Cancer Pharmacology: Analysis of Drug Penetration Through Avascular Tissue - Essay Example

MSc in Cancer Pharmacology Masters Dissertation Analysis of Drug Penetration through Avascular Tissue Supervisor: Module leader: Analysis of drug penetration through avascular tissue Abstract Index Title page 1 Abstract 2 Index 3 Acknowledgments 4 Introduction 6 Materials and methods 14 Results 24 Discussion 48 Conclusion 59 References 60 Acknowledgments I would like to take this opportunity to thank almighty God for helping me through this testing time in my life, god willing helping me to move positively forward. Secondly and by far means least, I would like to thank., without his guidance and support this project would not been possible. He has given me great inspiration through his knowledge of Science research, giving me ideas on what to do further on in life. Paul has been a pleasure to work with, the assets I have gained have been invaluable and it has been experience, one of which I will never forget. I would also like to acknowledge .. who has given me the encouragement and confidence throughout my year at University. The words thank you do not comprehend the way he has guided me through this year, including the highs and many lows I have encountered. In addition I am extremely grateful for the assistance, generosity, and advice I received from Hafiz. His encouragement and support throughout the project has been invaluable. As he encouraged me to develop my project and my personal independent thinking skills. He continuously encouraged me stimulate my analytical skills and greatly assisted me in the laboratories and showing me how to put my results together. This dissertation would not be possible without the fabulous technicians at , without their knowledge and expertise, also not forgetting, how should I put it, there alternative sense of humour, in particular ., and .. you are my stars. I would also like to thank my Family; Mum Dad and not forgetting my sisters and my brother!! Also my two gorgeous nieces, Komal and Armani who have taken my mind off many a stressful situation and have brought a smile to my face when thinking my final project was a drawing book. I would also like to thank who I have not mentioned above who have helped through my project. Introduction A fully functioning vasculature is important for the delivery of nutrients and removal of toxic metabolic wastes associated with normal cellular metabolism. All normal tissues acquire this immensely intricate vasculature from the blood vessels or circulatory tree in the vicinity. The human circulatory system, apart from these functions, also provide cells that can help immune functions and maintain hemostasis. Human body, although ultimately comprised of cells as the final functional unit, is organised into tissues that are richly supplied by the network of a circulatory system, where the architecture is planned in such a manner that the majority of these about 1013 cells are located almost in close proximity of about less than 100 micron to a feeding or draining blood vessel. This facilitates to and fro movements of substances bilaterally from the cells and blood vessels. Like nutrients, this serves as a significant vehicular pathway for drugs. Although tumor cells originate from the healthy and normal cells, they vary from the normal tissue cells in different counts. The vascular network for a growing tumor is indeed acquired at least in part by the incorporation of existing host blood vessels. Tumor expansion requires ultimately new blood vessels. Thus tumor growth and survival are critically linked to a parallel proliferation of endothelial cells comprising the tumor blood vessel network (Hahnfeldt et al., 1999). The process involved, neovascularization, is relatively uncommon in most normal tissues, but now recognized to be an important feature of solid tumors. These variations mean that the physiology of solid tumors differs from the physiology of normal tissues in a multiplicity of aspects, mostly stemming from the disparity between the two vascular structures (Brown & Giaccia, 1998) and these physiological characteristics play a significant role in drug penetration in tumors. In the introduction, how the characteristics of cancerous cells create the vasculature of solid tumors and how this in turn affects the tumor's physiology shall be explored. Furthermore, a detailed explanation into why the tumor's physiology further influences the penetration of drugs through the tumor and why the effective penetration of drugs through the tumor is essential to effective chemotherapy will be given. Certain properties of solid tumors that can influence drug distribution will be described, as will certain methods for studying drug penetration, especially those with particular relevance to the investigation. Finally, a brief description of the experiments undertaken and why they are valuable for studying drug penetration shall conclude the introduction. Cancer Cells Cancers are result of alterations in genes regulating cell growth and behaviour. Cell cycle research has been able to identify many molecules which have capability to drive and regulate the cell cycles. These are termed as cyclins, and with appropriate rise and fall during normal cell divisions, they regulate precise co-ordinating events that allow DNA duplication leading to cell division. This regulation guarantees maintenance of DNA fidelity and protects against a loss of genetic information. Research has established many direct links between disruption of cell cycle control and cancer. Most cancers are monoclonal, arising from a single cell that has accumulated key mutations leading to uncontrolled cell proliferation. Such mutations can cause gene function to be either enhanced or activated or lost, inactivated. Genes whose function becomes lost or inactivated in carcinogenesis are termed tumor suppressor genes. Genes whose function becomes enhanced are termed protooncogenes and their mutated form is an oncogene. Proto-oncogenes play an essential role in controlling cell proliferation and encoding growth factors, growth factor receptors, signal transducers, cytoplasmic regulators, and transcription factors. Tumor suppressor genes are also deactivated in cancer cells, leading to a loss of accurate DNA replication and control of the cell cycle amongst other normal cell functions (Osborne et al., 2004). This combination of changes in cells' DNA causes the formation of cancer and has many important and far-reaching implications. The development of cancer is a multi-step process characterized by repeated cellular insults resulting in the accumulation of mutations. Blood Vessels In any tumor mass, usually there are a huge number of blood vessels. Any tumor will have a stroma that will demonstrate a distinctive capillary network. As observed by Goldman (1907), there is a distinctive vasoproliferative response of the organ hosting a tumor. The normal pattern of angiogenesis is disturbed by the chaotic growth leading to a dilatation and spiralling of the affected vessels with remarkable capillary budding and new vessel formation. It must be remembered that the solid tumors are made up of neoplastic cells with blood vessels, stormal cells, infiltrating immune-competent cells, and a differentiated extracellular matrix. Tumor rapid growth leads to compression of tissue mass with pressure generated on different elements of the developing tumor tissue. Areas of cellular hypoxia and acidosis lead to neoangiogenesis to lead to rapid tumor growth. The process of neoangiogenesis incorporates host blood vessels, sprouting and growth of new blood vessels from the pre-existing ones and thus leads to leaky or malformed blood vessels. Solid tumor growth occurs by means of an avascular phase followed by a vascular phase. The avascular phase appears to correspond to the histopathological picture presented by a small colony of neoplastic cells that reaches a steady state before it proliferates and becomes rapidly invasive. The cells at the periphery of the tumor continue to reproduce, whereas those in the deeper portion die away. Malignant tumors can grow beyond the critical size of 2 mm at their site of origin by exploiting the host's pre-existing blood vessels. With rapid growth of the tumor cells, with peripheral extension, there is compression of blood and lymphatic vessel compression (Padera et al., 2004; Brown & Giaccia, 1998; Chaplin et al., 1987). The tumor cell proliferation creates these physiological characteristics, which in turn exacerbate the problem that certain areas of the tumor are avascular regions i.e. they are not supplied by blood vessels. This results in several diffusion gradients being established, including decreasing oxygen (resulting in hypoxic conditions), decreasing nutrients and ultimately decreasing drug concentration (figure 1). Other factors can also affect the diffusion and penetration of drugs into the tumour, which will now be described. (Taken from Minchinton & Tannock, 2006) Lymphatic vessels and extracellular matrix Lymphatics are present at the tumor periphery but not within the tumor mass. In solid tumours, functional lymphatics are often absent (Leu et al., 2000) or compressed by the invasive tumours. The heart pumps blood through the arterial network and the blood pressure forces fluid out of the arteries at capillaries and into the extracellular matrix, before the nutrients can enter the cells. Lymph vessels act to remove the interstitial fluid from between the cells in order to maintain the interstitial fluid pressure (IFP). The virtual absence of lymphatics within the stromal core of the solid tumor does not permit fluid reabsorption, and along with the above factors, create a progressive increase in tumor interstitial fluid pressure (IFP) from the periphery to the center of the tumor (Heldin et al. 2004). Thus it can be perceived that atypical vasculature, virtual lack of lymphatic vessels, tumor desmogenesis leading to fibrosis, collagen formation and contraction of the extracellular matrix (ECM) cause an increase in the interstitial fluid pressure (Heldin et al., 2004). Netti et al. (2000) showed that the composition of the ECM could also affect the movement of molecules from the blood vessels through the tumour, by preventing "the penetration of therapeutic agents" (Netti et al., 2000). As a direct consequence of the disorganised vascular structure in tumours, there is a concentration gradient of molecules through the tumour, meaning that cells distant (>100 microm) from functional blood vessels receive limited oxygen and other nutrients. This results in regions of the tumour being hypoxic (Coleman, 1998), leading to a gradient of decreasing cell proliferation with increasing distance from the tumour blood vessels (Tannock, 1968). Tissues in solid cancers generally have been demonstrated to possess extensive regions of hypoxia as opposed to the normal surrounding tissue (Vaupel 2004). Rapid proliferation of the tumor mass distances the cells from the oxygen-carrying vasculature has been attributed to this along with the formation of a distorted and irregular vasculature leading to inefficient and insufficient oxygen transport (Folkman et al. 2000). The degree of hypoxia is variable within a tumor mass leading to alteration in the metabolic process leading to accumulation of carbonic and lactic acids within that environment. This reduces the pH of the tumor tissues (Minchinton & Tannock, 2006). The gradient of molecules through the tumour is created not only by the disorganised and insufficient vasculature, but also by the increase in interstitial fluid pressure and the complexity of the ECM. If anticancer drugs cannot access all tumour stem cells at a sufficient concentration, the effectiveness of the drug will be reduced. This is because tumour stem cells (or clonogenic cells), if not killed by the anticancer drugs, could form the epicentre of a new tumour. As has been mentioned earlier, in the cell population of a solid tumor, there are cells which eventually grow out of the feeding vessels being supplied by virtually very distant blood vessels. These cells have the potential to be resistant to curative processes such as radio and chemotherapy for a number of reasons. Distant cells may be exposed to only low concentrations of anticancer drug due to limited penetration and access, caused by poor vascular network, increased IFP and complex ECM. Secondly, many anticancer drugs exert selective toxicity on cycling cells, meaning that rapidly cycling tumour cells are selectively targeted. Those cells which are remote from the blood vessels, with lower concentrations of oxygen and nutrients, proliferate and cycle much more slowly, meaning they have some resistance to the anticancer drugs. Finally, certain drugs are less active in hypoxic, acidic or nutrient deprived microenvironments (Brown & Giaccia, 1998). Remote avascular microenvironments of solid tumours therefore have several different reasons why their physiology makes them resistant to regular anticancer drugs. The extermination of tumour stem cells during treatment is paramount to curing the disease; if some stem cells are permitted to survive, the tumour can reform. The ultimate aim for any efficient anticancer drug is therefore one that can effectively penetrate solid tumour tissue and eradicate all tumour cells, clonogenic or otherwise (Phillips et al., 1998). Properties that influence drug distribution Drug distribution in tumour tissues will be dependent on three main factors as shown in figure 2: supply, flux and consumption (Minchinton & Tannock, 2006). Supply of a drug to a solid tumour tissue is dependent upon initial dose and the pharmacokinetics of the drug. Prior to introduction into human clinical practice, these pharmacokinetic properties of the drugs are measured. These parameters, such as volume of distribution (Vd), half-lives for disappearance from plasma, and clearance from the body are used to describe the behaviour of drugs (Minchinton & Tannock, 2006) and involve the relationship between drug absorption, distribution, metabolism and excretion with respect to time. Flux of drugs through the tissue can occur via transcellular pathways if lipid soluble, or via the ECM if water soluble, allowing for diffusion around cells. Penetration of tissues occurs by a net movement of fluid out of the blood vessels, eventually flowing back into the lymphatic system. As described above, solid tumours often lack functional lymphatics (Leu et al., 2000), leading to an increase in IFP, hindering molecule penetration of tumour tissues. As well as the role that tumour physiology plays in drug penetration, further physicochemical properties such as molecular weight, charge and solubility can affect rates of transmission through tissues. The successful penetration of a drug is heavily dependent upon its rate of consumption. Consumption can involve sequestration and preservation of the drug within tumour cells, cellular metabolism and binding to receptors in cellular membranes. Certain drugs have been developed in order to penetrate tumours efficiently, and to specifically target cells in hypoxic regions where the effectiveness of conventional chemotherapy would be compromised. This class of compounds are called bioreductive drugs, and are designed to destroy cells specifically within hypoxic microenvironments (Minchinton and Tannock, 2006) (Sartorelli, 1988). Bioreductive drugs are ideally presented via the vasculature as an inactive form of the drug and are activated in low oxygen environments by electron reductases (Phillips et al., 1998). Effective diffusion through the aerobic section of the tissue is therefore necessary for the bioreductive drug to reach the hypoxic microenvironment and to be reduced to an active cytotoxic form. As the drug diffuses out of the blood stream and through the tumour tissue cells however, the decreasing oxygen concentration causes an increase in metabolism of the anticancer drug. An equilibrium exists between penetration and metabolism; severely reduced metabolism results in too low concentrations of the active bioreductive and incomplete tumour tissue destruction, whilst too much metabolism renders too little of the drug left to effectively diffuse. Doxorubicin, the drug used in the investigation, is an anthracycline antibiotic. Although its workings are still unclear, it is thought to interact with the DNA strands by intercalating and then inhibiting Topoisomerase II. When the DNA strands have been separated, doxorubicin inhibits the enzyme and prevents it from replicating the DNA, therefore calling a halt on DNA replication. Doxorubicin has often been measured as having poor penetration through tumour tissue, due to DNA binding and uptake into acidic endosomes (Lee & Tannock, 2006). Kobayashi et al. (1992) have highlighted that the cytotoxicity of doxorubicin progressively decreases when tumor cell density increases. Moreover, pharmacologically, doxorubicin is less active in the acidic environment. There has been debate as to whether the cell density related decrease in cytotoxic activity is a result of the decreased pH is a rapidly developing solid tumor, since acidification of the medium could be a direct resultant of high cell density. Measurement of cellular drug levels revealed that with high cell density, cells would accumulate much smaller amounts of doxorubicin, indicating the drug concentration relative to cell density could be the major determinant of doxorubicin-induced cell killing (Kobayashi et al., 1992). It is clear that in order to achieve a therapeutic cell kill, the cytotoxic agents must reach tumors in optimal quantities, and to be able to do this, particularly in solid tumors, the agents must reach the cells through their imperfect blood vascular system, cross them into an avascular interstitium where there may be considerable avascular fibrous tissue planes, and penetrate multiple layers of disorganized tissues. Added to this, there might be reduced pH and very high cell density. Attention to alleviation of these factors through research in this area may reveal ways to improve drug penetration through multilayered cell population throwing new light into therapeutic efficacy of such agents. Extracellular pH has important roles to play in the cytotoxicity of anthracyclines, doxorubicin and epirubicin. Kleeberger et al. (1993) demonstrated that both of these drugs are less effective at lower pH. Extracellular pH is significantly lower in many solid tumors with rapid growth rate in comparison to healthy tissues. While considering doxorubicin therapy, the accumulation and toxicity of doxorubicin, which is a weak base, the various pH gradients through extracellular and intracellular spaces may determine pharmacokinetics. They demonstrated that doxorubicin accumulation and toxicity increased with increasing extracellular pH in both normal and low-pH dependent cells. "The difference in doxorubicin accumulation and cytotoxicity at the same extracellular pH was found to be dependent on the difference in the transmembrane pH gradient of the two cell types. As the cellular pH gradient differs between tumour and normal tissue" they concluded that this could serve as a basis for enhancing cellular drug uptake in either tissue type (Gerweck et al., 1999). Direct measurement of tumor pH through pH electrodes in tissues or tissue preparations has shown lower average pH in the tumors in comparison to healthy tissues. Gerweck et al. (1996) commented that the extent of ionization of a drug such as doxorubicin will ultimately be exponentially related to the pH of their milieu of action. The extent of ionization would in turn indicate the presence or absence of charge and hence the extent of lipophilicity. Therefore, slight differences in pH may markedly influence the ability of these drugs to pass through the cell membrane leading to variations in drug distribution (Gerweck and Seetharaman, 1996). They further have demonstrated that this becomes relevant to the pharmacokinetics of doxorubicin, that being a weak base. The principal barrier for the entry of a drug into the site of action is the cell membrane. Selective distribution of doxorubicin would hence, over a relevant pH range, vary according to the magnitude of pH gradient across the cell membrane. Therefore extracellular pH would be a determinant of doxorubicin distribution. Under basic pHe conditions, the ionized fraction of the drug confined to the extracellular space would predominate. When the pHe would approach the pKa of the drug, as expected, gradually increasing proportion of the drug would lose its charge leading to free diffusion of the fraction across the cell membrane. Intracellularly, the environment is relatively basic, and this would render most of the penetrated drug ionized leading thus an increased concentration of the drug in the intracellular compartment (Gerweck and Seetharaman, 1996). As indicated in the literature, the pH variations have important implications in the effectiveness of chemotherapy regimen induced cell kill. pH changes can determine the cytotoxic activities of anticancer drugs such as doxorubicin, which is weakly basic. In fact the pHe can be a determinant of doxorubicin transport across the cell membrane since uptake of drugs containing basic charged groups may be enhanced through passive diffusion at pHe that favours the nonionized form of the drug. Moreover, as expected the influence of pHe on in vivo activity of doxorubicin may be further complicated by the fact that the more acidic regions of the developing tumor would be located more distally from the blood vessels which are functional yet. Therefore, in a rapidly developing solid tumor where there are avascular fibrous tissue and more immature blood vessels from tumor angiogenesis, penetration of the drug doxorubicin may be poor (Tannock and Rotin, 1989). Methods for studying drug penetration Both in vivo and in vitro methods have been used to study drug penetration through tumour tissue. In vivo methods, have the advantage of nearly simulating the clinical environment and involve either using direct fluorescence or immunohistochemistry to detect drugs such as etoposide, mitoxantrone or doxorubicin (Henneberry & Aherne, 1992), or to detect fluorescent labelled antibodies to detect the effect the drug has on the cell. These methods helped to demonstrate that diffusion of drugs across solid tumour tissue was poor compared to that across healthy tissue. Whilst certain techniques such as window preparations - observing the developing tumour in vivo under varying physical conditions - have been successful in gleaning information concerning changes in the vascular network with relation to time and diffusion gradients of metabolites and molecules from tumour blood vessels to solid tumour tissue, in vivo studies have several disadvantages. For example, in vivo experiments require the use of drugs that have sufficient staining or fluorescence, or are radio-labelled, whereas most anticancer drugs, with a few notable exceptions, do not demonstrate sufficient staining, fluorescence, or radioactivity. Further problems with in vivo experiments are the requirement for a host organism, reducing the ease of the experiment, and in vitro experiments offering microenvironments with drug distribution free from complicating factors such as pharmacokinetics. For the investigation, in vitro methods shall be used for analysing drug penetration in tumours, due to ease of use and an environment unperturbed by obscuring factors found in the solid avascular tumour tissue. In vitro multicellular models are increasingly used to measure drug penetration across tumour tissue samples. Whilst they do not have the advantages of direct duplication of the clinical environment and the possibility of observing the body's response to the cancer growth, they can be used to test anticancer drug penetration in human cells without factors such as pharmacokinetics and metabolism, which can vary in different species, playing a role in drug penetration. There are several different in vitro models available to assay anticancer drug penetration in tumour cells. The most commonly employed, due to ease of use, is the monolayer culture, a single layer of cells. However, the simplicity of the model backfires when being used for cancer research; as shown previously, many microenvironments exist in solid tumours (Chaplin et al., 1987) and concentration gradients of oxygen, nutrients and the anticancer drug create a range of environments for tumour tissue cells. Cells in a monolayer have no microenvironment and are not exposed to varying concentrations of a drug and are therefore not particularly useful when assessing the effectiveness of new and existing cancer drugs. More useful than monolayer culture in observing drug penetration are multicellular models. These allow for the diffusion and convection of molecules to be measured across a tissue culture, so that drug distribution to avascular tumour cells can be analysed. They also include the cell contact effect; tumour cells growing in contact with each other leads to differential gene expression for some cells, an effect that occurs to tumours in vivo. Multicellular spheroids are aggregations of tumour cells, forming a sphere in which many characteristics of solid tumours are established, ranging from an ECM to concentration gradients of nutrients and cell proliferation, decreasing towards the centre of the spheroid (Sutherland, 1988). Although not necessarily a complete mirror of the geometry of cancerous tissue, they have nonetheless been very useful in describing the penetration of certain drugs used in the fight against cancer (Erlanson et al., 1992). Another method of in vitro analysis and the one used in the investigation was multilayered cell cultures, or layers (MCCs/MCLs). These are layers of tumour cells grown on a permeable membrane for support and to allow penetration. Originally created by Wilson and colleagues (Minchinton & Tannock, 2006), Cowan et al. (1996) developed a method of studying drug penetration by assaying the ability of drugs to cross a multicellular layer, which is in turn supported by a microporous membrane. Standard systematic methods, such as high performance liquid chromatography, can be used to measure drug concentration and to calculate the penetration of the drug with relation to time. In the investigation into the penetration of the anticancer drug, Doxorubicin, the Transwell apparatus will be used. This utilises a top chamber, containing a donating reservoir with the anticancer drug, and a bottom chamber filled with liquid media, between which is the MCC. Drug diffusion between the two chambers across the MCC is measured as a function of time. Techniques such as mass spectrometry or high performance liquid chromatography can be used to quantify the drug concentration within the final media. In the investigation, MCCs will be used as an analytical method for testing the diffusion and penetration of doxorubicin across a tumour tissue. Because Epirubicin is a stereoisomer of doxorubicin, it can be used as an internal standard. Variables such as pH, cell number and cell type can be tested in order to get a fuller picture of drug penetration into avascular tissue. To refute or substantiate the effects of variavle pH environments, different pH gradients will be used to mimic the relative hypoxic tumour microenvironment and its effect upon the penetration of doxorubicin, whereas MCF-7 cells and 3T3 cells will be used, sometimes in combination, so as to create the cellular composition of tumour more accurately; 3T3 cells are fibroblast cells that synthesise and maintain the extracellular matrix of cells. Materials and methods Test Compounds Doxorubicin was purchased from Sigma (Sigma Aldrich, Poole, UK) and Epirubicin which was used as an internal standard were provided by I.C.T drugss banks (University of Bradford, UK). Both Doxorubicin and Epirubicin were dissolved in DMSO and stored at - 20 C Doxorubicin was dissolved in sterile water and stored at -20 C. All chemicals used were of analytical grade (Sigma) and all solvents were HPLC grade (Fisher Scientific, Loughborough), UK) Monolayer Cell Culture The human breast cancer cell line and a line of fibroblast cells, MCF-7 and 3T3 were obtained from the ICT cell culture bank (University of Bradford, UK). RPMI-1640 Complete Medium (Sigma - Aldrich, Poole, UK), supplemented with 10% foetal bovine serum (FBS) (Costar), 1mM glutamine (Sigma - Aldrich, Poole, UK) and 2mM of sodium pyruvate (Sigma - Aldrich, Poole, UK). Cells were grown in T75 flasks and allowed to grown to between 70-80% confluence after which they were washed in Hank's balanced salt solution (HBSS) and lifted using 5ml 0.25%/ ethlyen3eiaminetetracetric acid (EDTA) solution (Sigma - Aldrich, Poole, UK) for 5mins at 37 C. Detached cells were resuspended in 10ml of fresh medium. The numbers of cells in suspension were determined by placing a 10l sample of the cell suspension into a haemocytometer chamber and taking a mean of cells counts taken from five grids of the haemocytometer chamber. Cell numbers were expressed as (mean cell count) x 104 / ml medium. A few drops of the resuspended cell solution was then reseeded in T75 flasks (Corning) with 20ml of fresh media for routine maintenance and incubated at 37C in 5% CO2 humidified atmosphere. Seeding of Transwell Apparatus A total of 2.5 x 105 cells in 200l of RPMI-1640 culture medium were added to the top chamber of Transwell-COL plastic insert (Corning Costar, High Wycomber UK). The top and bottom chambers were separated by collagen-coated, microporous membrane. Transwell vessels were incubated at 37 C for 3 hours to allow cells to attach to the membrane. Once seeded, the upper chamber of the Transwell apparatus was filled with 0.5ml complete medium, and the lower chamber was filled with 2 ml of complete medium, so that they were just over flowing in both the chambers. Cells were incubated at 37C in an atmosphere containing 5% CO2 for up to 4 days with daily changes of medium in both upper and lower chambers. Drug Penetration Assay Medium was removed from the top chamber of the Transwell and replaced with 100 l of medium (phenol red-free RPMI-1640 medium supplemented with 10% foetal calf serum). The Transwell was then inserted into one well of a 24-well plate containing 600l of medium incubated at 37c At various interval time intervals there after 290l was removed from the bottom chamber and added to 1ml of acetonitrile, mixed and stored at - 20C until required for analysis. The bottom chamber was then replaced with fresh 290ul of fresh medium. This procedure was repeated throughout the duration of the experiment. At all stages of the process, medium in the bottom chamber was agitated using a small magnetic stirrer. In the case of MCF-7 drug penetration was assessed on days 1, 2, 3 and 4 of the growth curve in order to determine the relationship between the thickness of the multicell layer and the rate of penetration. In case of pH drug penetration was completed using pH 6, pH 7 and pH 8 and at days 2 and day 4 using MCF-7 and 3T3 cells independently. None of the experiments were repeated due to time constraints. The stability of test compounds under different pH's as be described below. pH Stability Test Phenol Red Free medium pH was adjusted using hydrochloric acid and sodium hydroxide. The Phenol Red Free Medium was adjusted to pH 6, 7 and 8. 475l of the adjusted medium was placed in eppendorf tubes and a further 50l of Doxorubicin in DMSO of 100mM was added. At 20 minute intervals until 120 minutes, samples of 50l were taken and placed in 100l of Acentonitrile and injected straight into the HPLC machine. Sample Analysis High purity HPLC grade solvents (Fisher Scientific, Loughborough, UK), analytical grade chemicals (Sigma Chemical Co. Ltd. Poole, UK) and triple distilled water were used throughout. Each 10 ml sample taken for analysis was added to 290 ml of fresh culture medium and doxorubicin, extracted by solid phase extraction (SPE). Each SPE cartridge C18-(EC), 50mg, was primed by adding 1ml methanol followed by 1ml of 0.02% formic acid in to the cartridge. The analytical sample (300 ml) was added to each cartridge which was washed by adding 1ml of 0.02% formic acid solution, then dried under vacuum. Doxorubicin was eluted from the cartridge using 1.5ml isopropanol:methanol (3:1) and the effluent evaporated to dryness in a centrifugal evaporator at room temperature. The sample was reconstituted in 30 l of mobile phase A and transferred to glass vials for injection into the HPLC. Chromatographic analysis of doxorubicin used mobile phase A comprising 90%5 mM ammonium formate, adjusted to pH 3.5 with formic acid, 10% acetonitrile and mobile phase B comprising 40% 5mM ammonium formate, adjusted to pH3.5 with formic acid, 60% acetonitrile. The mobile phases were mixed in a ratio of 60% A - 40% B. Samples were separated using a using a Column: C18 (HIRPB-Hichrom) 250mm x 4.6mm. Samples were injected (50 l) using a Waters Acquity Separation Module and the total run time was 10 minutes . HPLC analysis was performed at a flow rate 1.5 mL/min. Doxorubicin and epirubicin were detected with the use of a scanning fluorescence detector. The excitation and emission wavelengths were 480 nm and 550 nm, respectively. Read More