new di-ester functional monomer containing acetal protecting amine groups. The methyl esters of the aspartic acid monomer underwent melt-transesterification with dodecane diols, an aliphatic hydrophobic di-functional alcohol to produce high molecular weight polyesters. The hydroxyl functionalised polyesters were synthesised by acid assisted deprotection of the acetal groups in each repeating units of the polymer. The free hydroxyl groups were further converted into carboxylic functional groups by ring opening of succinic anhydride. The hydrophilic hydroxyl and carboxylic groups along with the hydrophobic aliphatic backbone made these polymer amphiphilic in nature and enabled these to self-assemble into spherical nanoparticles in water. Successful and high amount of DOX loading was achieved and the cellular uptake and killing were demonstrated in MCF 7 cells. The carboxylic polymers were further covalently stitched to a second anticancer drug cisplatin and their killing was demonstrated in the cancer cells. These polyesters were further tailor-made into AIE active polymers by introducing custom designed tetraphenylethene units in the backbone during melt condensation process making these polymers nanoscaffolds luminescent in nature. The DOX loaded AIE active polymers were used to demonstrate FRET at the cellular level where aggregated TPE acted as donor and DOX acted as an acceptor. This thesis work put forward the direction for future research as a new class of amphiphilic polyesters were designed and developed based on L-aspartic acid residues. In the thesis, these polymers are new entries as enzymatic-biodegradable polymers in the literature; hence they would be expected to be very important nano-carriers for long-term application in the biomedical field. Further, the custom designed OPV-tagged and TPE-tagged fluorescent polymers are excellent nano-scaffolds for constructing wide range of FRET probes with drugs and fluorophores which could be employed for early diagnostics of cancer and other bio-imaging applications.