i Thermo-Responsive Small and Polymeric Amphiphiles for Drug Delivery

Abstract

Polymer based nanocarriers are emerging as important biomaterials for delivering anticancer drugs or genes to cancer tissues. Polymeric drug carriers such as micelles, vesicles and nanoparticles have been fabricated till date to achieve site-specific drug delivery. These nanocarriers have advantage of undergoing passive selective accumulation in tumor tissues through EPR effect. The advent of stimuli-responsive drug carriers has amplified the targeting ability and specificity of the nanocarriers. In a view of designing stimuli-sensitive polymeric vehicles, the unusual physiochemical environments of the cancer tissues such as high temperature (40-43C), acidic pH (6.1 to 6.8), over-expression of enzymesand hypoxic conditions have been used as a trigger for releasing drug at cancer site. Among all these stimuli-based materials, thermo-sensitive polymers provide unique opportunity for delivering chemotherapeutic agents to tumor tissues without affecting the micro-environment of tissues. Unfortunately, thermos-responsive polymeric materials are much less explored compared to other stimuli-based materials; hence, new smart amphiphilic designs (both small and polymeric) are required to accomplish better treatment for cancer therapy. This thesis work is focused on the design and development of an efficient nanocarrier for targeting tumor or cancer cells using temperature as a stimulus for releasing the loaded cargoes. Small amphiphilic molecules based thermo-responsive scaffolds were synthesized in order to understand the role of hydrophobic and hydrophilic segments on the thermal-behavior of their drug-release mechanism. These amphiphiles were designed with hydrophobic unit from renewable recourse 3-pendadecyl phenol which is one of the main constituent of cashew nut shell liquid. The self-assembled nanostructure formed from the amphiphile was further employed for loading fluorescent dyes and various anti-cancer drugs. Synopsis viii This thesis has been divided into five major chapters: 1. Introduction Chapter: The first chapter provides a complete literature survey on polymer based drug delivery and importance of stimuli-responsive nanocarrier in the field of drug delivery. 2. Thermo-responsive and shape transformable amphiphiles: A renewable resource based amphiphilic molecules having shape transforming ability upon exposure to temperature variation was developed.The drug loading capabilities and thermally-induced drug release kinetics from these unique core-shell nanoparticles was investigated in detail. 3. Thermal and enzyme dual responsive polymeric scaffold:New series of amphiphilic copolymers composed of hydrophobic acrylate unit and ethylene glycols were synthesized. These nanoparticles were loaded with anticancer drug doxorubicin and their cytotoxicity was studied in breast cancer (MCF 7) and cervical cancer (HeLa) cell lines. 4. The Hofmeister effect: An amphiphilic molecule with “super-LCST characteristics” was developed in order to study the influence of biologically relevant anions on the thermo-responsive properties of nanocarriers. 5. Multivesicular amphiphilic scaffolds: New amphiphiles were designed to produce small unilamellar vesicles (SUVs) and multi-vesicular bodies (MVBs). The drug loading and delivering capabilities of both SUVs and MVBs were investigated under physiological conditions (pH=7.4, PBS) and in presence of esterase enzyme. The chapter-1 provides a brief introduction to drug delivery with emphasis on advantages of polymeric materials as drug vehicles for treatment of cancer.A detailed literature survey on different types of small amphiphile based drug delivery systems which are commonly used as nanocarrier, their properties and limitations have been discussed.This chapter also provides a complete literature survey on importance of stimuli-responsive polymeric nano-vehicles and highlighting their application in the field of biotechnology and drug delivery. Thermo-responsive Synopsis ix nanocarriers have been discussed as an important approach for efficiently delivering the chemotherapeutic agents in tumor microenvironment. The second chapter describes the drug loading and delivering capabilities of temperature induced shape-transformable carrier’s at cancer tissue temperature. Amphiphilic molecule based on renewable resource hydrophobic 3-pendadecylphenol connected to hydrophilic oligoethylene glycol via hydrogen bonded amide linkage were tailor made through multi-step organic synthesis. The thermo-responsive behaviour and self-assembly of the amphiphiles were analysed. The three dimensional core-shell nanoparticles underwent temperature induced shape transformation in to one-dimensional rod-like structures. The temperature driven in-situ transformation of the amphiphilic scaffold from three dimensional core-shell at temperature below LCST to rod-like structures at higher temperature in water (or PBS at pH= 7.4). The thermo-responsive core-shell nanoparticles were employed for encapsulating anticancer drugs such as doxorubicin (DOX) and camptothecin (CPT). The release profile of the DOX under in-vitro conditions revealed that the DOX can be selectively release at cancer tissue temperature (40-43C) as compared to normal body temperature (37 C). The cytotoxicity studies of the nascent scaffold and drug loaded was carried out on cervical cancer (HeLa) cell lines using MTT assay method. The third chapter describes the role of dual responsive polymer nano-scaffolds for administrating anticancer drugs both at extracellular level in tumor tissues and intracellular compartments of cancer cells for improving drug efficacy. For this purpose, a new class of thermo and enzyme dual responsive polymeric amphiphiles was tailor-made through copolymerization of hydrophobic acrylate monomer from 3-pentadecylphenol (PDP, a renewable resource) and oligoethylene Synopsis x glycol acrylate (as hydrophilic monomer). The copolymers synthesized varied in the composition of hydrophilic and hydrophobic segment in their structure. The thermo-responsive behaviour of the various amphiphilic copolymers was investigated. The copolymers with 6 % hydrophobic unit in their backbone showed LCST close to cancer tissue temperature. These copolymers self-assembled to produce spherical core-shell nanoparticles in water at temperature below LCST. The dual responsive polymer scaffold was found to be capable of loading both hydrophobic dye (Nile red) and drug (DOX). The release profile of DOX at normal body temperature (below LCST,  37 C) revealed that DOX was preserved in the core-shell assemblies, while at temperature closer to cancer tissue (above LCST, ~ 43C), the polymeric scaffold underwent burst release to deliver 90 % of loaded drugs within 2 h. On the other hand, under conditions similar to in intra-cellular compartment (pH = 7.4, 37 C, esterase enzyme); the amphiphilic copolymer ruptured slowly leading to controlled release of drug (> 95 %) for 12 h. Thus, both burst release of cargoes at the tumor microenvironment and control delivery at intracellular compartments were accomplished. Cytotoxicity assay of the nascent and DOX loaded polymer were carried out in breast cancer (MCF-7 cells) and cervical cancer (HeLa cells). Among the two cell lines, the DOX loaded polymers showed enhanced killing in breast cancer cells. Confocal microscopic images confirmed that DOX loaded core-shell nanoparticles were taken up by MCF-7 cells, showing a distinctly perinuclear localization in cell. The fourth chapter deals with the design and development of super LCST thermo-responsive amphiphilic nanoparticle assembly for detection of adenosine triphosphate (ATP) through Hofmeister effect. A new diblock molecular based on hydrophilic polyethylene glycol and PDP as hydrophobic unit was designed to study Synopsis xi the role of biologically relevant anions on the thermo-responsive behavior of the nanocarriers. The amphiphile self-assembled as 150 nm micellar nanoparticle and showed super lower critical solution temperature (LCST) above 90 C. The effect of anions on the phase-transition temperature (LCST) of the amphiphile was in consistent with the “Hofmeister series” with higher selectivity for the recognition of ATP over its adenosine precursors such as ADP, AMP and inorganic phosphate (Pi). The preferential binding for ATP is attributed to the encapsulation in the hydrophobic pocket and modification of hydration shell at the periphery of the amphiphilic nanoparticles.The binding constants for the amphiphilic nanoparticle binding to ATP were determined by isothermal calorimetric measurements. The cytotoxicity of the super LCST amphiphile carried out on cervical cancer (HeLa) cells revealed that the amphiphile was non-toxic in cells. In the fifth chapterfluorophore encapsulation pathways and drug loading abilities in synthetic macromolecular amphiphiles sorting into mutivesicular bodies (MVB)s was reported. For this purpose, renewable resource based amphiphiles having hydrophobic units and flexible hydrophilic polyethylene glycols (PEG) were custom designed. To prove the existence of the strong inter-molecular interactions and the formation of uni-lamellar layer-like self-assemblies single crystal structure was resolved. Small uni-lamellar vesicles (SUV)s or MVBs were produced from these Synopsis xii amphiphilic AB amphiphiles through selective vesicular fission either by outward budding or inward invagination, respectively. The mechanistic aspects of the MVB andformations was studied by encapsulating environment sensitive fluorescent probe, pyrene. An un-usual non-linear trend was observed in the pyrene dynamic excimer formation with respect to the sorting of diblock membrane into MVBs. Doxorubicin, the anti-cancer drug was employed for studying the encapsulation capabilities of both MVBs and SUVs. The drug release profile of DOX loaded MVBs and SUVs under physiological conditions (pH = 7.4, PBS) revealed that DOX was stable in MVBs while SUVs released more than 90 % of the drug. MVBs showed two step DOX release profileswith respect to outer and inner vesicles cleavage in the presenceof esterase enzyme. The last chapter summarizes the overall outcome of thesis work and future directions