L-Tyrosine Based Polymer Brushes for Drug Delivery In-vitro and In-vivo

Abstract

Development of new polymers based on bioresources is essential to cater the ever-increasing demand in commodity plastics and also to meet the new demand for biocompatible and biodegradable nanocarriers for safe drug formulations in healthcare industry. This thesis work involves the design and development of L-amino acid-based polymer brushes and study their role of polymer topology on the enzyme-responsive drug delivery and deep-tissue penetration under in-vitro and in-vivo. For this purpose, L-tyrosine based propargyl-functionalized monomer is tailor-made and polymerized via solvent free melt polycondensation strategy to yield hydrophobic and clickable biodegradable poly(ester-urethane)s. Post-polymerization click chemistry strategy is applied to make well-defined amphiphilic one-dimensional rod-like and three-dimensional spherical polymer brushes by merely varying the lengths of PEG-azides in the reaction. These core-shell polymer brushes are found to be non-toxic, non-hemolytic and capable of loading clinical anticancer drug doxorubicin and deep-tissue penetrable near infrared biomarker IR 780. In vitro enzymatic-drug release kinetics and lysotracker-assisted real-time live-cell confocal bioimaging revealed that the rod-like polymer brush is superior than its spherical counterparts for faster cellular uptake and enzymatic-biodegradation at the endo-lysosomal compartments to release DOX at the nucleus. Further, in-vivo live-animal bioimaging by IVIS technique established that the IR-780 loaded rod-like polymer brush exhibited efficient deep-tissue penetration ability and emphasized the importance of polymer brush topology-control for biological activity. Polymer brushes exhibit good stability in the blood plasma for more than 72 h and they predominately accumulated in the digestive organs like liver, kidney and spleen and they were less toxic to heart and brain tissues. IVIS imaging of cryotome tissue slices of organs confirmed that deep-penetrating ability of the polymer brushes. The present investigation opens opportunity for bio-derived and biodegradable polymer brushes as next generation smart drug delivery scaffolds. L-amino acid and sugar containing biodegradable polymer brushes were developed using mannose and glucose as anchoring units. These poly(ester-urethane)s were characterized for structural, thermal properties, and self- assembled into 100±10 nm sized spherical nanoparticle in aqueous medium. The nano-assemblies showed excellent encapsulation capabilities for anticancer drug camptothecin (CPT), Topotecan (TPT), anti-inflammatory drug curcumin (CUR), biomarkers such as 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS), Cardiogreen (ICG), etc. these polymers are non-hemolytic and were checked for cytotoxicity and it found that these polymers are non-toxic to the MCF-7 cancerous cells, MDA-MB-468, as well as normal cells MEF, HEK-293 and pancreatic cells MIA PaCa-2 cells. Confocal laser scanning microscopy (CSLM) assisted time-dependent cellular uptake analysis directly evident for the of CPT loaded polymer NP endocytosis and their internalization for biodegradation. Further, In-vivo live animal bio-imaging for biodistribution was carried out using cardio green biomarker labelled polymer brushes. The polymer brushes exhibited longer blood circulation time as well and harvested organs showed larger accumulation of polymer brushes in major excretory organs after 24h. Finally, the L-tyrosine propargyl functionalized coumarin tagged fluorescent polymer brushes were developed to study the real-time drug delivery aspects. The fluorophore was introduced via a click reaction as side chain to study the role of the polymer topology on the drug or fluorophore loading and delivering capabilities. The photophysical characterization of the nanoparticle was done using absorbance and emission spectroscopy, TCSPC to establish FRET probe. It was clearly demonstrated that there is transfer of energy from Coumarin molecule to Nile red (NR) in the hydrophobic nanocavity of the nanoparticle which was further confirmed by determining the distance between donor and acceptor, FRET efficiency etc. These poly(ester-urethane)s based FRET probes were capable of enzyme-responsive behaviour which was supported by enzyme responsive release of NR. The presentation will give the overall importance of biodegradable polymer brushes in this field and, more specifically, in vitro and in vivo studies based on L-tyrosine based polymer system.