The ability of a novel fish oil emulsion antigen-delivery system administered orally and by immersion, to stimulate antibody responses in the dab, was measured in a further experiment. On this occasion, oral intubation of HGG (25 mg) in saline induced no detectable responses. Immersion in a bath containing HGG in lipid emulsion induced a transient ASC response in the blood only. Anal intubation of HGG (25 mg) in saline induced a slight ASC response in the gut and blood. Oral intubation of HGG (25 mg) in lipid emulsion induced ASC responses in the gut and transiently in the gill but no response (above background) in the head kidney. None of the above methods of immunisation induced serum antibody titres. Intraperitoneal injection of HGG (1 mg) in saline induced high numbers of ASC in the head, kidney, gut and gill as well as serum antibody. The ASC response in the head kidney was detected from week 5 to 10, peaking at week 5. The response in the gill was from week 3 to 10, peaking at week 6, and the response in the gut was from week 5 to 10, peaking at week 8. The results indicate that systemic stimulation induced ASC responses in both systemic and mucosal compartments. The orally protected HGG in lipid emulsion was more effective than oral HGG in saline and anal HGG in saline in inducing ASC responses in the gut and the gill without including serum antibody, suggesting that oral immunisation can induce a common mucosal response independently of the head kidney. Leucocytes were isolated from the perfused gill of rainbow trout (<I>Oncorhynchus mykiss</I>) and fractioned on a 40-70% Percoll gradient into two subpopulations, top and bottom cell fractions. On stimulation with calcium ionophore, the isolated gill cells, following nylon wool filtration, were shown to be capable of producing chemoattractants for head kidney leucocytes at a dilution of 1:8. Only the bottom cell fraction exhibited migration toward a 2% dilution of trout serum while dilutions of 0.25% and 0.5% rainbow trout serum were not chemoattractive for either head kidney or gill leucocyte populations. The highest migration index was achieved after 1.5 h and the optimal cell number for migration was 4.65x10⁷ cells/ml. Respiratory burst activity was undetectable with isolated gill cells. Mitogenic responses of isolated and fractionated gill cells to LPS and PHA suggested the presence of few B-cells and a preponderance of T-cells.

Currently, the most common route for vaccine delivery is by intramuscular injection with a needle and syringe. Injection has number of disadvantages, such as risk of infection at the i njection site, needle prick injuries, and needle phobia that leads to significant levels of patient non-compliance. Therefore, the focus of this thesis is the development of an alternative ultrasound-assisted transdermal vaccine delivery system. To do so, we target immunological Langerhans cells in the epidermal layer of the skin that efficiently provoke an immune response. The stratum corneum (SC) is a barrier that prevents conventional transdermal vaccine delivery. Methods such as microneedles, iontophoresis and thermal ablation are presented in literature for the permabilisation of this layer. Sonophoresis is the use of ultrasound to transport molecules through a medium. Previous studies have demonstrated that the key underpinning mechanism is inertial cavitation, which leads to permeabilisation of the SC and facilitates transdermal delivery. Most studies to date have pre-exposed the skin to ultrasound prior to delivery of a vaccine in liquid form as a droplet placed on the skin. This approach is not practical for widespread use, but more importantly fails to take advantage of the potential of cavitation-mediated micro streaming to enhance active transport of molecules beyond the permeabilised skin. The focus of the present work is the development of a complete system that enables storage of the vaccine in a readily useable gel form whilst promoting and monitoring cavitation activity to simultaneously permeabilise the skin and enhance transdermal vaccine transport. Through initial in vitro studies, we first demonstrated that inertial cavitation can be exploited to promote the active transport of molecular entities such as vaccine molecules from a gel into a biological medium. A gel vaccine dosage formulation is utilised in order to mimic current clinically approved and established clinical ultrasound coupling gel formulations. By comparing the effects mediated at two ultrasound frequencies (0.256 MHz vs 1 MHz) which preferentially promote cavitational microstreaming or acoustic streaming, ultrasound parameters most conducive to producing high levels of inertial cavitation were identified as 0.256 MHz and peak rarefactional pressures on the order of 1 MPa. Three vaccine loaded gels were then formulated with either micro- or nano-sized cavitation nuclei and assessed for the optimal acoustic and chemical characteristics at the predetermined ultrasound parameters. Nano-sized nuclei were shown to be most effective at lowering the inertial cavitation threshold, as well as instigating the highest and most sustained levels of inertial cavitation as indicated by broadband acoustic emissions at the ultrasound focus, without causing any structural damage to the vaccine molecules themselves. Ex vivo data has shown that nanoscale-nucleated inertial cavitation at the skin surface delivered a model vaccine Ovalbumin (OVA) to depths of 500 &mu;m into porcine skin. Novel nanoparticles produced in-house used to enhance and instigate cavitation at lower pressures penetrated to depths of up to 700 &mu;m, due to their small size and unique ability to self-propel. Delivery profiles were obtained using multi-photon microscopy of skin sections immediately after treatment. Analysis of acoustic emissions from the focus showed substantial correlation between high delivery dose and depth, and significant amounts of inertial cavitation (i.e. broadband acoustic emissions from the focus). In vivo studies showed that the delivery achieved to murine skin was significantly (p&LT;0.05) higher in the nanoparticle-assisted ultrasound transdermal vaccination group than the chemical penetration enhancer (positive control) group, with delivery of doses up to 1 &mu;g /treatment, compared to 400 ng in the positive control group. This dose was sufficient to trigger an antigen-specific immune response. Specific anti-OVA IgG antibody levels in the ultrasound-assisted vaccine delivery group were significantly (p&LT;0.05) higher than in all other control groups, and substantially higher than the current gold standard in transdermal delivery – chemical penetration enhancers. Although a low level antibody response was observed transdermally compared to the subcutaneous injection group (indicative of 100&percnt; delivery response), it is believed that optimisation of this system will lead to a viable and non-invasive delivery platform for vaccines that can be used both in a primary care setting, and eventually for self-vaccination at home.

L'administration nasale a un grand avantage pour stimuler l'immunité protectrice locale et systémique. Cependant, des systèmes d'administration et des adjuvants sont souvent nécessaires pour améliorer l'efficacité du vaccin intranasal. Nous avons appliqué la technologie des nanoparticules en tant que système universel de délivrance de vaccins contre la grippe dans le projet européen FP7 appelé UniVacFlu.Nous avons évalué différentes nanoparticules (NP) pour rechercher le meilleur nanovecteur. Pour cela, nous avons comparé 5 types de nanoparticules avec différentes charges de surface (anioniques ou cationiques) et diverses compositions internes comme vecteurs potentiels: des liposomes cationiques ou anioniques, des NP de PLGA cationique ou anionique (poly acide lactique co-glycolique) et une NP cationique composée de maltodextrine fonctionnalisée par un agent cationisant avec un coeur de lipides anioniques (NPL). Nous avons d'abord quantifié leur temps de résidence nasale après l'administration nasale chez la souris en utilisant l'imagerie in vivo et les NPL ont montré le plus long temps de résidence. L'endocytose in vitro sur des cellules muqueuses (cellules épithéliales des voies respiratoires, macrophages et cellules dendritiques) en utilisant des nanoparticules marquées a été réalisée par cytométrie de flux et microscopie confocale. Parmi les 5 nanoparticules, les NPL ont été majoritairement captées par 3 lignées cellulaires différentes représentatives d’un épithélium respiratoire et les mécanismes d'endocytose ont été caractérisés. Afin d’évaluer le meilleur vecteur en tant que véhicules, le chargement d'antigènes et la délivrance intracellulaire ont été évalués dans des cellules de la muqueuse des voies respiratoires (cellules épithéliales des voies aériennes, macrophages et cellules dendritiques) par cytométrie de flux. Nous montrons que les NPL sont les meilleurs candidats capables de délivrer la plus grande quantité de protéines dans les cellules. Pris ensemble, notre étude a révélé que parmi 5 nanoparticules, la NPL était le meilleur nanovecteur en termes de temps de résidence nasale, d'endocytose par les cellules et de délivrance de protéines dans l'épithélium des voies respiratoires. Les NPL ont donc été sélectionnées comme nanovecteurs pour le projet UniVac Flu.Les antigènes de la grippe CTA1-3M2e-DD et HA ont été formulés avec les NPL. Le CTA1-3M2e-DD est un antigène chimérique adjuvanté et ciblé. Il est composé de la sous-unité A1 de la toxine du choléra et un épitope conservé du virus grippal A (M2e), ainsi que le dimère de l'analogue synthétique de la protéine A de Staphylococcus aureus (DD) utilisé comme agent de ciblage des lyphocytes B. Pour améliorer l'effet antigénique, l’HA recombinant de H1N1 a été combinée avec CTA1-3M2e-DD. Ces formulations ont été évaluées chez la souris par le consortium UniVacFlu. Les résultats ont montré que CTA1-3M2e-DD et HA chargé dans les NPL formeraient un vaccin intranasal prometteur contre la grippe. Ce travail de thèse montre que les NPL sont des nanovecteurs d’intérêt pour le vaccin nasal. / Nasal administration has great advantage for stimulating the immune system, such as stimulating local and systemic protective immunity. However, delivery systems and adjuvants are often necessary to improve the efficacy of the intranasal vaccine. We applied nanoparticle technology to deliver a universal influenza vaccine via the nasal route in a European FP7 project called UniVacFlu.We evaluated different nanoparticles to search the best nanocarrier for an intranasal vaccine. Here we compared 5 types of nanoparticles with different surface charges (anionic or cationic) and various inner compositions as potential vectors: cationic and anionic liposomes, cationic and anionic PLGA (Poly Lactic co-Glycolic Acid) nanoparticles and zwitterionic maltodextrin nanoparticles (cationic surface with an anionic lipid core: NPL). We first quantified their nasal residence time after nasal administration in mice using in vivo live imaging and NPL showed the longest residence time. In vitro endocytosis on mucosal cells (airway epithelial cells, macrophages and dendritic cells) using labeled nanoparticles were performed by flow cytometry and confocal microscopy. Among the 5 nanoparticles, NPL were taken up to the greatest extent by the 3 different cell lines and the endocytosis mechanisms of NPL were characterized. In order to compare different nanoparticles as vaccine carriers, antigen loading and cell delivery were evaluated. In this study, we compared the loading and delivery of labeling ovalbumin with airway mucosa cells (airway epithelial cells, macrophages and dendritic cells) by flow cytometry. Our data showed that NPL were the best candidate that can payload with highest amount of protein and eventually the most efficient cellular protein delivery capacity. Taken together, our study revealed that among 5 nanoparticles, NPL were the best nanocarrier that own longer nasal residence time, efficiently uptake and deliver protein into airway epithelium. NPL were then selected as nanocarrier for the UniVac Flu project.The flu antigens CTA1-3M2e-DD and HA were formulated with NPL. The CTA1-3M2e-DD is an adjuvanted antigen composed of the A1 subunit of cholera toxin and a conserved epitope of influenza A virus (M2e), as well as the dimer of the synthetic analogue of Staphylococcus aureus protein A (DD) used to target B cells. To improve antigenic effect, recombinant HA from H1N1 was combined with CTA1-3M2e-DD. These formulations were evaluated in mice by the UniVacFlu consortium. We observed that CTA1-3M2e-DD and HA loaded into NPL could be a promising universal intranasal influenza vaccine.

acase@tulane.edu

Biopharmaceuticals, including proteins, DNA and vaccines, are one of the fastest growing segments of the overall pharmaceutical market. While the hypodermic injection, the most common delivery method for these molecules, is effective, it also has limitations, including low patient compliance, need for medically trained personnel and biohazardous sharps after delivery. The overall goal of this thesis was to develop a new delivery system for biopharmaceuticals, based on dissolving polymer microneedles, which is effective and more patient compliant than the hypodermic needle. Microneedles are microscopic needles that are large enough to insert into the skin to deliver drugs effectively, while being short enough to avoid the pain causing nerves deep in the skin. An additional benefit of polymer microneedles is that the needles completely dissolve in the skin, leaving behind no biohazardous sharps. There are significant material and fabrication issues that must be overcome in the development of this new device. The first part of this thesis focused on the development of a new fabrication process, based on in situ photopolymerization, for the creation of polymer microneedles. These microneedles were shown to successfully insert into the skin, dissolving within a minute to deliver the encapsulated cargo, and retain full activity of encapsulated proteins. Next, we applied the microneedle technology to the delivery of the influenza virus. We found that the reformulation process required to encapsulate the influenza virus in polymer microneedles did not affect the antigenicity or immunogenicity of the virus. In addition, we used coated metal microneedles to successfully immunize mice with the influenza virus, verifying the delivery capabilities of a microneedle system. Finally, we used the dissolving polymer microneedles to successfully immunize mice with the influenza virus, resulting in full protection against lethal challenge after one immunization. This immune response was equivalent to the control intramuscular injection. In conclusion, we have developed dissolving polymer microneedles as an effective and patient compliant delivery system for biopharmaceuticals. This system could be especially applicable to mass immunization efforts or home use, since it can be self-administered and allows for easy disposal with no biohazardous sharps.

Ongoing advances in tissue engineering with the goal to address the clinical shortage of donor organs have encouraged the design and development of biomaterials to be used in tissue-engineered scaffolds. Furthermore, biomaterials have been used as delivery vehicles for vaccines that aim to enhance the protective immunity against pathogenic agents. These tissue-engineered constructs or vaccines are usually combination products that combine biomaterial and biological (e.g. cells, proteins, and/or DNA) components. Upon introduction into the body, the host response towards these products will be a combination of both a non-specific inflammatory response towards the biomaterial and an antigen-specific immune response towards the biological component(s). Recently, the biomaterial component was shown to influence the immune response towards a co-delivered antigen. Specifically, poly(lactic-co-glycolic acid) (PLGA), but not agarose, scaffolds or microparticles (MPs) enhanced the humoral response to a model antigen, ovalbumin. This in vivo result echoed with the in vitro study that PLGA, but not agarose, supported a mature phenotype of dendritic cells (DCs), the most potent antigen-presenting cells. Therefore, it is hypothesized that the effect of biomaterials on DC phenotype may influence the adaptive immunity against a co-delivered antigen. Understanding how biomaterials affect DC response will facilitate the selection and design of biomaterials that direct a desired immune response for tissue engineering or vaccine delivery applications. The objectives of this research were to elucidate the correlations between material properties and DC phenotype, develop predictive models for DC response based on material properties, and uncover the molecular basis for DC response to biomaterials. Well-defined biomaterial systems, including clinical titanium (Ti) substrates and two polymer libraries, were chosen to study induced DC phenotype. Due to the time-consuming nature of conventional methods for assessing DC phenotype, a high-throughput (HTP) method was first developed to screen for DC maturation based on surface marker expression (CHAPTER 4). A 96-well filter plate-based HTP methodology was developed and validated for the assessment of DC response to biomaterials. A "maturation factor", defined as CD86/DC-SIGN and measured by immunostaining, was found to be a cell number-independent metric for DC maturation and could be adapted to screen for DC maturation in a microplate format. This methodology was shown to reproducibly yield similar results of DC maturation in response to biomaterial treatment as compared to the conventional flow cytometric method upon DC treatment in 6-well plates. In addition, the supernatants from each treatment could easily be collected for cytotoxicity assessment using glucose-6-phosphate dehydrogenase (G6PD)-based assay and cytokine profiling using multiplex technology. In other words, the 96-well filter plate-based methodology can generate three outcomes from one single cell culture: 1) maturation marker expression, 2) cytotoxicity, and 3) cytokine profile. To examine which material properties were critical in determining DC phenotype, a set of three clinical titanium (Ti) substrates with well-defined surfaces was used to treat DCs (CHAPTER 5). These Ti substrates included pretreatment (PT), sand-blasted and acid-etched (SLA), and modified SLA (modSLA), with different roughness and surface energy. DCs responded differentially to these substrates. Specifically, PT and SLA induced a mature DC (mDC) phenotype, while modSLA-treated DCs remained immature based on surface marker expression, cytokine production profiles and cell morphology. Both PT and SLA induced higher CD86 expression as compared to iDC control, while modSLA maintained CD86 expression at a level similar to iDC. PT- or SLA-treated DCs exhibited dendritic processes associated with a mDC phenotype, while modSLA-treated DCs were rounded, a morphology associated with an iDC phenotype. Furthermore, PT induced increased secretion of MCP-1 by DCs compared to iDCs, indicating that PT promoted a pro-inflammatory environment. SLA induced higher IL-16 production, which is a pleiotropic cytokine, by DCs, most likely as a pro-inflammatory response due to the enhanced maturation of DCs induced by SLA. In contrast, modSLA did not induced enhanced production of any cytokines examined. Principal component analysis (PCA) were used to reduce the multi-dimensional data space and confirmed these experimental results, and it also indicated that the non-stimulating property of modSLA co-varied with certain surface properties, such as high surface hydrophilicity, % oxygen and % titanium of the substrates. In contrast, high surface % carbon and % nitrogen were more associated with a mDC phenotype. Furthermore, PCA also suggested that surface line roughness (Ra) did not contribute to the expression of CD86, an important maturation marker, suggesting that roughness had little impact on DC response (CHAPTER 5). DC response-material property relationships were also derived using more complex materials from a combinatorial library of polymethacrylates (pMAs) (CHAPTER 6). Twelve pMAs were selected and were found to induce differential DC response using the HTP method described in CHAPTER 4. These pMAs resulted in a trend of increasing DC maturation represented by the metric CD86/DC-SIGN, which was consistent with the trends of the production of pro-inflammatory cytokine, TNF-α, and chemokine, IL-8. Interestingly, this set of pMAs induced an opposite trend of IL-16 production, which is most likely released as an anti-inflammatory cytokine in this situation. These polymers were characterized extensively for a number of material properties, including surface chemical composition, glass transition temperature (Tg), air-water contact angle, line roughness (Ra), surface roughness (Sa), and surface area. Similar to the results from the Ti study, PCA determined that surface carbon correlated with enhanced DC maturation, while surface oxygen was associated with an iDC phenotype. In addition, Tg, Ra, and surface area were unimportant in determining DC response. Partial square linear regression (PLSR), a multivariate modeling approach, was implemented using the pMAs as the training set and a separate polymer library, which contained methacrylate- and acrylate-based terpolymers, as the prediction set. This model successfully predicted DC phenotype in terms of surface marker expression with R2prediction = 0.76. Furthermore, prediction of DC phenotype was effective based on only theoretical chemical composition of the bulk polymers with R2prediction = 0.80 (CHAPTER 6). Nonetheless, one should note that a predictive model can be only as good as what it is trained on and cannot be used to predict the DC response induced by a type of materials different from the training set. Also, this model might not contain all the important material properties such as polymer swelling and cannot predict specific types of immune responses. However, these results demonstrated that a generalized immune cell response can be predicted from biomaterial properties, and computational models will expedite future biomaterial design and selection (CHAPTER 6). From the pMA library, pMAs that induced the two extremes of DC phenotype (mature or immature) were identified for elucidating the mechanistic basis of biomaterial-induced DC responses (CHAPTER 7). Two pMAs, polyhydroxyethylmethacrylate (pHEMA) and poly(isobutyl-co-benzyl-co-terahydrofurfuryl)methacrylate (pIBTMA), were selected because they induced the least and the most mature DC phenotype, respectively. These pMAs were used to elucidate the activation profiles of transcription factors in DCs after biomaterial treatment and were compared to the iDC and mDC controls. In addition, a combined treatment of pHEMA and LPS was also included to determine if pHEMA could maintain an iDC phenotype in the presence of LPS. Interestingly, pIBTMA induced DC maturation primarily through the activation of NF-κB, while pHEMA mediated suppression of DC maturation through multiple TFs, including the activation of ISRE, E2F-1, GR-PR, NFAT, and HSF. GR-PR and E2F-1 have been shown to be associated with the suppression of DC maturation; ISRE, E2F-1, and NFAT are linked to apoptosis induction; HSF regulates the production of heat shock proteins (HSPs) that induce DC maturation and inhibit apoptosis. The activation of HSF by pHEMA was most likely a natural defensive mechanism against the other apoptotic signals. Therefore, pHEMA suppressed DC maturation through the induction of apoptosis. Surprisingly, in the presence of pHEMA, the effect of LPS was completely eliminated, suggesting that biomaterials can override the effect of soluble factors. The morphology and surface marker expression of DCs treated with these different biomaterials or controls were consistent with TF activation profiles (CHAPTER 7). Overall, this research illustrates that biomaterial properties, within the chosen biomaterial space, can be correlated to DC phenotype and more importantly, can be used as predictors for relative levels of DC phenotype. Furthermore, the differential responses induced by different biomaterials were mediated through the distinct activation profiles of transcription factors. Together, these findings are expected to facilitate the design and selection of biomaterials that direct desired immune responses.

Infecções associadas a cepas da Escherichia coli enterohemorrágica (EHEC), podem causar manifestações clínicas sendo a Síndrome Hemolítica Urêmica (SHU), a complicação mais severa. SHU está relacionada a presença da toxina de Shiga do tipo 2 (Stx2) e até o momento não se dispõe de uma vacina ou tratamentos efetivos para uso em humanos. Assim, este trabalho teve por objetivo desenvolver uma vacina baseada no derivado atóxico contendo a subunidade B e a porção A2 da subunidade A denominado Stx2&#916;AB. Após expressão em linhagens de E.coli e tentativas iniciais de purificação, resultaram na formação de agregados proteicos. Ajustes nas condições de cultivo e purificação permitiram obter a proteína na forma de monômero da subunidade B, mas sem a presença da porção A2. O antígeno foi incorporado a lipossomas multilamelares (MLVs), combinados ao lipídio A e administrados por via subcutânea a camundongos. Animais imunizados desenvolveram anticorpos sistêmicos específicos contra Stx2 capazes de neutralizar a toxina in vitro e conferir proteção parcial a animais desafiados com dose letal da toxina. Em conclusão, o trabalho confirmou o potencial vacinal do antígeno e validou a estratégia baseada na incorporação do antígeno às MLVs como estratégia de imunização. / Infections associated with strains of enterohaemorrhagic Escherichia coli (EHEC), can cause clinical manifestations are the hemolytic uremic syndrome (HUS), the most severe complication. HUS is related to the presence of Shiga toxin type 2 (Stx2) and yet do not have a vaccine or effective treatments for use in humans. This work aimed to develop a vaccine based on non-toxic derivative containing the B subunit and the A2 portion of the subunit called Stx2&#916;AB. After expression in E. coli strains and initial purification attempts resulted in the formation of protein aggregates. Adjustments in the cultivation and purification conditions have enabled the protein as the monomer subunit B but without the presence of the A2 portion. The antigen was incorporated into multilamellar liposomes (MLVs), the combined lipid A and administered subcutaneously to mice. immunized animals develop systemic antibodies specific against Stx2 able to neutralize toxin in vitro and to confer partial protection when challenged with a lethal dose of toxin. In conclusion, the study confirmed the potential vaccine antigen and validated strategy based on antigen incorporation into MLVs as immunization strategy.

The prevention and treatment of cancer and infectious diseases requires vaccines that can mediate cytotoxic T lymphocyte-based immunity. A promising strategy is protein subunit vaccines composed of purified protein antigens and immunostimulatory adjuvants, such as Toll-like receptor (TLR) agonists. In this research, we developed two new biodegradable polymeric delivery vehicles for protein antigens and TLR agonists, as model vaccine delivery systems. This work was guided by the central hypothesis that an effective vaccine delivery system would have stimulus-responsive degradation and release, biodegradability into excretable non-acidic degradation products, and the ability to incorporate various TLR-inducing adjuvants. The first vaccine delivery system is a cross-linked polyion complex micelle which efficiently encapsulates proteins, DNA, and RNA. The micelle-based delivery system consists of a block copolymer of poly(ethylene glycol) (PEG) and poly(L-lysine), cross-linked by dithiopyridyl side groups to provide transport stability and intracellular release. The second delivery system consists of solid biodegradable microparticles encapsulating proteins, nucleic acids, and hydrophobic compounds. The microparticles are composed of pH-sensitive polyketals, which are a new family of hydrophobic, linear polymers containing backbone ketal linkages. Polyketals are synthesized via a new polymerization method based on the acetal exchange reaction and degrade into non-acidic, excretable degradation products. In addition, the technique of hydrophobic ion pairing was utilized to enhance the encapsulation of ovalbumin, DNA, and RNA in polyketal microparticles via a single emulsion method. Using in vitro and in vivo immunological models, we demonstrated that the micelle- and polyketal-based vaccine delivery systems enhanced the cross-priming of cytotoxic T lymphocytes. The model vaccines were composed of ovalbumin antigen and various TLR-inducing adjuvants including CpG-DNA, monophosphoryl lipid A, and dsRNA. The results demonstrate that the cross-linked micelles and polyketal microparticles have considerable potential as delivery systems for protein-based vaccines.

The major hurdle in the development of transdermal route as a versatile drug delivery method is the formidable transport barrier provided by the stratum corneum. Despite decades of research to overcome the stratum corneum barrier, limited success has been achieved. The objectives of this research were to develop and characterize two different strategies to overcome the stratum corneum barrier for transdermal delivery of biopharmaceuticals and vaccines. In the first strategy, coated microneedles (sharp-tipped, micron-sized structures) were developed to enable delivery of drugs directly into the skin by bypassing the stratum corneum barrier. In the second strategy, instead of bypassing the barrier, microdermabrasion was used to selectively abrade stratum corneum with sharp microparticles for topical drug application. Coated microneedles For developing painless microneedles, the first detailed study was performed to characterize the effect of microneedle geometry on pain caused by microneedle insertions in human volunteers. This study demonstrated that microneedles are significantly less painful than a 26-gage hypodermic needle and that decreasing microneedle length and numbers reduces pain. Next, the first in-depth study of microneedle coating methods and formulations was performed to (i) develop a novel micron-scale dip-coating process, (ii) test the breadth of compounds that can be coated onto microneedles, and (iii) develop a rational basis to design novel coating formulations based on the physics of dip-coating. Finally, a plasmid DNA-vaccine was coated onto microneedles to immunize mice, to provide the first evidence that microneedle-based skin immunization can generate a robust in vivo antigen-specific cytotoxic-T-lymphocyte response using similar, or lower, DNA doses on microneedles as when using the gene gun or intramuscular injection. Microdermabrasion We demonstrated for the first time that microdermabrasion in monkeys and humans can selectively, yet completely remove the stratum corneum layer. Using a mobile mode of microdermabrasion, an increase in the number of treatment passes led to greater tissue removal. Furthermore, topical application of Modified Vaccinia Ankara virus after microdermabrasion induced virus-specific antibodies in monkeys. In conclusion, both coated microneedles and microdermabrasion were developed to enable delivery of biomolecules into the skin, indicating their potential for transdermal delivery of a wide range of biopharmaceuticals and vaccines.

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.