Design & Fabrication of Bio-responsive Drug Carriers Based on Protamine & Chondroitin Sulphate Biopolymers

The present thesis focuses on the fabrication of bio-stimuli responsive micro- and nano-carriers for drug delivery applications. In particular, the objective of this work is to investigate the possibility of using polypeptide drug protamine and glycosaminoglycan drug, chondroitin sulphate as stimuli responsive components in the design of bioresponsive carriers. These biopolymers are biocompatible, biodegradable and clinically used for various applications. Two designs that incorporate these stimuli responsive components have been studied in this thesis. The first design involves hollow micro and nanocapsules that have been fabricated by incorporating the stimuli responsive biopolymers as wall components. Upon exposure to biological triggers, these hollow capsules disintegrate releasing the encapsulated drug. The second design consists of mesoporous silica nanoparticles-biopolymer hybrids. The mesoporous silica nanoparticles act as a gated scaffold that carries the drug molecules. The mesopores of these drug loaded nanoparticles are then blocked with the bioresponsive polymers. Upon exposure to the bio-triggers which consist of enzymes over-expressed in conditions such as cancer and inflammation, these “molecular gates” disintegrate allowing the drug trapped in the mesoporous silica nanoparticles to escape into the surroundings. The thesis has been divided into five chapters: Chapter 1 is an introduction to bio-responsive drug delivery. The broad classification of stimuli used in responsive drug delivery systems are visited. A brief discussion on the various types of bio-stimuli that can be utilized in designing bio-responsive systems is also included in this chapter. Chapter 2 defines the aims and scope of the thesis which is followed by an overview of the various design parameters involved in the fabrication of systems presented in this work. The major stimuli responsive components and the architectures incorporating these elements are discussed in detail here. A literature review of the various carrier designs involved in the study is provided , with special emphasis on stimuli responsive drug delivery. Chapter 3 gives an overview of the various materials and methods involved in this work. A summary of the various characterisation techniques used in the thesis is also included along with the details of the experiments that has been carried out. Chapter 4 provides an overview of the results and discussions of the thesis. The chapter has been divided into six sections: Chapter 4.1 deals with the fabrication of a hollow microcapsule system incorporated with protamine as the stimuli responsive element for bio-responsive drug delivery. The hollow microcapsules that were fabricated by Layer by Layer assembly of protamine and heparin display pH responsive variations in permeability and disintegrate in the presence of the enzyme trypsin that degrades protamine. The biologically triggered enzyme responsive drug release from these microcapsules is also demonstrated using enzymes secreted by colorectal cancer cells. Chapter 4.2 presents nanocapsules fabricated from protamine and heparin. The pH and enzyme responsive drug release of this systems is evaluated in vitro. A wall crosslinking strategy has been tested to control the rate of drug release under physiological pH conditions in the absence of the trigger. The cellular interactions of these nanocapsules loaded with an anticancer drug, doxorubicin was studied using cancer cell lines. Bioavailability studies of doxorubicin encapsulated in these nanocapsules were performed using a BALB/c mice model. Chapter 4.3 discusses the fabrication of a hollow microcapsule system that can disintegrate in response to dual biological stimuli. These carriers have been fabricated by incorporating protamine and chondroitin sulphate as the wall components. Due to the incorporation of two separate stimuli responsive components in the walls, these capsules are expected to be sensitive to the enzymes trypsin or hyaluronidase I. Chapter 4.4 deals with the fabrication of dual enzyme responsive hollow nanocapsule which can be targeted to deliver anticancer agents specifically inside cancer cells. The enzyme responsive elements integrated in the hollow nanocapsule walls can undergo degradation in presence of either of the enzymes trypsin or hyaluronidase I leading to the release of encapsulated drug molecules. The drug release from these nanocapsules which were crosslinked and functionalised with folic acid, is evaluated under varying conditions. The cellular uptake and intracellular drug delivery by these nanocapsules were evaluated in cervical cancer cell lines. Chapter 4.5 introduces a mesoporous silica nanoparticle − protamine hybrid system. The system consists of a mesoporous silica nanoparticle support whose mesopores are capped with protamine which effectively blocks the outward diffusion of the drug molecules from the mesopores of the mesoporous silica nanoparticles. Upon exposure to the enzyme trigger, the protamine cap disintegrates opening up the molecular gates and releasing the entrapped drug molecules. The drug release from this system is evaluated in different release conditions in the presence and absence of the enzyme trigger. The ability of these particles to deliver hydrophobic anticancer drugs and induce cell death in colorectal cancer cells has also been demonstrated. Chapter 4.6 discusses the fabrication of another mesoporous silica nanoparticles based bio-responsive drug delivery system consisting of mesoporous silica and chondroitin sulphate hybrid nanoparticles. The ability of the system to modulate drug release in response to hyaluronidase I is demonstrated. By utilizing a cervical cancer cell line, we have demonstrated the cellular uptake and intracellular delivery of hydrophobic drugs encapsulated in these particles. Interestingly, the system showed ability to enhance the anticancer activity of hydrophobic drug curcumin in these cancer cells. Chapter 5 gives a summary of the general conclusions drawn from the thesis work