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A REPORT ON STUDY OF INDIAN MEDICINAL PLANT EXTRACT AS A POSSIBLE ADJUVANT FOR VACCINES IN LIPSOME MEDIATED DRUG DELIVERY Submitted in fulfilment of the Laboratory Oriented Projects BITS C313 BY AMRUTA VASUDEVAN (2008B1A3568H) UNDER THE SUPERVISION OF Dr. Kumar Pranav Narayan Assistant Professor, Biological Sciences Group BIRLA INSTITUTE OF TECHNOLOGY & SCIENCE, PILANI HYDERABAD CAMPUS March, 2012 1 A REPORT ON STUDY OF INDIAN MEDICINAL PLANT EXTRACT AS A POSSIBLE ADJUVANT FOR VACCINES IN LIPSOME MEDIATED DRUG DELIVERY Submitted in fulfilment of the Laboratory Oriented Projects BITS C313 BY AMRUTA VASUDEVAN (2008B1A3568H) UNDER THE SUPERVISION OF Dr. Kumar Pranav Narayan Assistant Professor, Biological Sciences Group BIRLA INSTITUTE OF TECHNOLOGY & SCIENCE, PILANI HYDERABAD CAMPUS March, 2012 2 CERTIFICATE This is to certify that the report entitled, Study of Indian Medicinal Plant extract as a possible adjuvant for vaccines in liposome drug delivery, and submitted by Amruta Vasudevan ID No. 2008B1A3568H in fulfilment of the requirements of BITS C313 Laboratory Oriented Projects embodies the work done by her under my supervision. Signature of the supervisor Name: Designation: Date: 3 ACKNOWLEDGEMENTS I would like to thank Prof. B.N. Jain, Vice-chancellor of BITS - Pilani, and Prof. V.S. Rao for encouraging me to undertake this study. My sincere thanks are due to Dr. Balaji Gopalan, Instructor In-charge Laboratory Oriented Projects at Hyderabad Campus for this unique opportunity. I d like to thank Dr. Kumar Pranav Narayan, Biological Sciences Group for so graciously accepting me in his tutelage and the many patient discussion sessions that gave me directions to conduct this study. I m also highly grateful to Ms. Priyanka Sharma, for her immense support and guidance she has provided me. Amruta Vasudevan (2008B1A3568H) 4 ABSTRACT In the modern day, several novel methods of gene delivery into human/mammalian cell lines have been invented and proven to be very effective. These methods find particular importance in the production of novel cancer vaccines. The method of Liposome-mediated drug delivery is the most promising technique of direct (target specific) gene delivery. However, the efficiency of this method also depends greatly on the readiness of the uptake of the antigen by the target cells. Saponin, an amphipathic glycoside, found abundantly as a secondary metabolite in plant leaves and roots, has been experimented upon and proved to be a very good adjuvant for vaccines (antigens) in novel drug delivery systems. Saponin is widely considered the ideal adjuvant for the purpose of vaccine/drug delivery because of its large size and the ability to illicit a balanced and prolonged immune response from the body (by activating both the Humoral and Cell-mediated Immunity simultaneously). This project aims to study Indian medicinal plants like Ocimum sanctum and : Obtain their saponin extract, optimise the procedure for its extraction and purification. Carry out Lipofection of the relevant vaccine/drug using the saponin extract as an adjuvant Testing of the procedure using mice as model organisms 5 CONTENTS: Cover Page........................................................................................................................1 Title Page..........................................................................................................................2 Certificate..........................................................................................................................3 Acknowledgement.............................................................................................................4 Abstract..............................................................................................................................5 Contents..............................................................................................................................6 List of Tables and Figures...................................................................................................7 Tissue Culture Techniques..................................................................................................8 History of Tissue Culture..............................................................................................8 Advantages of Tissue Culture.......................................................................................9 Limitations of Tissue Culture........................................................................................10 Equipments required in Tissue Culture.........................................................................12 Maintaining aseptic environment and sterile handling..................................................14 Cell lines and their maintenance.........................................................................................16 Primary culture...............................................................................................................16 Subculture and Cell lines...............................................................................................17 A549 Cell line................................................................................................................18 Counting the cells...........................................................................................................19 Revival of the cell culture...............................................................................................20 Preservation and subculturing of the cell lines...............................................................20 Plating the cells...............................................................................................................24 Liposomes.............................................................................................................................27 Introduction.....................................................................................................................27 Structure of liposomes and their classification................................................................28 Components of Liposomes ..............................................................................................30 Preparation of Liposomes ................................................................................................31 6 Mechanism of transfection using liposomes ..................................................................34 Cell viability and Cytotoxicity assays...................................................................................35 Microtitration Assays (Metabolic cytotoxicity)..............................................................35 MTT-based cytotoxicity assay..................................................................................36 Beta-Galactosidase Assay...............................................................................................38 Saponin-based adjuvants.......................................................................................................39 Preparation of ethanolic extract.......................................................................................42 Plasmid DNA extraction........................................................................................................43 History of plasmid extraction..........................................................................................43 List of reagents used........................................................................................................44 Procedure.........................................................................................................................45 DNA Nanodrop.....................................................................................................................48 Results...................................................................................................................................49 Conclusion.............................................................................................................................51 Future plan of work...............................................................................................................52 References.............................................................................................................................53 7 LIST OF TABLES AND FIGURES: Figure1: Applications of Tissue Culture Techniques.....................................................9 Figure 2: Issues concerning sterile handling..................................................................15 Figure 3: Cell Morphology, courtesy: ATCC.................................................................19 Figure 4: Hemocytometer cell counting.........................................................................20 Figure 5: TEM micrographs of lipoplexes.....................................................................28 Figure 6: Size based classification of liposomes............................................................28 Figure 7 Process of Liposome Preparation.....................................................................30 Figure 8: Structures involved in preparation process.....................................................31 Figure 9: Mechanism of transfection using liposomes...................................................32 Figure 10: Flow chart showing sample preparation for Soxhlet apparatus.....................41 Table 1: MTT Assay.......................................................................................................42 Table 2: Beta-Gal Assay (1st run)...................................................................................42 Table 3: Beta-Gal Assay (2nd run)..................................................................................42 8 A. TISSUE CULTURE TECHNIQUES[1] I. History Of Tissue Culture Techniques Tissue culture was devised at the beginning of the twentieth century as a method for studying the behavior of animal cells free of systemic variations that might arise in vivo both during normal homeostasis and under the stress of an experiment. Disaggregation of explanted cells and subsequent plating out of the dispersed cells was first demonstrated by Rous, although passage was more often by surgical subdivision of the culture by Fischer, Carrel, and others, to generate what were then termed cell strains. L929 was the first cloned cell strain, isolated by capillary cloning from mouse L-cells. It was not until the 1950s that trypsin became more generally used for subculture, following procedures described by Dulbecco to obtain passaged monolayer cultures for viral plaque assays, and the generation of a single cell suspension by trypsinization, which facilitated the further development of single cell cloning. The term tissue culture is used as a generic term to include organ culture and cell culture. The term organ culture implies a three-dimensional culture of undisaggregated tissue retaining some or all of the histological features of the tissue in vivo. Cell culture refers to a culture derived from dispersed cells taken from original tissue, from a primary culture, or from a cell line or cell strain by enzymatic, mechanical, or chemical disaggregation. There is much information that can be gleaned from carrying out tissue culture experiments. They can be used to study several things such as indicated in figure 1. The demonstration that human tumors could also give rise to continuous cell lines, such as HeLa, encouraged interest in human tissue, helped later by the classic studies of Leonard Hayflick on the finite life span of cells in culture and the requirement of virologists and molecular geneticists to work with human material. 9 Figure 1: Applications of Tissue Culture Techniques. II. Advantages of Tissue Culture: There are many advantages that tissue culture techniques can give an experiment. Depending on the problem at hand and the system to be studied, they may be of varying value to the experiment design, but are all equally important for a successful run. Some of them are: a) Control of Environment: The two major advantages of tissue culture are the ability to control the physiochemical environment (pH, temperature, osmotic pressure, and O2 and CO2 tension), which has to be controlled very precisely, and the physiological conditions, which have to be kept relatively constant. b) Characterization and Homogeneity of Samples: Tissue samples are invariably heterogeneous. Replicates, even from one tissue, vary in their constituent cell types. After one or two passages, cultured cell lines assume a homogeneous (or at least uniform) constitution, as the cells are randomly mixed at each transfer and the selective pressure of the culture conditions tends to produce a homogeneous culture of the most vigorous cell type. Hence, at each subculture, replicate samples are identical to each other, and the characteristics of the line may be perpetuated over several generations, or even indefinitely if the cell line is stored in 10 liquid nitrogen. Because experimental replicates are virtually identical, the need for statistical analysis of variance is simplified. c) Economy, Scale, and Mechanization: Cultures may be exposed directly to a reagent at a lower, and defined, concentration and with direct access to the cell. Consequently less reagent is required than for injection in vivo, where >90% may be lost by excretion and distribution to tissues other than those under study. Screening tests with many variables and replicates are cheaper, and the legal, moral, and ethical questions of animal experimentation are avoided. New developments in multi-well plates and robotics also have introduced significant economies in time and scale. III. Limitations of Tissue culture: Like all things good, tissue culture is not all sugar-spice-and-everything-nice. The flip side to the incredible customization that the techniques can render to an experiment, we also have to deal with the wide array of problems that can disrupt the functionality. We see the main ones here: a) Expertise: Culture techniques must be carried out under strict aseptic conditions because animal cells grow much less rapidly than many of the common contaminants, such as bacteria, molds, and yeasts. Furthermore, unlike microorganisms, cells from multicellular animals do not normally exist in isolation and consequently are not able to sustain an independent existence without the provision of a complex environment simulating blood plasma or interstitial fluid. These conditions imply a level of skill and understanding on the part of the operator in order to appreciate the requirements of the system and to diagnose problems as they. Also care must be taken to avoid the recurrent problem of cross-contamination and to authenticate stocks. Hence tissue culture should not be undertaken casually to run one or two experiments, but requires proper training, strict control of procedures, and a controlled environment. 11 b) Environmental Control: In tissue culture labs, stringency of protocol is matched only that of laboratory environment control. It is of utmost importance for the successful experiment design that we build the lab in an isolated location and away from a general purpose lab where microbiology work is done. Further, there is a great advantage to be had b y building the lab with specifications that allow for positive air-pressure, body suits and air-showers. Also of great importance is the containment and disposal of biohazards. c) Differentiation and Selection: In the course of culture maintenance, it is possible to get a loss of the phenotypic characteristics typical of the tissue from which the cells had been isolated. This effect has been blamed on dedifferentiation, a process assumed to be the reversal of differentiation but later shown to be largely du e to the overgrowth of undifferentiated cells of the same or a different lineage. The development of serumfree selective media has now made the isolation of specific lineages possible, and it can be seen that under the right conditions, many of the differentiated properties of these cells may be restored. d) Origin of Cells: If differentiated properties are lost, for whatever reason, it is difficult to relate the cultured cells to functional cells in the tissue from which they were derived. Stable markers are required for characterization of the cells; in addition the culture conditions may need to be modified so that these markers are expressed. Regrettably, many cell lines have been misidentified due to cross-contamination or errors in stock control in culture or in the freezer. This makes it essential to have the technology, or access to it, to ensure authentication of each cell line that is used. 12 IV. Equipment Required In Tissue Culture Labs: The need to maintain asepsis distinguishes the tissue culture laboratory from most others, so it is important it be dust free and have no through traffic. The introduction of laminar flow hoods has greatly simplified the problem and allows the utilization of unspecialized laboratory accommodation, provided that the location is suitable. If possible it is preferable for the tissue culture lab to be separated from the preparation, washup, and sterilization areas, while still remaining adjacent. The design of the lab and the equipment in it can vary a lot, however certain things are absolutely essential, and we list them here: 1. Aseptic Area: a) Vertical Flow Laminar Hood: For most laboratories, which are typically busy and overcrowded, the simplest way to provide aseptic conditions is to use a laminar-flow hood. Usually one hood is sufficient for two to three people. A horizontal-flow hood is cheaper and provides the best sterile protection for your cultures, but it is really suitable only for preparing medium (without antibiotics) and other nontoxic sterile reagents and for culturing nonprimate cells. It is particularly suitable for dissecting nonprimate material for primary culture. For potentially hazardous materials (any primate, including human, cell lines; virus-producing cultures; radioisotopes; and carcinogenic or toxic drugs), a Class II or Class III microbiological safety cabinet should be used. In practice, most laboratories now use a Class II microbiological safety cabinet as standard. b) Sterile Liquid Handling Pipetting: These devices originated from Eppendorf micropipettes used for dispensing 10 to 200 L. As the working range now extends up to 5 mL or more, the term micropipette is not always appropriate, and the instrument is more commonly called a pipettor. Only the tip needs to be sterile, but the length of the tip then limits the size of vessels used. If a sterile fluid is withdrawn from a container with a pipettor, the nonsterile stem must not touch the sides of the container. Reagents volumes of 10 to 20 mL may 13 be sampled in 5- L to 1-mL aliquots from a sample tube such as a universal container or in 5- to 200- L volumes from a bijou bottle or similar small vial but withdrawing liquids from larger containers will risk contamination unless extended length sterile tips are used. 2. Inverted microscope: A simple inverted microscope is essential. It cannot be overemphasized that it is vital to look at cultures regularly to detect morphological changes and the possibility of microbiological contamination. It should have a stage that is large enough to accommodate large roller bottles, if required, between it and the condenser. Long working-distance phase-contrast optics (condenser and objectives) are required to compensate for the thickness of plastic flasks. 3. Centrifuge: Periodically cell suspensions require centrifugation to increase the concentration of cells or to wash off a reagent. A small bench-top centrifuge is sufficient for most purposes. Refrigeration is not necessary, although it can be used, set at room temperature, to prevent cell samples overheating. Cells sediment satisfactorily at 80 to 100 g; higher g may cause damage and promote agglutination of the pellet. 4. Cell Counting using Hemocytometer slide: The simplest direct method uses an engraved graticule slide with a thick coverslip. It is the cheapest option and has the added benefit of allowing cell viability to be determined by dye exclusion. If used routinely, it is better to issue one slide per person each with multiple coverslips. 5. CO2 Incubator: CO2 incubators are more expensive, but their ease of use and superior control of CO2 tension and temperature justify the expenditure. A controlled atmosphere is achieved by using a humidifying tray and controlling the CO2 tension with a CO2-monitoring device, which draws air from the incubator into a sample chamber, determines the concentration of CO2, and injects pure CO2 into the incubator to make up any deficiency. Air is circulated around the incubator by natural convection or by using a 14 fan to keep both the CO2 level and the temperature uniform. Fan-circulated incubators recover their CO2 levels after opening, although natural convection incubators can still have a quick recovery and greatly reduce the risks of contamination. Frequent cleaning of incubators particularly humidified ones is essential, so the interior should dismantle readily without leaving inaccessible crevices or corners. Flasks or dishes, or boxes containing them, which are taken from the incubator to the laminar-flow hood, should be swabbed with alcohol before being opened. V. Maintaining Aseptic Environment and Sterile handling: The sterility of the work area and the aseptic environment in the lab must be always maintained at all times for any experiment to be successful. There can be many aspects of this and can include personal hygiene and regular cleaning of the work area. a) Personal Hygiene: The hands must be washed with an alkaline soap before entering the aseptic area and also should be washed with 70% ethanol before handling any cell samples, culture bottles, CO2 incubator or the laminar hood. The nails must be neatly trimmed. If you have long hair, tie it back. When working aseptically on an open bench, do not talk. Talking is permissible when you are working in a vertical laminar-flow hood, with a barrier between you and the culture, but should still be kept to a minimum. If you have a cold, wear a face mask, or, better still, do not do any tissue culture during the height of the infection. b) Sterile handling: Swabbing: Swab down the work surface with 70% alcohol before and during work, particularly after any spillage, and swab it down again when you have finished. Swab bottles as well, especially those coming from cold storage or a water bath, before using them, and also swab any flasks or boxes from the incubator. Swabbing sometimes removes 15 labels, so use an alcohol resistant marker. Isopropyl alcohol ( rubbing alcohol, IPA) can be used instead of ethanol or methanol and is available as a proprietary spray or as prepacked swabs. The other issues concerning sterile handling are covered in the following figure: Figure 2: Issues concerning sterile handling 16 B. CELL LINES AND THEIR MAINTENANCE[1] A primary culture is that stage of the culture after isolation of the cells but before the first subculture after which it becomes a cell line. We now look at each of these in a sharper focus. I. Primary Culture. The cells that are to be studied in an experimental setting are acquired from the proper organ of the desired organism and cultured. The process is called a primary culture and it s initiation involves the following four processes: (1) acquisition of the sample, (2) isolation of the tissue, (3) dissection and/or disaggregation, and (4) culture after seeding into the culture vessel. After isolation, a primary cell culture may be obtained either by allowing cells to migrate out from fragments of tissue adhering to a suitable substrate or by disaggregating the tissue mechanically or enzymatically to produce a suspension of cells, some of which will ultimately attach to the substrate. The enzymes used most frequently for tissue disaggregation are crude preparations of trypsin, collagenase, elastase, pronase, Dispase, DNase, and hyaluronidase, alone or in various combinations. Although each tissue may require a different set of conditions, certain requirements are shared by most of them: (1) Fat and necrotic tissues are best removed during dissection. (2) The tissue should be chopped finely with sharp scalpels to cause minimum damage. (3) Enzymes used for disaggregation should be removed subsequently by gentle centrifugation. 17 (4) The concentration of cells in the primary culture should be much higher than that normally used for subculture because the proportion of cells from the tissue that survives i n primary culture may be quite low. (5) A rich medium, such as Ham s F12, is preferable to a simple medium, such as Eagle s MEM, and if serum is required, fetal bovine often gives better survival than does calf or horse. Isolation of specific cell types will probably require selective serum-free media. II. Subculture and Cell Lines. The first subculture represents an important transition for a culture. The need to subculture implies that the primary culture has increased to occupy all of the available substrate. Hence cell proliferation has become an important feature. From a very heterogeneous primary culture, containing many of the cell types present in the original tissue, a more homogeneous cell line emerges. However, there are a number of less desirable consequences of generating a cell line. While propagation and cryopreservation extends the lifetime of a culture and its availability, it also increases the risk of cross-contamination. The potential sources of contamination are: 1. contamination could be due to poor pipetting techniques, 2. sharing media and pipettes among cell lines, 3. or the generation of aerosols when flasksor media bottles from more than one cell line are open simultaneously. 4. Contamination accidents may also occur at subculture or during cryopreservation from mislabeling, seeding the wrong flask, or poor inventory control in the freezer leading to a cell line becoming misidentified. The biggest threat of cross-contamination is that if the accidentally introduced cells have a faster growth rate, it will overgrow and eventually replace the original cell line. The following practices help avoid cross-contamination: (1) Obtain cell lines from a reputable cell bank. 18 (2) Do not have culture flasks of more than one cell line, or media bottles used with them, open simultaneously. (3) Handle rapidly growing lines, such as HeLa, on their own and after other cultures. (4) Never use the same pipette for different cell lines. (5) Never use the same bottle of medium, trypsin, or other substances, for different cell lines. Dedicate one set of medium and other reagents to each cell line. (6) Do not put a pipette back into a bottle of medium, trypsin, or other substances, after it has been in a culture flask containing cells. (7) Add medium and any other reagents to the flask first, and then add the cells last. (8) Do not use unplugged pipettes, or pipettors without plugged tips, for routine maintenance. (9) Check the characteristics of the culture regularly, and suspect any sudden change in morphology, growth rate, or other phenotypic properties. Cross-contamination or its absence may be confirmed by DNA STR profiling. III. The A549 Cell Line[9] A549 is a human lung epithelial carcinoma cell line that is aneuploid and synthesizes surfactant. These are adherent carcinomatous cells that were initially cultured in 1972 from a 58 year old Caucasian male by Dr. D.J. Gliard et al. using an explants transfer. Further studies by M. Lieber, et al. revealed that A549 cells could synthesize lecithin with a high percentage of desaturated fatty acids utilizing the cytidine diphosphocholine pathway. The cells are positive for keratin by immunoperoxidase staining. 19 Figure 3: Cell Morphology, courtesy: ATCC The base medium for this cell line is ATCC-formulated F-12K Medium. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10%. The culture requires 95% air and 5% carbon dioxide (CO2), maintained at at a temperature of 37.0 C. IV. Counting the Cells: Quantitation in cell culture is required for the characterization of the growth properties of different cell lines, for experimental analyses and to establish reproducible culture conditions for the consistency of primary culture and the maintenance of cell lines. The concentration of a cell suspension may be determined by placing the cells in an optically flat chamber under a microscope. The cell number within a defined area of known depth (i.e., within a defined volume) is counted, and the cell concentration is derived from the count. 20 Figure 4: Hemocytometer cell counting V. Revival of The Culture:[10] The original cell sample of A549 is procured as a vial having cells suspended in 1 ml of DMEM. In order to revive the culture, we need to add 1ml of Complete Media (DMEM + serum) into the vial, and then centrifuge the contents to get the cell pellet. We then discard the supernatant, and re-suspend the cells in fresh media. VI. Preservation and Subculturing of the Cell Line:[1] The protocol for subculturing of the A549 cell line is detailed at the ATCC website and is reproduced here: 21 PROTOCOL: Subculturing A549 Cells 1. Remove and discard culture medium. 2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor. 3. Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes). Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37 C to facilitate dispersal. 4. Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting. 5. Add appropriate aliquots of the cell suspension to new culture vessels. Cultures can be established between 2 X 103 and 1 X 104 viable cells/cm2. Do not exceed 7 X 104 cells/cm2. 6. Incubate cultures at 37 C. We need to maintain the culture at a cell concentration that is between 6 X 10(3) and 6 X 104 cells/cm2. Instead of counting the cells each time using a hemocytometer, it is instead easier to follow the rule of thumb to subculture when the cells appear about 75%-80% confluent under the microscope. Each time we subculture, a subcultivation ratio of 1:3 to 1:8 is recommended. ATCC prescribes a medium renewal 2 to 3 times per week. When we subculture the cells 1:3, the other two culture flasks are preserved for batch building and future uses. To preserve them, we add 5% DMSO (v/v) to the complete media and store in liquid nitrogen vapour at -80 C. 22 ISSUES : 1. In the laboratory, we grow the A549 cells in a serum- free media. This requires alteration in the subculture protocols. After trypsin-mediated subculture, the addition of serum inhibits any residual proteolytic activity. Consequently protease inhibitors such as soya bean trypsin inhibitor or 0.1 mg/mL aprotinin must be added to serum free media after subculture. 2. Because crude trypsin is a complex mixture of proteases, some of which may require different inhibitors, it is preferable to use pure trypsin followed by a trypsin inhibitor. Alternatively, one may wash cells by centrifugation to remove trypsin, although it may still be advisable to include a trypsin inhibitor in the wash. In recent years, the use of serum in culture media has surely been on the decline. It is expensive, and is not always present in surplus quantities and the purification process is tedious. So it is beneficial to not use serum in the media. Also, using a serum free media is further beneficial in following ways: 1. Simplified and better defined media composition, 2. A reduced risk of contamination, 3. A reduced risk of exposure to infectious agents this is especially true in case of exposure to Bovine Spongiform Encephalopathy through the use of BSA. 4. Reduced cost, 5. Unlike regular serum, it does not interfere with the transfection assay reagents. However, despite these advantages, the presence of the serum- whether derived directl y from animal sources or synthesized in the lab- is important and preferred for a number of reasons. The most important of them being: 1. First, an investment in time is required to adopt a particular cell line to serum-free medium. The cells will have to be weaned from serum slowly. 2. Moreover, some cell lines may require the addition of growth factors specific to that cell type to overcome a deficiency in the particular medium employed. It is advisable to begin with a serum-free medium which has a source of growth factors, such as pituitary extract, which can be incrementally removed if necessary. 23 3. The low protein concentration of serum-free medium while an advantage for reducing potential sources of contamination removes proteins which play a role in shear protection and attachment to growth substratum. For example, the BSA present in serum protects cells grown in suspension from shear damage. The addition of Pluronic F68 or polyethylene glycol may be needed in place of BSA. BSA also provides other transport functions and the replacement of native sources of albumin with recombinant sources is not straightforward. 4. Attachment-dependent cell lines require an extracellular matrix on the growth substratum. Serum provides some the components for this matrix. Therefore, when using serum-free medium the substratum (plastic dishes) should be pre-coated with a fibronectin, laminin or another suitable alternative such as FNC Coating Mix (a fibronectin/collagen mixture manufactured by AthenaES, Baltimore, MD), Pronectin (a synthetic fibronectin polymer manufactured by Sanyo Chemical Industries, Kyoto, Japan) or Matrigel. The protocol has taken care of all of the above concerns. The adherent cell strain A549 binds to the D-Lysine coated T-25 flasks that are used. That layer also provides it with the shear protection the cell line needs for its successful growth. Transformation causes several changes in cells that include loss of contact inhibition, anchorage independence (usually, not always) and a reduced serum dependenc y. Transformed cells have lower serum dependence than their normal counterparts, due, in part, to the secretion of growth factors by tumor cells. These factors have been collectively described as autocrine growth factors. Implicit in this definition is that: (1) the cell produces the factor, (2) the cell has receptors for the factor, and (3) the cell responds to the factor by entering mitosis. Some of these factors may have an apparent transforming activity on normal cells (e.g. TGF ) binding to the EGF receptor and inducing mitosis, although, unlike true transformation, this type of transformation is reversible. These factors also cause non-transformed cells to adopt a transformed phenotype and grow in suspension. This is the reason why most of the 24 tissue culture work has dealings with carcinoma cells. They just thrive well in the tissue culture conditions, better than do regular non-transformed cells. VII. Plating the Cells: Any meaningful work on the cells will require the cells to be transferred to a microtiter plate after which the requisite reagents can be added to the wells in the required quantities. The plate arrangement provides a very convenient way to conduct experiments which need visualization of colour development and serial dilutions etc. The plating of cells is done keeping in mind the number of cells that are present in the culture already. As a good rule of thumb, it is never advisable to put more than 10 X 4 cells in a well. Given this constraint, it becomes important to control the number of ells that actually enter into the final plating media. Given below is the protocol adopted in the lab and is based on the following assumptions: a. We plate the cells in a 96-well microtiter plate. b. Each well is going to contain 0.1 ml culture solution that has the required cell concentration of 10 X 4 cells. c. The neubauer chamber has the standard dimensions of having 1 ml of solution in an area of 1 cm2 . 25 PROTOCOL: Plating cells 1. Count the number of cells as was explained in the protocol above. 2. Now based on the number of cells obtained conduct the following calculations: As we are plating 96 wells and filling each of them with 0.1 ml of culture solution, we definitely need 9.6 ml of culture. To be safer, we take 10.5 ml. These 10.5 ml of the culture must have the required cell count of 10 X 5 cells per ml. Each cell has 0.1 ml = 10 X 4 cells. So 10.5 ml of the plating sample must have 10.5 X 5 cells. Now based on the cell count we do the further calculations. Suppose the cell count is C cells per ml. C cells are present in 1 ml of sample. So 10.5 X 5 cells will be present in { (10.5 X 5 ) / C } ml. 3. Take the above volume of the original culture and make up the volume to 10.5 ml using pure DMEM media. 4. Using a pipettor dispense 0.1ml to each well. Now wait the required amount of time for the cells to grow, or as the experiment requires. 26 C. LIPOSOMES I. Introduction A liposome is a tiny bubble (vesicle), made out of the same material as a cell membrane. Membranes are usually made of phospholipids, which are molecules that have a head group and a tail group. The head is attracted to water, and the tail, which is made of a long hydrocarbon chain, is repelled by water. In nature, phospholipids are found in stable membranes composed of two layers (a bilayer). In the presence of water, the heads are attracted to water and line up to form a surface facing the water. The tails are repelled by water, and line up to form a surface away from the water. In a cell, one layer of heads faces outside of the cell, attracted to the water in the environment. Another layer of heads faces inside the cell, attracted by the water inside the cell. The hydrocarbon tails of one layer face the hydrocarbon tails of the other layer, and the combined structure forms a bilayer. When membrane phospholipids are disrupted, they can reassemble themselves into tiny spheres, smaller than a normal cell, either as bilayers or monolayers. The bilayer structures are liposomes. The monolayer structures are called micelles. Liposomes are composite structures made of phospholipids and may contain small amounts of other molecules. Though liposome s can vary in size from low micrometer range to tens of micrometers, unilamellar liposomes are typically in the lower size range with various targeting ligands attached to their surface allowing for their surface-attachment and accumulation in pathological areas for treatment of disease. Liposomes are generally formed by the self-assembly of dissolved lipid molecules, each of which contains a hydrophilic head group and hydrophobic tails. These lipids take on associations which yield entropically favorable states of low free energy, in some cases forming bimolecular lipid leaflets. Such leaflets are characterized by hydrophobic hydrocarbon tails facing each other and hydrophilic head groups facing outward to associate with aqueous solution. At this point, the bilayer formation is still energetically unfavorable because the hydrophobic parts of the molecules are still in contact with water, a problem that is overcome through curvature of the forming bilayer membrane upon itself to form a vesicle 27 with closed edges. This free-energy-driven self-assembly is stable and has been exploited as a powerful mechanism for engineering liposomes specifically to the needs of a given system. II. Structure of Liposomes Lipid molecules used in liposomes are conserved entities with a head group and hydrophobic hydrocarbon tails connected via a backbone linker such as glycerol. Cationic lipids commonly attain a positive charge through one or more amines present in the polar head group. The presence of positively charged amines facilitates binding with anions such as those found in DNA. The liposome thus formed is a result of energetic contributions by Van der Waals forces and electrostatic binding to the DNA which partially dictates liposome shapes.Because of the polyanionic nature of DNA, cationic (and neutral) lipids are typically used for gene delivery, while the use of anionic liposomes has been fairly restricted to the delivery of other therapeutic macromolecules. Based on their sizes, liposomes are classified in the following categories: CLASSIFICATION SIZE Multilamellar Vesicles 500 to 10000 nm Smal Unilamellar Vesicles < 50 nm Large Unilamellar Vesicles > 50 nm Giant Liposomes 10000 to 1000000 nm 28 Figure 5: Transmission electron micrographs of lipoplexes prepared at 5/1 (+/ -) charge ratio. DOTAP/DOPE/DNA (A), DOTAP/DOPE/DNA in 60% FBS (B), DOTAP/CHOL/DNA (C), DOTAP/CHOL/DNA+Tf (D). Figure 6: Size based classification of liposmes 29 III. Components of Liposomes: Liposomes can be synthesized using a large number of different phospholipids that give them a range of physico-chemical properties, which ultimately affect their transfection success rate. Since their discovery in the 60 s the liposomes are traditionally synthesized using neutral lipids such as prostaglandins, diacyl glycerol and its derivatives etc. However, they are not uniformly successful in transfection efforts. As discussed above, the liposomes that are formed using a cationic lipid is helpful in transfection experiments since it readily complexes with the polyanionic DNA that needs to be inserted into the cell. On the other hand, neutral/anionic lipids are used for inserting other macromolecules into the cells. The choice of lipid doesn t only depend on the purpose to which the liposome is to be put, but is also dependent on several other factors such as: 1. Desired final size of the liposome, 2. lamellarity, 3. structure, 4. fusogenicity, 5. charge ratio, 6. and charge density To further complicate the matter, these factors influence each other in many ways, making any definite implications seem absurd. Recent research has shown that the addition of a helper lipid in addition to the cationic lipid increases the stability of the liposome and hence increases the stability of any subsequent lipoplexes that may be formed. The most commonly used helper lipid is cholesterol, and it is claimed that it sort of plugs the liposome and not only increases the stability, but also makes the structure more rigid. This is makes the liposome more robust and better suited to carry bigger drugs in its intra-vesicular space, which otherwise would not have been possible due to potential leakages . 30 We use DODEAC as the primary cationic lipid in our experimental setup. The helper lipid is cholesterol and they are mixed in a 1:1 ratio to make the final liposome concentration to 1 mM. IV. Preparation of Liposomes: Properties of lipid formulations can vary depending on the composition (cationic, anionic, neutral lipid species), however, the same preparation method can be used for all lipid vesicles regardless of composition. The general elements of the procedure involve preparation of the lipid for hydration, hydration with agitation, and sizing to a homogeneous distribution of vesicles. a) Preparation of Lipid for Hydration: 1. When preparing liposomes with mixed lipid composition, the lipids must first be dissolved and mixed in an organic solvent to assure a homogeneous mixture of lipids. Usually this process is carried out using chloroform or chloroform : methanol mixtures. The intent is to obtain a clear lipid solution for complete mixing of lipids. Typically lipid solutions are prepared at 10-20mg lipid/ml organic solvent, although higher concentrations may be used if the lipid solubility and mixing are acceptable. 2. Once the lipids are thoroughly mixed in the organic solvent, the solvent is removed to yield a lipid film. For small volumes of organic solvent (<1mL), the solvent may be evaporated using a dry nitrogen or argon stream in a fume hood. For larger volumes, the organic solvent should be removed by rotary evaporation yielding a thin lipid film on the sides of a round bottom flask. The lipid film is thoroughly dried to remove residual organic solvent by placing the vial or flask on a vacuum pump overnight. 3. Dry lipid films or cakes can be removed from the vacuum pump, the container close tightly and taped, and stored frozen until ready to hydrate. 31 b) Hydration of Lipid Film/Cake: Hydration of the dry lipid film/cake is accomplished simply by adding an aqueous medium to the container of dry lipid and agitating. The temperature of the hydrating medium should be above the gel-liquid crystal transition temperature (Tc or Tm) of the lipid with the highest Tc before adding to the dry lipid. After addition of the hydrating medium, the lipid suspension should be maintained above the Tc during the hydration period. Once a stable, hydrated LMV suspension has been produced, the particles can be downsized by a variety of techniques, including sonication or extrusion. Figure 7:Process of liposome preparation 32 a) Sizing of Lipid Suspension: Disruption of LMV suspensions using sonic energy (sonication) typically produces small, unilamellar vesicles (SUV) with diameters in the range of 15-50nm. The most common instrumentation for preparation of sonicated particles are bath and probe tip sonicators. Probe tip sonicators deliver high en-ergy input to the lipid suspension but suffer from overheating of the lipid suspension causing degradation. Sonication tips also tend to release titanium particles into the lipid suspension which must be removed by centrifugation prior to use. For these reasons, bath sonicators are the most widely used instrumentation for preparation of SUV. Sonication of an LMV dispersion is accomplished by placing a test tube containing the suspension in a bath sonicator (or placing the tip of the sonicator in the test tube) and sonicating for 5-10 minutes above the Tc of the lipid. Figure 8: The structures obtained after: (A) evaporating the solvent, (B) hydrating the Lipid layer, and the other structures obtained in the solution upon sonication. 33 V. Mechanism of transfection using Liposomes: The liposomes are prepared using a cationic lipid (having a positive polar head, DODEAC here) and a neutral helper lipid (in our case, cholesterol). Because of this the overall charge on the liposome is positive. This means that when we mix it with the DNA sample that needs to be transfected, the liposome will electrostatically bind to the negatively charged DNA(DNA is poly-anionic). This means that the overall negative charge is now shielded by the positive charge on the liposome, and the aggregate is overall either neutral or positively charged based on the charge ratio of the liposome being employed. It is because of this, the overall positively charged lipoplex binds to the negatively charged cell membrane. When granules of lipid are present on the surface of the plasma membrane, it initiates the endocytic mechanism. It is claimed that the helper lipid, which forms granules inside the liposome bilyaer, is the major factor in inititating the endocytosis.Whether this proceeds through direct fusion of the liposome with the plasma membrane or other mechanisms is not clearly understood. Now when the DNA enters the cytoplasm, it untangles itself from the endocytic vesicles using mechanisms that have not been clearly elucidated. Figure 9: mechanism of transfection using liposomes. 34 D. CELL VIABILITY AND CYTOTOXICITY ASSAYS The choice of assay will depend on the agent under study, the nature of the anticipated response, and the particular target cell. In vitro assays can be divided into five major classes: (1) Viability. An immediate or short-term response, such as increased and uncontrolled membrane permeability or a perturbation of a particular metabolic pathway correlated with cell proliferation or survival. (2) Survival. The long-term retention of self-renewal capa-city (5 10 generations or more). (3) Metabolic. Assays, usually microtitration based, of inter-mediate duration that can either measure a metabolic response (e.g., dehydrogenase activity; DNA, RNA, or protein synthesis) at the time of, or shortly after, exposure. Making the measurement two or three population doublings after exposure is more likely to reflect cell growth potential and may correlate with survival. (4) Genotoxicity and Transformation. Survival in an altered state (e.g., one or more genetic mutations with resultant alterations in growth control or malignant transformation). (5) Irritancy. A response analogous to inflammation, allergy, or irritation in vivo; as yet difficult to model in vitro, but may be possible to assay by monitoring cytokine release in organotypic cultures. I. MICROTITRATION ASSAYS (METABOLIC CYTOTOXICITY) The introduction of multiwell plates revolutionized the approach to replicate sampling in tissue culture. These plates are economical to use, lend themselves to automated handling, and can be of good optical quality. The most popular are 96-well microtitration plates or microplates, each well having 28 to 32 mm2 of growth area, 0.1 or 0.2 mL medium, and up to 1 105 cells. Microtitration offers a method by which large numbers of samples may be 35 handled simultaneously, but with relatively few cells per sample. With this method, the whole population is exposed to the agent, and viability is determined subsequently, usually by measuring a metabolic parameter such as the ATP or NADH/NADPH concentration. The end point of a microtitration assay is usually an estimate of the number of viable cells, if the assay is done after the removal of the toxin. Although this result can be achieved directly by cell counts or by indirect methods, such as isotope incorporation, cell viability as measured by MTT reduction[14] is widely used as the endpoint[15]. MTT is a yellow water-soluble tetrazolium dye that is reduced by live, but not dead, cells to a purple formazan product that is insoluble in aqueous solutions. However, a number of factors can influence the reduction of MTT[16]. The assay described below, provided by Jane Plumb of the Cancer Research UK Centre for Oncology and Applied Pharmacology, University of Glasgow, Scotland, UK, has been shown to give the same results as a standard clonogenic assay[17]. It illustrates the use of microtitration in the assay of anticancer drugs, but would be applicable, with minor modifications, to any cytotoxicity assay. a. MTT-BASED CYTOTOXICITY ASSAY Principle: Cells in the exponential phase of growth are exposed to a cytotoxic drug. The duration of exposure is usually determined as the time required for maximal damage to occur, but is also influenced by the stability of the drug. After removal of the drug, the cells are allowed to proliferate for two to three population-doubling times (PDTs) in order to distinguish between cells that remain viable and are capable of proliferation and those that remain viable but cannot proliferate. The number of surviving cells is then determined indirectly by MTT dye reduction. The amount of MTT-formazan produced can be determined spectrophotometrically once the MTT-formazan has been dissolved in a suitable solvent. Outline: Incubate monolayer cultures in microtitration plates in a range of drug concentrations. Remove the drug, and feed the plates daily for two to three PDTs; then f eed the plates again, and add MTT to each well. Incubate the plates in the dark for 4 h, and then remove the medium and MTT. Dissolve the water-insoluble MTT-formazan crystals in DMSO, add a buffer to adjust the final pH, and record the absorbance in a plate reader. 36 Materials: Sterile: Growth medium Trypsin (0.25% + EDTA, 1 mM, in PBSA) MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide (Sigma), 50 mg/mL, filter sterilized Microtitration plates (Iwaki) Pipettor tips, preferably in an autoclavable tip box Petridishes(non TC-treated) Universal containers or tubes, 30 mL and 100 mL Nonsterile: Plastic box (clear polystyrene, to hold plates) Multichannel pipettor Dimethyl sulfoxide (DMSO) and Methanol solution DMSO dispenser (optional); such as Labsystems Microplate Dispenser (Cat No 5840 127, from Thermo Fisher; see also Fig. 4.7) ELISA plate reader (Molecular Devices, with SOFTmax PRO; see also Fig. 4.7; Appendix II: Plate Readers) Plate carrier for centrifuge Estimation of surviving cell numbers: Feed the 96 well microtitre plate with 50 L of fresh medium at the end of the growth period, and add 10 L of MTT to all of the wells. Wrap the plates in aluminum foil, and incubate them for 4 h in a humidified atmosphere at 37 C. Note that 4 h is a minimum incubation time, and plates can be left for up to 8 h. Remove the medium and MTT from the wells (centrifuge for nonadherent cells), and dissolve the remaining MTT-formazan crystals by adding a solution of DMSO: MeOH (1:1) to all of the wells. 37 Record absorbance at 570 nm immediately, because the product is unstable. The wells which contain medium and MTT but no cells are the blank wells. b. B-GAL ASSAY Protocol: 1. In the 96 well microtitre plate, use the first row of 12 wells for liposome solution and the second row for DNA solution as directed below. Cells are plated in triplicate mode in order to pinpoint the possibility of contamination. 2. The first well (A1:by general nomenclature) contains 56 l of DMEM and 44 l of liposome solution (the total volume adds up to 100 l.). The remaining wells of Row1 contain 50 l of DMEM. The row of wells vertically below it (Row2) contains 41 l of DMEM and 9 l of DNA solution in each well. (Total volume:50 l) 3. 50 l of solution in A1 is added to the solution in A4(A2 and A3 are replicates of A1). The remaining 50 l of liposome solution is added to its horizontally adjacent well, preparing a dilution of the solution. 4. 50 l of solution in A4 is now added to solution(media) in A7(A5 and A6 are replicates of A4). The same procedure is repeated for solution in A10. Thus serial dilutions of the liposome solution are formed, viz., 8:1, 4:1, 2:1 and 1:1. These solutions are then added to the DNA solutions in the wells vertically below them respectively, forming lipoplexes of varying component ratios. 5. The titre plate is left on a rocker for shaking for 20 minutes. After that, we add 210 l CM1X, serum-containing media to each well. The plate is now incubated at 37 C and transfection occurs over a time period of 24-48 hrs. This entire procedure is called treatment of cells for the assay. 38 6. After 48 hrs, the media from each of these wells is removed and 50 l of Lysis buffer is added to them.(100 l in blank). After 20 minutes of shaking, 50 l of Assay buffer is added to the wells. Its incubated at 37 C for 20 minutes. 7. -galactosidase can be assayed by measuring hydrolysis of the chromogenic substrate, o-nitrophenyl- -D-galactoside (ONPG). The amount of onitrophenol formed can be measured by determining the absorbance at 420 nm. (Apparatus used: Biotek Elisa reader, Gen5 software) E. SAPONIN-BASED ADJUVANTS The most widely used adjuvants, alum and oil-in-water emulsions, were introduced into clinical practice long before their mode of action was elucidated. These adjuvants were found to be effective in inducing a humoral immune response and to be non antigenic. There are several reasons for searching for additional adjuvants, including the desire to induce (1) broader immune responses capable of covering multiple serotypes, (2) strong T-cell responses required against infections such as hepatitis C virus and human immunodeficiency virus (HIV) or to form the basis of therapeutic vaccines, (3) responses which overcome immunological senescence or stimulate an immune response early in life, (4) potent mucosal immunity, (5) immune responses to poorly immunogenic antigens or (6) to allow manufacturers to reduce the dose of antigen, thus reducing cost and increasing the number of doses of vaccine that can be produced. Advocates of novel adjuvants aim at tailoring the vaccine-induced immune responses to achieve maximal efficacy, through optimization of B-cell responses (in terms of level, quality, duration and memory) and generating appropriate T-cell responses (in terms of effector functions and memory). Their expectation is that understanding which innate immune receptors are involved in the response to different classes of adjuvants, and which signals convert dendritic cells (DC) and monocytes into immunostimulatory antigen presenting cells (APCs), will provide a scientific rationale for their use, and reduce the risk of unintended adverse reactions. the discovery of pathogen-associated molecular patterns 39 (PAMPs) such as lipopolysaccharide (LPS) and CpG motifs, as powerful activators of the immune system through toll-like receptor (TLR) interaction, prompted a variety of TLR agonists to be used to modulate the immune system. Immunopotentiators act on APCs, mainly DC, which express these TLRs for screening the environment for pathogens. TLR activation leads to an increased recruitment of innate immune cells at the infection site, along with production of pro-inflammatory cytokines and chemokines, finally resulting in the induction of antigen-specific adaptive immune responses[18]. The way immunopotentiators are delivered can affect their immunogenicity. New formulations have been designed to increase the antigen availability at the injection site, thus facilitating antigen uptake by APCs. Monophosphoryl Lipid A (MPL), a non-toxic bacterial LPS-derivative, was the first TLR targeting adjuvant (dependent on TLR4) approved for human use and is the basis of GSK s adjuvant AS0 4 (MPL & alum), which is used in the licensed hepatitis B vaccine[19] and human papilloma virus vaccine[20]. AS0 4 stimulates the migration and activation of DC and monocytes in draining lymph nodes, leading to the induction of the NF-kB pathway. It results in an early and transient local cytokine response, which is more efficient in stimulating adaptive T and B-cell responses, including a high level of memory B cells, than alum[21,22]. It should be noted that the antigen (virus-like particles) and the adjuvant need to be co-localized in lymph nodes to have a beneficial adjuvant effect on relevant APCs. The importance of formulation was further indicated with adjuvants involving saponins, which are derived from the bark of the quillaja tree. The starting material, Quil A , is reactogenic and can cause toxicity in man including haemolysis[23], properties which are lost when the material is mixed with cholesterol. While several saponin-based adjuvants are currently in advanced clinical development, their mechanism of action is still being investigated. Thus far, saponins-based adjuvants were shown to possess antigen delivery and immunomodulatory capabilities. These include induction of a balanced TH1/TH2 response with antibody production[24], as well as cytotoxic CD8 lymphocytes[25]. Saponin complexes have been shown to utilize a MyD88-dependent (TLR-independent), and IL-18 receptor signalling pathway. CSL s saponin-based adjuvant, ISCOMATRIX , induces rapid and transient cytokine production, thus leading to a transient influx of innate cells (e.g. natural killer (NK) cells, NKT, neutrophils and macrophages) to the draining lymph nodes[26]. In addition, APCs (specifically migratory DC (CD205+CD8-) and CD8 + DC) 40 are activated to induce cross-presentation and prolonged antigen presentation in draining lymph nodes. 41 I. Preparation of Ocimum sanctum ethanolic extract: Leaves are dried in shade and powdered to a very fine consistency Powder (say 75gms) was extracted with 700ml of 95% ethanol in a soxhlet apparatus at 6075 C and concentrated. Figure 10: Flow chart showing sample preparation for Soxhlet apparatus 42 F. PLASMID DNA EXTRACTION: I. HISTORY OF PLASMID ISOLATION In the 19th century, biochemists initially isolated DNA and RNA (mixed together) from cell nuclei. 2 types ribose and deoxyribose. It was this subsequent discovery that led to the identification and naming of DNA as a substance distinct from RNA. 1869 : Friedrich Miescher (1844 1895) discovered a substance he called "nuclein" in. He isolated a pure sample of the material now known as DNA from the sperm of salmon 1889: Richard Altmann named it "nucleic acid". This substance was found to exist only in the chromosomes. 1919 : Phoebus Levene at the Rockefeller Institute identified the components (the four bases, the sugar and the phosphate chain) and he showed that the components of DNA were linked in the order phosphate-sugar-base. He called each of these units a nucleotide and suggested that they are the 'backbone' of the molecule. 1921: Torbjorn Caspersson and Einar Hammersten showed that DNA was a polymer. 1979 : The universal method for plasmid DNA extraction was invented by Birnboim and Doly. 43 II. LIST OF REAGENTS USED 1. Alkaline Lysis soln. 1: 50mM glucose 25mM Tris-HCl (pH 8.0) 10mM EDTA (pH 8.0) 2. Alkaline Lysis soln. 2: 0.2% NaOH 1% (w/v) SDS 3. Alkaline Lysis soln. 3: 5M KOAc Glacial Acetic Acid Autoclaved water 4. Lysozyme 5. STE Buffer 6. 10N NaOH 7. 5M KOAc ,5M NaCl, 5M LiCl 8. 1M Tris-HCl 9. 10X TE buffer 10. 3M NaOAc (pH:7.0) 11. 1.6M NaCl + 13% PEG-8000 (W/V) 12. Isopropanaol 13.70% Ethanol 44 III. PROCEDURE 45 46 47 G. DNA NANODROP The five nucleotides that comprise DNA and RNA exhibit widely varying 260/280 ratios. The following represent the 260/280 ratios estimated for each nucleotide if measured independently: Guanine: 1.15 Adenine: 4.50 Cytosine: 1.51 Uracil: 4.00 Thymine: 1.47 The resultant 260:280 ratio for the nucleic acid being studied will be approximately equal to the weighted average of the 260/280 ratios for the four nucleotides present. It is important to note that the generally accepted ratios of 1.8 and 2.0 for DNA and RNA respectively, are "rules of thumb". The actual ratio will depend on the composition of the nucleic acid. RNA will typically have a higher 260/280 ratio due to the higher ratio of Uracil compared to that of Thymine. Other forms of stray contaminants, viz., CHO contaminants, can be observed at an absorption wavelength of 230nm. The A260/A230 absorbance ratio comes in handy for the detection of contaminants other than proteins or RNA in the DNA solution. 48 RESULTS: Table 1: MTT Assay Table 2: Beta-Gal Assay (1st run) Table 3: Beta-Gal Assay (2nd run) 49 Concentration of plasmid DNA obtained after plasmid extraction, as measured by UVVisible spectrophotometer: A260 /A230 = 2.68 Expected 260/230 ratio values are commonly in the range of 2.0-2.2. A value lower than this usually indicates an appreciable amount of CHO contamination. 50 CONCLUSION: From the results obtained above, it is easy to deduce that there must have been some form of contamination in the cell media, owing to which there are discrepancies in the results. A triplicate set of wells must usually have similar (very close) absorbance values. As we can see from the above tables, for e.g. Table 1: well nos. B1, B2, B3 and B10, B11, B12 show huge variation in absorbance values among themselves, although all conditions are identical, which is a sure sign of contamination. The result of plasmid DNA extraction tells us that there is hardly any CHO contamination in the extracted plasmid DNA sample, which implies that the protocol followed provides a good yield. 51 FUTURE SCOPE OF WORK: 1. Extraction and Purification of Saponin from crushed leaves of Ocimum sanctum (Tulsi). 2. Protoplast culture techniques (callus growth). 3. Lipofection of vaccine (antigen) into mammalian cell lines, with Saponin as adjuvant, tested on mice as model organisms. 52 REFERENCES 1. R. Ian Freshney, Culture of Animal Cells: A Manual of Basic Technique and Specialised Applications, 6 th edition, (John Wiley & Sons, Inc., Hoboken, New Jersey, 2010) 2. Biochemistry 1997 Membrane Fusion with Cationic Liposomes: Effects of Target Membrane Lipid Composition Austin L. Bailey* and Pieter R. Cullis. 3. Biophysical Journal Biophysical Journal 1996 The Role of Helper Lipids in Cationic Liposome -Mediated Gene Transfer Sek Wen Hui, Marek Langner, Ya-Li Zhao, Patrick Ross, Edward Hurley, and Karen Chan. 4. Mechanism of Cationic Lipid -mediated Transfection . Invitrogen.com 5. 6. 7. Chesnoy, S. and Huang, L. (2000) Annual Review of Biophysical Biomolecular Structure 29: 27-47. Hirko, A. et al. (2003) Current Medicinal Chemistry 10: 1185-1193. Liu, D. et al. (2003) Current Medicinal Chemistry 10: 1307-1315. 8. Letters in Drug Design & Discovery, 2009 . Structural and Morphological Studies of Cationic Liposomes DNA Complexes Yan Sun, Itziar Migu liz2 , Gemma Navarro and Conchita Tros de Ilarduya. 9. ATCC Catalog Search A549 cells. ATCC number: CCL-185 10. Formulation for Dulbecco s Modified Eagle s Medium (DMEM) ATCC 30 -2002 FROM ATCC.COM 11. Pakistan Journal of Pharmaceutical Sciences ,1996 LIPOSOMES PREPARATION METHODS MOHAMMAD RIAZ 12. Health Administrator 8 - MANAGING CANCER THROUGH TARGETED DRUG DELIVERY Dr. Subhas Bhowmick 13. Preparation of Liposomes Avanti Polar Lipids, Inc. http://www.avantilipids.com/index.php?option=com_content&view=article&id=1384&Itemid=372 14. Mosmann, 1983 15. Cole, 1986; Alley et al., 1988 16. Vistica et al., 1991 17. Plumb et al., 1989 18. Iwasaki A, Medzhitov R. Toll-like receptor control of the adaptive immune responses. Nat Immunol 2004;5:987 e 95. 19. Baldridge JR, Crane RT. Monophosphoryl lipid A (MPL) formulations for the next generation of vaccines. Methods 1999;19:103 e 7 20. Verstraeten T, Descamps D, David MP, Zahaf T, Hardt K, Izurieta P, et al. Analysis of adverse events of potential autoimmune aetiology in a large integrated safety database of AS04 adjuvanted vaccines. Vaccine 2008;26:6630 e 8 53 21. Giannini SL, Hanon E, Moris P, Van Mechelen M, Morel S, Dessy F, et al. Enhanced humoral and memory B cellular immunity using HPV16/18 L1 VLP vaccine formulated with the MPL/aluminium salt combination (AS04) compared to aluminium salt only. Vaccine 2006;24:5937 e 49. 22. Didierlaurent AM, Morel S, Lockman L, Giannini SL, Bisteau M, Carlsen H, et al. AS04, an aluminum salt- and TLR4 agonist-based adjuvant system, induces a transient localized innate immune response leading to enhanced adaptive immunity. J Immunol 2009;183:6186 e 97 23. Kensil CR. Saponins as vaccine adjuvants. Crit Rev Ther Drug Carrier Syst1996;13:1 e 55 24. Maloy KJ, Donachie AM, Mowat AM. Induction of Th1 and Th2 CD4 T cell responses by oral or parenteral immunization with ISCOMS. Eur J Immunol 1995;25:2835 e 41. 25. Lipford GB, Wagner H, Heeg K. Vaccination with immunodominant peptides encapsulated in Quil A containing liposomes induces peptide-speci fi c primary CD8 cytotoxic T cells. Vaccine 1994;12:73 e80. 26. Windon RG, Chaplin PJ, Beezum L, Coulter A, Cahill R, Kimpton W, et al. Induction of lymphocyte recruitment in the absence of a detectable immune response. Vaccine 2000;19:572 e8. 54

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