The past few years have witnessed significant work being directed towards drug delivery systems with layer-by layer (LbL) technique prominently featured as one of the most sought after approach. However, majority of the studies were focused on the fabrication of microcapsules which produced numerous drawbacks resulting in reduced applicability. This has spurred research into nanocapsules which has proved to overcome most of the drawbacks that plagued microcapsules by being able to evade the reticulo-endothelial system, exhibit enhanced permeability and retention in tumours etc. The capsules fabricated by the LbL technique requires a suitable combination of cationic and anionic polyelectrolytes which ensures that it is able to effectively protect the cargo it encapsulates as well as enhance its bio-applications. With numerous advantages such as biocompatibility and biodegradability to name a few, chitosan has proved to be an ideal cationic polyelectrolyte. Thus, this thesis focuses on the various therapeutic applications of LbL fabricated chitosan based nanocapsules. The first work focuses on the targeted delivery of the somatostatin analogue, Octreotide conjugated nanocapsules to over expressed somatostatin receptors. These LbL fabricated nanocapsules composed of chitosan and dextran sulfate (CD) encapsulate the anti cancer drug, doxorubicin and are found to attain site specificity as well as enhanced anti-proliferative activity. The results indicated that the nanocapsules were biocompatible and when conjugated with octreotide was found to have an enhanced internalization into SSTR expressing cells, thereby making it a viable strategy for the treatment of tumors that has an over expression of somatostatin receptors such as pancreatic carcinoma, breast carcinoma etc. The objective of the second work was to develop an efficient drug delivery system such as CD nanocapsules for encapsulation of Ciprofloxacin in order to combat infection by Salmonella, an intracellular and intra-phagosomal pathogen. In vitro and in vivo experiments showed that this delivery system can be used effectively to clear Salmonella infection. The increased retention of ciprofloxacin in tissues delivered by CD nanocapsules as compared to the conventional delivery proved that the same therapeutic effect was obtained with reduced dosage and frequency of Ciprofloxacin administration. The third work deals with the probiotic, Saccharomyces boulardii which is found to be effective against several gastrointestinal diseases but had limited clinical application due to its sensitivity to acidic environment. However, encapsulation of S. boulardii with chitosan and dextran sulfate ensured enhanced viability and selective permeability on exposure to acidic and alkaline conditions experienced during gastro intestinal transit. The final work involves the fabrication of novel pH responsive nanocapsules composed of chitosan-heparin which facilitate the intracellular delivery of a model anti-cancer drug, doxorubicin.

Chapter–I Introduction Shock waves appear in nature whenever the different elements in a fluid approach one another with a velocity larger than the local speed of sound. Shock waves are essentially non-linear waves that propagate at supersonic speeds. Such disturbances occur in steady transonic or supersonic flows, during explosions, earthquakes, tsunamis, lightening strokes and contact surfaces in laboratory devices. Any sudden release of energy (within few μs) will invariably result in the formation of shock wave since it is one of the efficient mechanisms of energy dissipation observed in nature. The dissipation of mechanical, nuclear, chemical, and electrical energy in a limited space will result in the formation of a shock wave. However, it is possible to generate micro-shock waves in laboratory using different methods including controlled explosions. One of the unique features of shock wave propagation in any medium (solid, liquid or gases) is their ability to instantaneously enhance pressure and temperature of the medium. Shock waves have been successfully used for disintegrating kidney stones, non-invasive angiogenic therapy and osteoporosis treatment. In this study, we have generated a novel method to produce micro-shock waves using micro-explosions. Different biological applications were developed by further exploring the physical properties of shock waves. Chapter – II Bacterial transformation using micro-shock waves In bacteria, uptake of DNA occurs naturally by transformation, transduction and conjugation. The most widely used methods for artificial bacterial transformation are procedures based on CaCl2 treatment and electroporation. In this chapter, controlled micro-shock waves were harnessed to develop a unique bacterial transformation method. The conditions have been optimized for the maximum transformation efficiency in E. coli. The highest transformation efficiency achieved (1 × 10-5 transformants per cell) was at least 10 times greater than the previously reported ultrasound mediated transformation (1 × 10-6 transformants per cell). This method has also been successfully employed for the efficient and reproducible transformation of Pseudomonas aeruginosa and Salmonella Typhimurium. This novel method of transformation has been shown to be as efficient as electroporation with the added advantage of better recovery of cells, economical (40 times cheaper than commercial electroporator) and growth-phase independent transformation. Chapter – III Needle-less vaccine delivery using micro-shock waves Utilizing the instantaneous mechanical impulse generated behind the micro-shock wave during controlled explosion, a novel non-intrusive needleless vaccine delivery system has been developed. It is well established, that antigens in the epidermis are efficiently presented by resident Langerhans cells, eliciting the requisite immune response, making them a good target for vaccine delivery. Unfortunately, needle free devices for epidermal delivery have inherent problems from the perspective of patient safety and comfort. The penetration depth of less than 100 µm in the skin can elicit higher immune response without any pain. Here the efficient utilization of the device for micro-shock wave mediated vaccination was demonstrated. Salmonella enterica serovar Typhimurium vaccine strain pmrG-HM-D (DV-STM-07) was delivered using our device in the murine salmonellosis model and the effectiveness of the delivery system for vaccination was compared with other routes of vaccination. The device mediated vaccination elicits better protection as well as IgG response even in lower vaccine dose (ten-fold lesser), compare to other routes of vaccination. Chapter – IV In vitro and in vivo biofilm disruption using shock waves Many of the bacteria secrete highly hydrated framework of extracellular polymer matrix on encountering suitable substrates and get embedded within the matrix to form biofilm. Bacterial colonization in biofilm form is observed in most of the medical devices as well as during infections. Since these bacteria are protected by the polymeric matrix, antibiotic concentration of more than 1000 times of the MIC is required to treat these infections. Active research is being undertaken to develop antibacterial coated medical implants to prevent the formation of biofilm. Here, a novel strategy to treat biofilm colonization in medical devices and infectious conditions by employing shock waves was developed. Micro-shock waves assisted disintegration of Salmonella, Pseudomonas and Staphylococcus biofilm in urinary catheters was demonstrated. The biofilm treated with micro-shock waves became susceptible to antibiotics, whereas the untreated was resistant. Apart from medical devices, the study was extended to Pseudomonas lung infection model in mice. Mice exposed to shock waves responded well to ciprofloxacin while ciprofloxacin alone could not rescue the mice from infection. All the mice survived when antibiotic treatment was provided along with shock wave exposure. These results clearly demonstrate that shock waves can be used along with antibiotic treatment to tackle chronic conditions resulting from biofilm formation in medical devices as well as biological infections. Chapter – V Shock wave responsive drug delivery system for therapeutic application Different systems have been used for more efficient drug delivery as well as targeted delivery. Responsive drug delivery systems have also been developed where different stimuli (pH, temperature, ultrasound etc.) are used to trigger the drug release. In this study, a novel drug delivery system which responds to shock waves was developed. Spermidine and dextran sulfate was used to develop the microcapsules using layer by layer method. Ciprofloxacin was loaded in the capsules and we have used shock waves to release the drug. Only 10% of the drug was released in 24 h at pH 7.4, whereas 20% of the drug was released immediately after the particles were exposed to shock waves. Almost 90% of the drug release was observed when the particles were exposed to shock waves 5 times. Since shock waves can be used to induce angiogenesis and wound healing, Staphylococcus aureus skin infection model was used to show the effectiveness of the delivery system. The results show that shock wave can be used to trigger the drug release and can be used to treat the wound effectively. A brief summary of the studies that does not directly deal with the biological applications of shock waves are included in the Appendix. Different drug delivery systems were developed to check their effect in Salmonella infection as well as cancer. It was shown for the first time that silver nanoparticles interact with serum proteins and hence the antimicrobial properties are affected. In a nutshell, the potential of shock waves was harnessed to develop novel experimental tools/technologies that transcend the traditional boundaries of basic science and engineering.