Inflammatory airway disease (IAD) is a common performance limiting pulmonary disorder in young racehorses in training. Although the precise aetiopathogenesis is poorly understood, proposed mechanisms include opportunistic bacterial infections and/or suboptimal air-hygiene. Since alveolar macrophages (AMs) are the first line defence in the lungs of mammalian species, they may constitute an appropriate therapeutic target cell in the treatment and the prevention of opportunistic airway infections. This thesis aimed to investigate the basic biology of the equine AM. A series of experiments were conducted to investigate the function and phenotype of this cell and comparisons made with equine macrophages derived from other anatomical sites and macrophage datasets derived from other species. The lung environment is unique, and may direct a unique phenotype and function compared with macrophages derived from other sites. Macrophages were isolated from the lungs, peritoneal cavity and other regions of healthy horses. Excellent cell recovery was demonstrated and associated with good viability, RNA yield and a demonstrable response to several stimuli, both when fresh and following cryopreservation. AMs produced tumor necrosis factor alpha (TNFα) when stimulated with lipopolysaccharide (LPS), polyinosinic-polycytidylic acid (Poly IC) and heat-killed Salmonella typhimurium and were actively phagocytic. By comparison, peritoneal macrophages (PMs) did not respond to these inducers and lacked phagocytic activity. In contrast to AMs, which showed high expression of the specific macrophage markers cluster of differentiation (CD) 14, CD163 and toll-like receptor 4 (TLR4), PMs lacked CD14. Moreover, gene expression analysis revealed an alternative macrophage activation for AMs, whereas PM showed a hybrid macrophage activation potentially attributed to the phenomenon of endotoxin tolerance. The response of equine AMs to LPS was analysed using microarrays. There was significant change in the expression of 240 genes. Those that were upregulated included well known inflammatory genes such as TNFα, IL1A and CXCL6. The pattern of response more closely resembled human and pig macrophages than mouse, including the LPS-induced expression of STAT4, IDO, IL7R genes and the failure to produce nitrite in response to LPS. These data suggest that the horse may represent a suitable animal model for human macrophage-associated lung inflammation, and conversely that data from humans may translate to horses. A final aim of this study was to investigate the effect of exercise on equine AM function. Therefore, AMs were isolated from bronchoalveolar lavage samples obtained from Standardbred racehorses at rest and during the training period and microarray analysis performed. Despite important limitations of the study, a few mechanisms at the molecular level were detected which may be involved in the development of either training-associated symptoms of, or susceptibility to IAD. Overall, this thesis aims to improve our understanding of equine macrophage biology and to provide useful information regarding the role of AMs in exercise-associated inflammation. Moreover, the findings presented here may help to inform future preventative pharmacological and/or managemental interventions for IAD.

Infants with bronchopulmonary dysplasia (BPD), showed impaired body growth when compared to control infants. In terms of changes in the biochemical composition of the lung, BPD infants had higher DNA, soluble protein, collagen and desmosine contents as well as increased concentrations of DNA, collagen and desmosine in their lungs when compared to the growth patterns obtained for the lungs of control infants. Pathologically BPD was classified into 4 grades. Grade I BPD, was a phase of acute lung injury, grades II and III were proliferative phases. In grade IV BPD, lung structure returned towards normal. Evidence of fibrosis was seen by a significant increase in collagen concentration in grades II and III while desmosine concentration was seen to increase in grades III and IV suggesting that the increase in collagen and desmosine contents in the lungs of BPD infants may be controlled by two different mechanisms. Collagen type I/III ratio was seen to decrease progressively from grade II to grade IV BPD in comparison to age matched controls, indicating a higher proportion of type III collagen in the lungs of infants with BPD. From the clinical analysis and the results obtained from discriminant analysis procedure, it was seen that there was a high degree of correlation between the continuation of the disease and collagen accumulation in the lungs suggesting that pulmonary fibrosis with excessive collagen accumulation is an integral part of BPD. This fibrotic process seemed to correlate significantly with assisted ventilation and high oxygen supplementation received by the infants, but it was difficult to assess the individual contribution of the two treatments in the pathogenesis of BPD. Other variables such as severity of the initial disease and the length of survival of the infants, made the assessment of individual contribution much more difficult. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

This study examined the effects of epinephrine, norepinephrine, AVP and ACh on fluid movement by the lungs of the late-term guinea pig fetus. Catecholamines and AVP are secreted in high amounts by the fetus during delivery, and could be important with respect to fetal lung fluid removal; this event is vital at the time of birth. The lungs were supported in vitro for a duration of three hours, and production rates were measured using a dye-dilution technique. The average resting production rate in terms of ml/kg‧h declined with gestational age (54-67 days gestation; n=171). There was a lesser decline in the average resting production rate in terms of ml/h. The average production rate of untreated preparations in the first hour was 1.60 ± 0.26 ml/kg body weight per hour, and rates did not change significantly during the remaining two hours of experimentation (n=30). This rate is comparable to those reported from chronically catheterized fetal sheep. Treatment was administered during the second hour of experimentation, following an ABA design. Lungs (n=36) were transferred to fresh Krebs-Henseleit saline containing one of the following concentrations of epinephrine: (a) 10‾⁵ M; (b) 10‾⁶ M; (c) 10‾⁷ M; (d) 5 x 10‾⁸ M; (e) 10‾⁸ M; and (f) 10‾⁹ M. With the exception of the top dose, epinephrine treatment caused an immediate reduction in fluid secretion, or fluid reabsorption. Sodium followed the movement of water in all cases. The effect of epinephrine at 10‾⁷ M was maximal, and the threshold dose for epinephrine was calculated at 1.78 x 10‾¹¹ M. Phentolamine and propranolol had no effect in control preparations. However, phentolamine completely blocked the effect of epinephrine, whereas propranolol was ineffective. Isoproterenol had no effect on pulmonary fluid production. Alpha-adrenergic receptors apparently mediate the effect of epinephrine on pulmonary fluid movement in the fetal guinea pig lung. This conclusion is different from that obtained in fetal sheep, in which beta-adrenergic receptors are utilized. A possible synergism between epinephrine and AVP was examined. Lungs (n=12) were transferred to fresh Krebs-Henseleit saline containing either (a) 0.6 mU/ml AVP, or b) 0.6 mU/ml AVP combined with epinephrine at 10‾⁷ M. Treatment with AVP caused a slow, prolonged reduction in fluid production. Treatment with AVP together with epinephrine did not demonstrate synergism. The effect of norepinephrine (NE) was examined. Lungs (n=36) were transferred to fresh Krebs-Henseleit saline containing one of the following concentrations of NE: (a) 1.24 x 10‾⁵ M; (b) 1.24 x 10‾⁶ M; (c) 1.24 x 10‾⁷ M; (d) 5.24 x 10‾⁸ M; (e) 1.24 x 10‾⁸ M; and (f) 1.24 x 10‾⁹ M. In all preparations, treatment with NE resulted in an immediate reduction in fluid production, and reabsorptions were observed at the higher doses. Sodium followed the movement of water in every case. The threshold dose was calculated at 3.16 x 10‾¹⁰ M. Phentolamine blocked the effect of NE, reinforcing the importance of pulmonary alpha-adrenergic receptors in the fetal guinea pig. There was no relationship between age and degree of response with treatment of either epinephrine or NE, but fetuses under 78.0 g did not respond to NE. The effect of ACh was examined. Lungs (n=24) were transferred to fresh Krebs-Henseleit saline containing one of the following concentrations of ACh: (a) 10‾⁴ M; (b) 10‾⁵ M; (c) 10‾⁶ M; and (d) 10‾⁸ M. At the three top doses, immediate and powerful reabsorptions of pulmonary fluid were observed in older fetuses (60 days gestation and above); significant falls were observed in the younger fetuses. This result was unexpected, as it was hypothesized that ACh would stimulate fluid production. The threshold dose for ACh was between 10‾⁶ M and 10‾⁸ M. Phentolamine blocked the effect of ACh. This result suggested that reabsorption is a result of an indirect effect of ACh acting through pulmonary alpha receptors. The results in this study show that epinephrine, NE, AVP and ACh are all important promoters of fetal pulmonary fluid removal in the fetal guinea pig. Pulmonary alpha-adrenergic receptors mediate the effects of epinephrine, NE and ACh (indirectly). The conclusions drawn from this study emphasize the importance of species' comparison in fetal research. LIST OF ABBREVIATIONS AVP Arginine Vasopressin NE Norepinephrine DOPA dihydroxyphenylalanine PNMT Phenylethanolamine n-methyltransferase ACh Acetylcholine / Science, Faculty of / Zoology, Department of / Graduate

The generation of oxygen free radicals by the cytosolic enzyme, xanthine oxidase (XO), has been implicated in post-ischemic or reperfusion damage in several organs. XO catalyzes the conversion of hypoxanthine to urate with the concomitant production of superoxide anion free radical (0₂̅˙) and hydrogen peroxide (H₂O₂). Oxygen free radical-mediated injury has also been demonstrated in inflammatory lung disease. The possible involvement of XO in oxidative injury in the lung has not yet been studied. Therefore, this research project was designed to determine whether XO is present in the lung and to investigate its characteristics in porcine, bovine, rat and human lung and other tissues. Immunochemical analysis of xanthine oxidase in the tissues employed on polyclonal antibody raised to bovine milk XO. Proteins were separated by SDS-polyacrylamide gel electrophoresis of tissue homogenates. Proteins were transfered from the gels to nitrocellulose filters by Western blotting. After incubating the filters with a antisera containing the antibody to the purified bovine XO. XO on the filter was detected by its reaction with an enzyme-conjugated second antibody. XO was immunologically detectable in bovine lung and milk. Rat lung, kidney and liver all showed XO reactivity. XO was detectable in porcine liver but not detectable in porcine lung or kidney. Thus, the antibody to bovine XO was cross-reactive with porcine and rat XO. XO protein was not immunologically detectable in human lung possibly because the antibody was not cross reactive with the bovine antibody. In vivo, xanthine oxidase exists predominantly as a dehydrogenase rather than an oxidase. In this form as xanthine dehydrogenase (XDH) the enxyme does not produce either 0₂̅˙ or H₂O₂. The activity of both XDH and XO was measured in several tissues using a fluorometric assay which uses an artifical substrate, pterin which is catalytically converted to the fluorescent product isoxanthopterin (IXP). XO activity in porcine liver was of 1.1 x 10⁻³ µg IXP/mg protein/min although XO activity was not detectable in porcine lung and kidney, in rat lung of 1.7 x 10⁻² µg IXP/mg protein/min, rat kidney of 1.5 x 10⁻² µg IXP/mg protein/min, and rat liver of 2.2 x 10⁻² µg IXP/mg protein/min. Seven human lung biopsy samples were obtained after lung resection and initially tested for viability by determination of NADH oxidase activity and then assayed for XO-XDH. Three of these samples showed NADH oxidase activity indicating tissue viability, but only one of these three showed measurable XO activity of 5.35 x 10⁻⁶ µg IXP/mg protein/min. Irreversible conversion of XDH to XO is thought to be the result of limited proteolysis by a Ca²⁺/calmodulin activated protease, whereas reversible conversion of the enzyme occurs by oxidation of critical thiol groups. Studies on the rate and nature of fluorescence assay to detect catalytic activities of both enzyme forms. Incubation of lung homogenates with trypsin for 60 min caused irreverisble conversion of 90% of the XDH to XO. In contrast, incubation of homogenates at 15°C for 10 hours caused conversion of 100% of the XDH to XO. This conversion was reversible to the extent of 80% by reduction of thiol groups with dithiothreitol (DTT). The effects of free Ca²⁺ on the conversion of XDH to X0 was examined by using EDTA, a chelator of Ca²⁺ and other divalent cations; and EGTA, a more specific chelator of Ca²⁺. The presence of these chelating agents during homogenization of either normoxic or ischemic rat lung tissue did not inhibit reversible enzyme conversion. Increased XO activity was reversible by DTT. In the normoxic rat lung, homogenates prepared with EDTA and EGTA showed a similar conversion of 95% of XDH to XO which was reversible to 70% with DTT. In the ischemic rat lung, samples prepared with EDTA and EGTA showed a'conversion of 80% and 95% XDH to XO which was similar to control samples. The extent of reversibility to XDH was 75% with DTT incubation. In addition, perfusion of rat lungs with EDTA and DTT via a pulmonary artery cannula prior to 60 min of ischemia and homogenization did not affect the extent of XDH to XO conversion. These results indicate that irreversible Ca²⁺-mediated proteolytic conversion of XDH to XO does not occur to a great extent in the rat lung during either normoxia or ischemia. However, reversible conversion of XDH to XO does occur, suggesting that reversible thiol dependent conversion may play a role in the lung under both physiological and pathophysiological states. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

The respiratory system represents a major interface between the body and the external environment. Its design includes a tree-like network of conducting tubules (airways) that carries air to millions of alveoli, where gas exchange occurs. The conducting airways are characterized by their great diversity in epithelial cell types with multiple populations of secretory, multiciliated, and neuroendocrine cells. How these different cell types arise and how these populations are balanced are questions still not well understood. Aberrant patterns of airway epithelial differentiation have been described in various human pulmonary diseases, chronic bronchitis, asthma, neuroendocrine hyperplasia of infancy, and others. The goal of this thesis is to investigate mechanisms of regulation of airway epithelial cell fate in the developing lung epithelium. More specifically, these studies focus on Notch signaling and address a long unresolved issue whether the different Notch ligands (Jagged and Delta) have distinct roles in the epithelial differentiation program of the extrapulmonary and intrapulmonary airways. Moreover, these studies investigate the ontogeny of the bHLH transcription factor Ascl1 and identify its targets in the developing airways as potential regulators of neuroepithelial body (NEB) size and maturation. My studies provide evidence that the Notch ligand families Jag and Dll are required for the specification and formation of different cell lineages in the developing airway epithelia. Jag ligands regulate multiciliated versus secretory (club) cell fates but also controls abundance of basal cell progenitors in extrapulmonary airways. Dll ligands regulate pulmonary neuroendocrine versus club cell fates in intrapulmonary airways. Analysis of mouse mutants showed that loss of Jag ligands has minimal impact on the size or abundance of NEBs and their associated secretory cells while loss of Dll ligands results in an expansion of NEB size and associated cells. To gain additional insights into the potential mechanisms of how neuroendocrine cells develop and undergo aberrant hyperplasia, I characterized the global transcriptional profile of embryonic lungs from mice deficient in Ascl1, which lack NEBs and neuroendocrine cells and identified a number of genes associated with neuroendocrine cell development, maturation, and the NEB microenvironment. Among these genes, components of the catecholamine biosynthesis pathway, such as tyrosine hydroxylase (Th), a key enzyme for catecholamine production, were downregulated in Ascl1 null lungs. Subsequent functional analysis using a pharmacological inhibitor of this pathway in lung organ cultures showed expansion of pulmonary neuroendocrine cells and NEB size, an observation of potential relevance in human diseases in which neuroendocrine cells are aberrantly expanded. Together these studies highlight the distinct role of Notch ligands and further implicate Ascl1 targets, as illustrated by catecholamine pathway components, in regulating epithelial cell fate. Further examination of these pathways may provide insights into the pathogenesis and ultimately therapeutic approaches for airway diseases.

Pneumoconiosis in rural Transkeian women termed "Transkei Silicosis" has been thought to be caused by silica inhaled while grinding maize by traditional methods (Palmer and Daynes, 1967). This study was undertaken to investigate the features and causes of hut lung. The range of clinical, radiologic, histologic, pulmonary physiologic and broncho-alveolar lavage features in patients meeting the following criteria was assessed: i) rural women practising traditional cooking methods ii) with a diffuse nodularity on chest x-ray iii) and lung biopsy evidence of pneumoconiosis iv) and without occupational exposure v) or evidence of active tuberculosis. Smoke and dust levels were measured in rural dwellings during cooking and maize grinding and ground maize and grinding rocks were analysed. 25 patients were studied. 17 were non-smokers, 5 were pipe smokers and 3 smoked 10 or less cigarettes per day. 7 had evidence of previous tuberculosis. The radiological findings ranged from a diffuse fine miliary pattern through coarse nodules with coalescence, to extensive fibrosis resembling PMF. The histologic features revealed simple "anthracosis" in 12, anthracosis with macules in 6 and mixed dust fibrosis in 7, of which 2 had silicotic nodules and 1 PMF. No such findings were observed in the control lung biopsy specimens obtained at post-mortem from city dwelling Xhosa females. Mild to moderate airflow limitation (defined as an FEV1/FVC ratio of < 65% and/or RV> 145% of predicted) was present in 73% while a reduced T'LCO (< 80% predicted) was found in 76% of the patients. Cell numbers and differential counts in BAL fluid were normal but> 80% of the macrophages were heavily laden with inorganic inclusions. The mean smoke level during indoor open fire cooking was 30mg/m³. Respirable dust and quartz concentrations ranging from 3,03 to 5, 82mg/m³ and 0,097 to 0,186mg/m³ respectively were found during hand grinding with sandstone (100% quartz), but were lower (ranging from 2,62 to 3,40mg/m³ and 0,024mg/m³ respectively) when non-quartz containing dolerite was used. Calculated cumulative equivalent time-weighted average respirable dust concentrations were shown to be similar to those found in an average South African gold mine while calculated equivalent respirable quartz concentrations were well below those found in the worst exposed gold miners and well within the recommended threshold limit values of the National Institute for Occupational Safety and Health (NIOSH) and the World Health Organisation (WHO). Respirable quartz exposure alone was not sufficient to explain the changes found. Respirable non-quartz containing nuisance dust and intense smoke exposure were shown to be significant. It was concluded that: i) hut lung can be defined as a domestic pneumoconiosis that occurs in rural women who practise primitive cooking methods ii) hut lung typically occurs in rural maize grinding Transkeian women but can occur in other rural women iii) there is a wide clinical, spectrum radiological and histologic iv) the pulmonary physiological changes are predominantly those of airflow limitation with some CO transfer factor reduction v) cigarette and pipe smoking do not contribute to the aetiology or pulmonary physiological abnormalities vi) the bronchoalveolar lavage features may help differentiate this condition from miliary tuberculosis vi) the aetiology of hut lung is multifactorial with exposure to respirable quartz and non-quartz containing dust together with smoke particles from biomass fuelled fires all playing a significant role while previous tuberculosis may be a contributing factor.

The impact of Chronic Obstructive Pulmonary Disorder is material in the dentistry field which not only centers its efforts on managing the oral health of the afflicted patients, prescribing proper medications, but also on raising awareness mainly through dental hygiene and smoking cessation. This literature review will explore the status and accomplishments of the field of dentistry with regards to battling COPD. The first section of the paper discusses the nature and dynamics of the disease which ranks between the third and fourth most common cause of death in the United States alone. Certain data trends from 1970 until 2002 showed double the death rates caused by the disease, and that exposure to environmental or occupational risk factors (e.g. dust, fumes, etc.) must be seriously considered for the effective management of COPD. The pressure of COPD management is great among dental practitioners who must ensure the safety of patients by providing the most suitable drug regimen and having a well-coordinated dental practice (i.e. with other health practitioners) to avoid exacerbation of the disease which often leads to hospitalization. The latter is often the result of oral devices or materials that are often comprised of small objects which might be swallowed or aspirated into the oropharynx of the patient when proper positioning during dental check-ups is not properly performed. Dentists must also make sure that they are able to gain the patient’s confidence and to avoid patient reluctance during dental care. Numbers and statistics also reveal that an estimated 24 million of the population in the United States is afflicted by COPD, and that is from 2015 alone. It is also prevalent among the male population which has a death rate of 83 per 100,000, while the death rate among women is 57 per 100,000. Immediate treatment of COPD is also emphasized to prevent hospitalization rates from rising, and as far as dental practice is concerned, they are often aware of CODP patients which number approximately 130 out of 2000. Smoking cessation is considered the best preventive measure in COPD management. Educating patients on the real dangers involved with smoking will help encourage them to quit while promoting better oral health. The field of dentistry is constantly responding to the challenges of COPD, and significant breakthroughs have already been achieved due to research efforts on COPD management. Through professional coordination with other fields in the healthcare system, it is with great hope that dental practitioners will contribute more to the alleviation of patient conditions and providing improved quality of life.

Thesis (M.Sc. (Biochemistry)) -- University of Limpopo, 2020 / Despite major advancements in the development of various chemotherapuetic agents, treatment for lung cancer remains costly, ineffective, toxic to neighbouring normal noncancerous cells and still hampered by high level of remissions (Wistuba et al., 2018; Tana et al., 2016; Schiller et al., 2002). Synthesis of novel quinoxalines with a wide spectrum of biological activities has recently received considerable attention with promising anticancer drug activity since most of them do not affect non-cancerous cells and are derived from readily available less costly raw materials (Srivastava et al., 2014). Since combination treatment has been shown to augment and improve single drug treatment, trace elements were employed in this study in combination with quinoxalines derivatives (Gomez et al., 2016; Kocdor et al., 2015; Ku et al., 2012; John et al., 2010; Killile and Killilea, 2007). Zinc is an essential element that is integral to many proteins and transcription factors which regulate key cellular functions such as the response to oxidative stress, DNA replication, DNA damage repair, cell cycle progression, and apoptosis (Dhawan and Chadha, 2010). Owing to the importance of these two approaches, the aim of this study was to provide in vitro preliminary anticancer activity data on A549 lung cancer cells using combination of zinc and quinoxaline derivatives. An assessment of the quinoxaline derivatives ferric reducing power and DPPH free radical scavenging activity was performed. The cytotoxic and anti-proliferation activity of these derivatives and zinc on cancer cell lines was determined using the MTT assay. The ability of the quinoxaline derivatives and zinc to modulate oxidative stress was evaluated using the H2DCFDA fluorescence assay. Cell cycle arrest stages were analysed by flow cytometry through propidium iodide cell cycle analyses. The ability of the quinoxaline derivatives to induce apoptosis in cancer cells was assessed using DAPI/PI, Acridine Orange/Ethidium Bromide (AO/EB) and Annexin V-FITC/Dead Cell assays. Western blot was used to investigate the Bcl/Bax expression ratios in A549 lung cancer cells after treatment with quinoxaline derivatives, zinc and in combination. Of the four quinoxaline derivatives tested, 3-(quinoxaline-3-yl) prop-2-ynyl methanosulphate (LA-39B) and 3-(quinoxaline-3-yl) prop-2-yn-1-ol (LA-55) produced significant anticancer properties against A549 lung cancer cells at minimal concentrations of 25μM. Both quinoxaline derivatives displayed antioxidant properties and did not induce cell death in non-cancerous Raw 267.4 macrophage cells. Cytotoxicity was observed in A549 lung cancer, HeLa cervical cancer and MCF-7 breast cancer cells albeit inhibition was more pronounced in A549 lung cancer cells. Treatment of cancer cells with zinc also resulted in pronounced cytotoxicity at a minimal concentration of 25μM. Although reduced oxidative stress was observed in Raw 264.7 macrophages, in A549 lung cancer cells both compounds were able to increase ROS production which was accompanied by high levels of apoptosis when treated with derivatives and zinc alone but when in combination an improved higher level of apoptosis is observed. The improved anti-cancer activity of this drug combination treatment was further accompanied by lower Bcl/Bax expression ratios with upregulation of Bax in A549 lung cancer cells. The results of the study suggest that 3-(quinoxaline-3-yl) prop-2-ynyl methanosulphate and 3-(quinoxaline-3-yl) prop- 2-yn-1-ol are potential candidates drug for treatment of lung cancer. The use of these quinoxaline derivatives in combination with zinc can offer alternative treatment options for lung cancer.

Lung cancer has a high incidence rate globally and the leading cause of cancer related mortalities. In 2018, lung cancer has been estimated to cause 1.76 million deaths worldwide (18.33% of total cancer mortalities). In Hong Kong lung cancer has been a leading cause of cancer related deaths, and in 2016 caused 3780 deaths (26.6% of total cancer mortalities). Non-small cell lung cancer (NSCLC) is the major (~85%) lung cancer type, and five-year survival rate for lung cancer has estimated to be 18%. Thus, an efficient lung cancer treatment with lesser adverse effects is need of the hour. In this connection, active targeting of overexpressed receptors at lung tumor site with a ligand functionalized drug delivery system is the current approach, and pulmonary administration could augment chemotherapeutic effect of the drug through localized administration, minimizing the off-target effects by retention of the drug in lungs.Quercetin (QR), a natural flavonoid present in edible fruits and vegetables possess anticancer activity i.e. inhibits lung cancer growth. However, the application of QR in lung cancer therapy has been restricted by various factors i.e. low water solubility (2.15 µg/ml at room temperature), low bioavailability and rapid plasma clearance. To overcome the issues, we have formulated various QR-loaded liposomes surface functionalized with transferrin receptor (TFR) targeting peptides i.e. T7 (HAIYPRH) and T12 (THRPPMWSPVWP) in two research projects with active targeting ability, prolonged circulation time, and sustained release behavior for lung cancer specific QR delivery. In first research project, T7 targeted liposomes with different peptide densities i.e. 0.5%, 1% and 2% and QR-lip (non-targeted) were formulated. TFRs are over expressed (~100 folds) in various cancers including lung cancer and have low expression in most normal cells. T7 surface-functionalized liposomes (2% T7-QR-lip) demonstrated significantly enhanced cytotoxicity (~3-folds), cellular-uptake, S-phase cell cycle arrest and apoptosis in A549 cells. However, in MRC-5 (normal-lung fibroblast) cells no significant difference was observed after treatment with T7-QR-lip and QR-lip in cytotoxicity and cellular uptake studies. In tumor spheroid penetration and inhibition studies, T7 targeted liposomes showed deeper penetration and pronounced inhibition. In vivo biodistribution study via pulmonary administration of T7-DiR-lip has demonstrated liposomes accumulation in the lungs and sustained-release behavior upto 96h. Further, T7-QR-lip significantly enhanced anticancer activity of QR and life-span of orthotopic lung-tumor bearing mice (**p < 0.01, compared with control) via pulmonary administration. In second research project, T12 surface-functionalized liposomes with 0.5%, 1% and 2% T12 peptide densities and QR-lip have been formulated with ~95 % encapsulation efficiency. In vitro drug release study showed sustained release of QR from T12-QR-lip and QR-lip. In vitro experiments showed A549 cells treatment with 2% T12-QR-lip enhanced cellular-uptake, in vitro cytotoxicity, induced apoptosis and S-phase cell cycle arrest due to TFR mediated endocytosis. No significant variation has been observed in cellular-uptake and cytotoxicity after MRC-5 cells were treated with T12-QR-lip and QR-lip. Further, T12-Cou6-lip showed significantly deeper penetration i.e. 120 µm in 3D lung tumor-spheroids. Biodistribution study showed retention of T12-DiR-lip and DiR-lip mainly in the lungs upto 96h after pulmonary administration, as compared to free DiR. Pulmonary administration of T12-QR-lip showed the strongest tumor growth inhibition and survival time of orthotopic lung tumor implanted mice without any systemic toxicity as compared to QR-lip and free-QR. In summary, in vitro and in vivo results of the two research projects suggest that surface functionalization of the liposomes with TFR targeting peptides i.e. T7 and T12 is a promising approach for lung cancer therapy through active targeting and receptor mediated endocytosis of QR at lung tumor site. Moreover, T7 and T12 functionalized liposomes provides a potential drug delivery system for a range of anticancer drugs to enhance their therapeutic efficacy by localized i.e. pulmonary administration and targeted delivery.

No description available.