Development of Fluorescent Polymeric Nano-Assemblies for Studying Organelle Interactions in Intracellular Drug Release and Accumulation

Abstract

Drug delivery via nano-carriers enabled the translation of promising therapeutics into successful therapies. Nanotechnology evolved over the years and led to the development of various nano- delivery scaffolds. Much research has been dedicated towards the design optimization of nano carriers to bring in the maximum drug efficacy. However, the current design paradigm is mainly focused from materials perspective and little attention is given to the biological aspects. Administered nanoparticles face plethora of biological obstacles which rather limit their ability to reach their desired targets. These obstacles are prevalent both at the macro and the micro level. Macro level obstacles comprise of organ level barriers to sub-organ level barriers, whereas micro level barriers are generally encountered at the intracellular level. The inability of the drug to reach and populate at the target site renders 1 out of 9 newly discovered drug delivery systems ineffective by authorities, leading to high attrition rates of such delivery systems in the later stage clinical trials. Most of the clinical trials of these drug delivery systems fails due to reduced efficacy which perhaps stems from a heterogenous patient response. The relatively low success rate of these drugs has prompted a need to better understand the underlying pharmacology mechanism at single cell level. This thesis aims to visualize the delivery aspects of nano carrier at cellular level to appreciate the barriers operational at micro level which then modulates the therapeutic efficacy of a drug delivery system. The thesis is divided into 4 chapters whose details are mentioned below:  Chapter 1: The first chapter extensively discussed the materials and biological aspects of drug delivery. Material aspects such as type of therapeutic and type of delivery system have been discussed. Emphasis is also given on biological barriers which stands tall in hampering the efficacy of drug delivery system. Intracellular barrier of lysosome drug sequestration plays a significant role in the reducing the therapeutic effect of the small molecule drugs by sequestering the therapeutic away from target site.  Chapter 2: The success of nano-carrier-mediated drug delivery following endocytosis hinges on the efficacious drug delivery to the target site. The second chapter of this thesis focuses on understanding the real-time visualization of delivery aspects of a fluorescent nano-carrier to deliver a fluorescent drug or dye molecule by employing FRET as a versatile imaging tool. Following the nano-carrier’s endocytosis and localization to endosomes, inter-organelle interaction with lysosomes mediates its cellular fate, which generally terminates at lysosomes where the encapsulated drug is released. Fluorescence-abled visualization of nano-carrier can help track their organelle residence, determining its drug efficacy.  Chapter 3: Cellular organelles such as Lysosomes and endosomes are known to sequester weakly basic therapeutics such as doxorubicin from target nuclear site. Alterations or defects in lysosome-endosome fusion can abate the therapeutic efficacy of weakly basic drugs. Although comprehending this intricate process can help understand drug sequestration leading to poor drug efficacy and concomitant poor prognosis, visualizing it remains challenging due to the difficulties with probe designs. Towards this, the third chapter of the thesis is focused on structural engineering of pH-responsive ESIPT nano-emitter that would be proficient in understanding the inter-organelle interaction in sequestration of the drug molecules at the single cell level. This requires the labelling of the endosomes and lysosomes in two fluorescent different colors that would able its differential visualization while live monitoring cells with a confocal microscope. Therefore, the engineered probe should be two color emitting in repose to the pH conditions in endosomes and lysosomes. In this chapter efforts were taken to develop and structurally engineer a nano-emitter fluorescent probe that was single-wavelength excitable and two-color emitting, which could possibly label organelles of different acidity.  Chapter 4: After optimizing the pH responsive nano-emitter, in-depth analysis was done to study their ability to annotate the endocytic vesicles i.e. endosomes and lysosomes in two different fluorescent colors. The lysosomes were labeled with green fluorescence and endosomes were labelled with green fluorescence that abled the monitoring of their dynamic interaction under single wavelength excitation. This probe was leveraged to understand the dynamic interactions between endosomes and lysosomes that were instrumental in sequestering of weakly basic doxorubicin drug.