Cancer usually involves the disruption of the normal processes that regulate cell fate decision and homeostasis. This can either be in the form of disrupted cell signaling mechanisms or mutations in the genetic make-up of the cell. Developing systems that will specifically direct and deliver drugs to particular regions of the cell will prove to be of great potential in the treatment of various cancers that will eventually lead to better prognosis and patient outcome. The minute sizes of nanoparticles and their aptitude to overcome both physiological and biological obstacles has made it possible for them to pass through narrow-sized capillaries and subsequently be taken up by cells. This can lead to their possible accumulation at target sites were they can be used to visualized tumors and also observe their desired therapeutic effects on the tumors. Compared to other conventional methods of cancer treatment, such as monoclonal antibodies conjugated to cytotoxic compounds, important advantages in using these nanoparticles as drug delivery systems have been noted due to their ability to transport therapeutically relevant amount of drugs and also, their surfaces can be deliberately modified in order to specifically affect biodistribution or conjugate specific ligands that will ensure that they will be specifically directed to target tissues reducing unwanted side effects by the healthy cells of surrounding organs.
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Tumor onset and progression has usually been linked to the disruption of some crucial signaling pathway such as the Notch signaling pathway. The Notch signaling pathway may be one of the most important signaling mechanisms involved in animal development. This signaling mechanism is effectuated through a series of membrane bound ligands and receptors called Notch ligands and receptors respectively.
Disruption of the Notch signaling pathway has been seen to be related to the development and the progression of certain solid tumours such as cervical, head and neck, endometrial, renal, lung, pancreatic, ovarian, breast and prostate carcinomas. Notch has been shown to be involved in the passage of differentiated tumor cells from an epithelial morphology to a highly invasive and migratory phenotype a process also known as epithelial-mesenchymal transition (EMT), thereby contributing to metastasies of the primary tumor.
The striking relationship between Notch and some aspects of tumor progression definitely places the Notch pathway in the spot light as an alluring therapeutic target. Due to the fact that the Notch signaling pathway has been found to be involved in cell-fate decisions such as the self-renewal of adult stem cells, tissue renewal in the gut and lumen and the differentiation of progenitor cells, the unspecific use of Notch inhibitors such as γ-secretase like DAPT, as potential therapies in cancer treatment will lead to the production of multiple unwanted side effects on these systems.
In this study, we shall seek to specifically target the Notch signaling pathway as a clear-cut targeting of Notch with γ-secretase inhibitors to cells of the primary tumors will maybe lead to a reduction of tumor metastasies associated with Notch and subsequently also reduce unwarranted side effects that are otherwise associated with its unspecific inhibition. This proposed method of specific targeting shall be achieved by the use of mesosporous silica nanoparticles conjugated with the biospecific targeting ligand folic acid (FA) and DAPT.
Folic acid has generally been considered as an effective targeting agent to be conjugated to nanoparticles. The folate receptor has been found to be up regulated in numerous cancers such as breast, cervical and prostate cancer. Folic acid is a very attractive targeting ligand due to its short side chains, its small size and its high affinity for its receptors facilitates the internalization of nanoparticles that are functionalized with it. Functionalizing mesoporous silica nanoparticles with FA as targeting ligand will ensure that not only are the particles taken up by target cells but also they will be delivered to tumor sites.
We shall also seek in the course of this study to investigate how characterizing and optimizing mesoporous silica nanoparticles structure and chemical nature will improve the cellular uptake and targeting potential of the nanoparticle. Does particle surface modification affect its biodistribution, protein absorption and drug releasing capacities? What targeting ligands and their relative densities can be functionalized to mesoporous silica particles without affecting their uptake by the target cells? What are the drug releasing potential and particle degradation kinetics of differently modified mesoporous silica nanoparticles under particular conditions such as pH, and the presence or absence of serum? In order to answer the above questions, particles of various designs, ligand densities, surface chemistry, charge, size and different drug loads will be tested in cell culture systems to measure cellular uptake, particle compartmentalization and intracellular routing.
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Mammalian breast carcimoma cells (MDA-MB-231, T47D, SK-BR3 and MDA-MB-468) and cervical cancer cells (HeLa) served as in vitro cancer models used during the course of this study. They were chosen based on their over-expression of folate receptors on their surfaces and also on their high specific uptake of folic acid conjugated nanoparticles.
Human embryonic kidney epithelial cells (HEK293) served as a negative control for this study as they posses relatively less folate receptors on their surfaces and also, their uptake of FA conjugated MSN was significantly lesser than the above four cancer cell lines.
For protein absorption studies, nanoparticle uptake and ligand density studies, twenty-five differently functionalized particles were used: FA, MTX, SiO2, PEI, PEG, GA, thiol, NH2-PEG, hydrophobized particles, glucose, succinylated particles, dextran, streptavidin, sugar acid 2.5%, sugar acid 1%, sugar acid 5%, C16 hexa acid, C6 diacid (adipic) and C10 diacid (sebacic), FA 0.2% wt DMF, FA 0.2% wt THF, PEI soaked in MES, PEI soaked in DMF, PEI soaked in THF and FA 0.2% soaked in MES.
Review of Literature
Overview of the Notch signaling Pathway
The Notch signaling pathway is one of the most important pathways involved in the normal animal development processes. It plays a crucial role in the regulation of cell-fate decisions and is an important mediator in cell-cell contact processes. It also has a general role in the continuous renewal of tissues such as those lining the gut. Notch has been observed to promote differentiation in a certain population of cells while maintaining others in an undifferentiated state a process known as lateral inhibitation. Aberrant Notch signaling can cause diseases such as cancer and other genetic disorders like Spondylocostal dystosis, Tetralogy of fallot, Syndactyly, and familial aortic diseases. The Notch 1 receptor type has been found to be upregulated in T-cell acute lymphoblastic leukemia (T-ALL) and is being reviewed as a potential therapeutic target for this disease. Simply put, the Notch signaling cascade begins with the binding of a ligand to the receptor and upon ligand biding, the receptor undergoes two successive cleavages S2 and S3 releasing the intracellular domain of the receptor (NICD) which translocates to the nucleus and activates targets genes. Notch signaling activates a series of downstream target genes that play a critical role in differentiation, apoptosis, proliferation and stem cell maintenance depending on the cell type and tissue. The principal actors in this pathway are the Notch ligands and receptors.
Notch and Cancer (Does it have a role?)
Too broad focus on Notch in cancer with specific focus on breast cancer ! background neees to be relevant for your aims.
Activation mechanism (gamma secretase complex - druggable with GSI, DAPT)
Problem cell fate regulator in most tissues - side effects.
Try to make it more physiological and les mechanistical as the mechanistic part of your thesis will be the characterization of the nanos.
Volume 2, 4th edition. Academic Press., 2006. p. 289-293). Intriguingly it has been demonstrated that Notch can crosstalk with other signaling pathways such as the TGF-β through Smad3 (Blokzijl et al., 2003), Wnt, JAK/STAT, Ras/MAP kinase, and cellular hypoxia.
Structure of Notch signaling pathway.
Nanoparticles as drug delivery systems in cancer therapy.
Nanobiotechnology can be defined as the use of nanomaterials in biomedical applications such as the detection, diagnosis and treatment of human cancers as well as other diseases. Nanoparticles are nanomaterials that range in size from 1 to 1000nm. Nanoparticles can be divided into two groups depending on the materials from which they are made up of: organic and inorganic nanoparticles. Examples of which are liposomes, dendrimers and micelles. They are composed of organic molecules that make up the major building material of the structure.
Inorganic nanoparticles are those that contain a typical core /shell structure. This core can be made up of metals (iron oxide, gold, silica and Cadmium/Selenium) that confer the nanoparticles their characteristic fluorescence, optical, electronic and magnetic properties. The outermost layer or shell is usually composed of metals or organic polymers that help protect the core from chemical interactions with the external environment. It can also sometimes be used as a substrate on which biomolecules such as antibodies, proteins, oligonucleotides and other molecules such as fluorosphores and drugs can be conjugated and this way can be used for imaging purposes, tumour diagnosis and also as a drug delivery system.
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Cancer is one of the most challenging diseases of all times and the treatment usually involves the use of highly invasive techniques such as chemotherapy and radiotherapy. Also, some of the drugs utilized in cancer therapy posses a very narrow therapeutic index and can cause damages to surrounding healthy cells and also serious side effects. Detection of the disease at an early stage has also proven to be problematic due to the fact that diagnostic methods such as mammography sometimes lead to false positive or negative results which can prove fatal. Clearly, there is a need for an efficient drug delivery and diagnostic system that will ensure safe and adequate delivery of anti-cancer drugs directly to tumour sites and which also can be used to detect the tumour in vivo to facilitate tumour resection.
During the development and progression of cancer, certain genetic mutations in tumor cells often leave molecular signatures. This signatures can be in the form of the overexpression of certain proteins and receptors which can then be used as biomarkers for tumor diagnosis and therapeutics thus nanoparticles conjugated with antibodies and ligands targeted against over expressed proteins or receptors can be used potentially as a sensitive and specific way to track and monitor the progression of cancers and also measure and observe the effects of therapeutic treatments.
A drug delivery system is one that should be able to transport biologically relevant agents and deliver them to a specific site and at a controllable rate. The principal function of such as system is to monitor drug dosage and the duration of the effect of the drug without harmful effects to the patient.
For a nanoparticle to be considered as an adequate drug delivery system (DDS), it must possess the following characteristics:
It must be possible to attach targeting ligands to the outermost layer of the nanoparticles to ensure that they will be adequately taken up by their target cells by receptor mediated endocytosis.
They must show a high level of specificity to their target cells.
They must exhibit low cytotoxic effects.
Prevent early leakage of drugs from the carrier system before it is taken up by the target cells.
Biodegradable and not accumulate in the kidney.
Incorporate therapeutically relevant amount of drugs.
Portray low levels of protein absorption that might influence the targeting ability of the particles due to the fact that the targeting proteins become inaccessible and they may also be recognized as foreign objects by the cells of the immune system.
2.1 Mesoporous silica nanoparticles and their future potential as drug delivery systems.
Mesopoorous silica particles were first synthesized by Kuroda and co workers in 1990. They reported that the synthesis of mesoporous silica was composed of a homogenous pore size distribution from layered polisilicate kanemite. In 1992, a significant discovery in silica nanoparticle synthesis was reported by researchers of the Mobil Corporation by the discovery of the M41S family of materials. These structured materials are divided into 4 main groups: disordered rods, MCM-41, MCM-48, and lamellar phase. Only MCM-41 and MCM-48 have been observed to be thermally stable and has been used in many applications.
Recent studies have demonstrated the potentials of mesoporous silica particles as promising drug delivery systems which can be attributed to several unique characteristics in the structure of the nanoparticles such as its large pore volume, its narrow pore size distribution and its high specific surface area which makes its possible to easily incorporate large amounts of payload to it, by surface functionalization of molecules of interest. Also, the core of this nanoparticle is composed of amorphous silica which is biodegradable, non-toxic and can be readily excreted in urine. The particle diameter is perfectly tunable and can range from about 50-300nm permitting their endocytosis by the cell while minimizing particle cytotoxicity. The structural nature of the particle predisposes it to several functions such as its ability to simultaneously carry fluorescent dyes, peptides, antibodies, oligonuclotides, ligands, for targeting a specific cell population and drugs for therapeutic purposes. The ability to functionalize MSN with all these biologically relevant agents makes it a good candidate for a huge variety of biomedical applications such as cellular and tumor imaging and drug delivery.
Tumor imaging and drug delivery are two recent areas of research were the potentials of MSNs as effective drug delivery and tumor imaging systems are being evaluated. Cancer drugs usually induce severe unwanted side effects due to their non specific uptake by healthy cells that are adjacent to tumor cells. There is thus a need for adequate cancer drug delivery systems in order to minimize this unspecific uptake problem. Also, tumor visualization is often difficult as the tumor sites are often in inaccessible areas therefore, developing fluorescent MSNs conjugated with a targeting ligand will ensure that tumors can be imaged by fluorescent techniques which can clearly lead to the improvement of surgical removal techniques of tumors. The method of targeted drug delivery is one that is being evaluated for the use of MSNs as cancer targeting and drug deliver nanosystems.
Targeted drug delivery can be attained through passive targeting via the enhanced permeability and retention effect (EPR) or actively via receptors. Tumor growth is usually associated with rapid angiogenesis to serve the fast growing cancer tissues which often lead to the formation of weird vessels that have a leaky and defective architecture lacking a basement membrane and a damaged lymphatic drainage system. This leaves room for large drug carrier systems to selectively reach tumor sites while avoiding normal healthy tissue. The defective lymphatic drainage system will impede their passage into the circulation system thus promoting their retention within tumor sites.
Active targeting requires the use of targeting ligands that are functionalized either on the outermost surface of MSNs or in the interior of its pores. This is based on the fact that tumors usually over express certain peptides and receptors on their surfaces making it easier to specifically target cells bearing this over expressed proteins. The targeting ligand promotes the uptake of the conjugate to be taken up by receptor mediated endocytosis and because glycoproteins have limited or no ability to eliminate MSNs that have entered the cells by endocytosis, this targeting methods provides a means of reducing multi-drug resistance.
Folate receptors (FR) are being viewed as interesting candidates in receptor mediated endocytosis approach. They have been seen to be over expressed in tumor cells of several malignancies such as breast, ovarian, cervical and lung cancers. Confocal microscopy together with flow cytometry analysis clearly demonstrate that particles conjugated with FA were selectively taken up by cancer cells rich in FR than non-cancerous cells with low folate receptor expression. These results clearly demonstrate the potential of the FR in receptor mediated endocytosis.
Another interesting targeting ligand that is being evaluated for targeting purposes is metothrexate (MTX) that is a structural analogue of FA. Curiously enough, metothrexate has been used as an anticancer drug for decades. Its mode of function is to inhibit the enzyme dihydrofolate reductase which is important in DNA synthesis. It can thus simultaneously act as a targeting ligand and an anticancer drug. Flow cytometry analysis of tumor cells expressing high levels of the FR on their surfaces and non cancerous cells with low levels of the FR incubated with MTX-PEI functionalized nanoparticles demonstrate that not only are these particles actively taken up by the cell, but also they have the ability to
Particle size and surface chemistry of MSN are key determinants in the fate of the particles in biological systems and also play key roles in the protein absorption and particle aggregation properties of the particle.
In general, mesoporous silica nanoparticles are synthesized by mixing tetramethoxysilane with 3-aminopropyltrimethoxysilane (APS) under inert atmospheric conditions and an alkaline solution containing a structure directing agent is added to the above mixture. The overall solution is stirred overnight at room temperature and the structure directing agent removed by ultrasonication in an acidic alcoholic solution.
Polyethylene imine (PEI) is grown unto the surface of the mesoporous silica particle by hyperbranching surface polymerization. The principal function of PEI is to protect particle payload from enzymatic degradation during the drug delivery process. It can also increase the bioavailability of a drug that is otherwise poorly soluble and permeable. It has a large amount of amino terminal groups that can be used for the covalent attachment of functional groups such as targeting ligands, fluorescent dyes or even drugs. Mesoporous silica nanoparticles that are coupled with PEI contain a highly positive surface charge density that can be modified for example y succinylation to give negative or neutral surface charges.
2.2 Functionalization of MSN
MSN surface conjugation can be effectuated via three procedures: grafting, imprint coating and co-condensation.
Co-condensation is a direct method of synthesis whereby the silica precursors are incorporated into an alkaline aqueous solution during the condensation step. Examples of functional groups that can be functionalized to MSN by co-condensation are: aminoalkyls, ureidoalkyls, allyls, cyanoalkyls and mercaptoalkyls.
This process involves the covalent functionalization of MSNs with organotrialkoxysilanes or organotrichlorosilanes. This method can be disadvantageous in that materials functionalized in this way have an inhomogeneous surface coverage. Silanols found on the exterior and inner pore openings are more kinetically stable than those located in the interior of the pores. This method of conjugating MSNs can also be advantageous in that functional groups can be selectively functionalized at the external surface.
Aims of the project
The overall goal of this project is to specifically target the Notch signaling pathway with mesoporous silica particles conjugated with a γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) to observe how targeting this pathway reduces tumour metastasies in breast cancers and also using differently conjugated mesoporous silica particles as drug delivery systems to tumour sites.
Specifically, this project aimed to:
Analyse by FACS nanoparticle uptake in breast cancer cells / primary breast cancer cells.
Analyse by FACS apoptosis in breast cancer cells induced by the uptake of nanoparticles conjugated with metothrexate at three time points.
Utilize luciferase reporter assays to determine the effect on Notch signaling on targeting MDA-MB-231 cells with DAPT nanoparticles.
Study the in vivo targeting of breast tumours with DAPT functionalized particles.
Study protein absorption on the drug carrier using differently functionalized particles.
Materials and methods
This study was conducted with the use of Human mammary cancer cell lines of epithelial origin (MDA-MB-231, T47D, SK-BR3 and MDA-MB-468), cervical cancer (HeLa cells) and the kidney epithelial cell lines (293).
MDA-MB-231 cells were first extracted from a patient in 1973 at the M.D. Anderson Center. They present an epithelial morphology with a spindle shape and at the in vitro level, exhibit an invasive phenotype and can form mammary fat pad tumours in mice.
MDA-MB-468 cells were isolated from the pleural effusions of a patient suffering from metastatic adenocarcinoma of the breast in 1977 at the M.D. Anderson Center. These cells are of epithelial origin and display aggressive lymphatic metastasies when grafted to nude mice for cancer studies in vivo and thus permit the recreation of a mouse model exhibiting metastatic adenocarcinoma of the breast.
HeLa cells were first isolated from a patient in 1952 suffering from glandular cancer of the cervix. They are human epithelial cells transformed by the human papillomavirus 18 which adhere to surfaces and maintain contact inhibition which is a typical characteristic of oncogenic cells. They are immortalized cell lines with a typically elongated structure, exhibiting high levels of telemorase activity, hemyzygously expressing glucose-6-phosphate deshydrogenase and can grow rapidly and propagate at an unlimited number of times which makes it adequate for cancer research at the in vitro level.
Human embryonic kidney 293 cells were derived from transformed human embryonic kidney cells with a sheared adenovirus 5 DNA in tissue culture by Alex Van der Eb and transformation of the cell line was performed by Frank Graham in the 1970's.
Drug delivery potential of Mesoporous Silica Nanoparticles.
In this study, we sought to ascertain the potential of mesoporous silica nanoparticles as effective vehicles for the intracellular delivery of drugs to target cells. This was done by measuring the differential uptake of folic acid conjugated nanoparticle in four different breast cancer cell line (MDA-MB-231, SK-BR-3, T47D and MDA-MB468), measuring the targeted delivery of MTX and FA conjugated nanoparticles to HeLa and 293 cells, quantifying the rate of apoptosis in HeLa and 293 cells induced by the anticancer drug MTX conjugated with a mesosporous silica nanoprticle.
Targeting the Notch signaling Pathway in MDA-MB-231 cells with DAPT conjugated nanoparticles; a γ-secretase inhibitor.
In order to target the Notch signaling pathway with DAPT functionalized nanoparticles, this experiment was performed along a five day strip which entailed three phases: Transfection of cells with 12xCSL and β-gal plasmids, targeting of cells with DAPT functionalized nanoparticles and measuring Notch signaling activity by luciferase reporter assays.
Transfection is a process whereby DNA of often foreign origin is introduced into mammalian cells by non-viral methods and this consist of the temporal formation of pores at the surface of the cell membrane which will permit the uptake of the DNA of interest by the host cell. The DNA of interest is often first incorporated into a structure called a plasmid (an autonomously replicating circular DNA molecule of bacterial origin) by recombinant DNA techniques and subsequently introduced into the target cell were the fragment of DNA of interest can be incorporated in the genetic makeup of the cell and bring about a change in the behavior of the host cell (e.g. Transformation of a normal cell into a potentially oncogenic one). Transfection of animal cells can be achieved by a variety of methods including, calcium phosphate, dendrimers and liposomes the latter of which will be used throughout this study.
Transfection was performed using the Lipofectamine 2000 reagent kit (Invitrogen life technologies, USA) that uses a liposome formulation of the polycationic lipid 2, 3-dioleyloxy-N [2(sperminecarboxamido) ethyl]-N, N-dimethyl-1-propanaminium and the neutral lipid dioleoyl phosphatidylethanolamine (DOPE) in membrane-filtered water trifluoroacetate (DOSPA) and opti-MEM reduced serum media. The plasmid DNA and lipofectamine volume varied according to the diameter of the culture dishes and the confluence of the cultured cells as stated in the lipofectamine 2000 transfection protocol of MDA-MB-231 cells. According to the protocol, the plasmid DNA volume was 1μg, the volume of the lipofectamine reagent varied between 2.5μl-4.5μl and the number of cells per well was 2.5x105. However, the volume of some of the reagents in this study was slightly modified from the manufacturer's instructions (Invitrogen life technologies, USA). For each well of a 12 well Cellstar plate (Greiner Bio-one, USA), 2μg of 12xCSL and β-gal plasmid DNA, 4μl of lipofectamine 2000 reagent and 200μl of opti-MEM reduced serum media (Invitrogen life technologies, USA) was used. Opti-MEM is a modified version of Eagle's Minimum Essential Modified Media, buffered with HEPES and sodium bicarbonate and supplemented with hypoxanthine, thymidine, sodium pyruvate, L-glutamate, trace elements and growth factors. It does not contain polyanionic fators present in normal media that will otherwise inhibit lipofectamine transfection of the plasmid DNA. These polyanionic components supposedly compete for binding sites on cationic lipid vesicles with polynucleotides and can thus inhibit transfection. They may also alternatively or simultaneously bind to and neutralize the charge on the cationic lipid / polynucleotide complexes making them less subject to be taken up by the cell surface.
Prior to the transfection phase of this experiment, 6 wells of the 12 well plate were coated with 500ml of Recombinant protein G (50μg/ml in Phosphate Buffered Saline) (Sigma-Aldrich co, USA) and the solution was incubated at room temperature for 24 hours. The recommended blocking time of the plates with 10mg/ml of BSA in PBS at room temperature was changed from two hours to one hour. The coated plates were washed three times with PBS solution and blocked with 500ml of 10mg/ml of BSA diluted in PBS solution for one hour at room temperature on a shaker and washed again three times with PBS after the blocking step. Delta-like ligands were obtained from the media that 293 DLL cells were grown it. 1ml of Dubelco's Modified Eagle's Media supplemented with 10% fetal calf serum (FCS), L-glutamine and penicillin/streptomycin in which these cell lines were previously cultured was then added to the newly washed plates and incubated at room temperature on a shaker for two hour. The recommended incubation time for this section of the experiment was set in between 2-4 hours hence this incubation time period was respected. During the two hour incubation period, the transfection phase of this experiment was performed using a 12XCSL-Luc and β-gal plasmid DNA. It entailed firstly, diluting 2μg of both 12XCSL-Luc and β-gal plasmid DNA in 100μl of opti-MEM solution in an eppendorf tube. 4μl of lipofectamine 2000 reagent was diluted in 100μl of opti-MEM solution and incubated for about five minutes at room temperature and then the solution containing the lipofectamine 2000 reagent plus opti-MEM solution was added to the DNA-opti-MEM solution to a total volume of 200μl and incubated for a total time of 20 minutes as stated in the protocol in order to allow for the DNA-lipid complexes to form.
In order to optimize the transfection efficiency and reduce cytotoxic effects of the lipofectamine 2000 reagent, it was advisable to vary the ratio of the DNA: lipofectamine 2000 between 2: 3 and 2: 4 and also to use very highly confluent cells which in this case was about 70-80% confluent. About two days before the transfection process, a fresh batch of MDA-MB-231 cells were grown in a humidified incubator at 37ËšC, 95% O2 and 5% CO2 in a 10mm Cellstar culture plate (Greiner Bio-one, USA) in Dubelco's Modified Eagle's Media (DMEM) supplemented with 50ml of 10% FCS, 200mM of 5mL L-Glutamate, 4000 Units/mL of penicillin and 10,000mg/mL streptomycin antibiotics. The cells were allowed to reach a confluency of about 80-90% after which they were harvested by trypsinising them with EDTA, centrifuging them in a centrifuge for 4 minutes at 37ËšC at a speed of 1000rpm and then resuspending the cell pellets in 9.6mL additive free DMEM supplemented with 4.5mL of 10% FCS, 200mM of 450μl L-glutamate. 800μl of the cell solution was added to each well in the 12 well plate and 200μl of the DNA-lipid complexes was added to the cell solution to a final volume of 1mL and the cells incubated in a humidified atmosphere of 95% O2 and 5% CO2. In order to minimize the cytotoxic effects of the lipofectamine 2000 reagents on the cells, the transfection medium was removed after 6 hours of incubation and replaced with DMEM supplemented with 50ml of 10% FCS, 200mM of 5mL L-Glutamate, 4000 Units/mL of penicillin and 10,000mg/mL streptomycin antibiotics.
This experiment was performed in such a way that the cells were incubated in all the wells of a Cellstar 12 well plate (Greiner Bio-one, GmbH Germany) which was equally divided into 2 sections, the first serving as negative control contained whilst the other served as a positive control for the experiment. In the first section, the first row contained 3 wells of MDA-MB-231 cells that were not grown on Delta-like ligands whilst the others in the second row were grown on the ligands. This was to ensure that Notch signaling activity was enhanced in cells grown on ligands while Notch was maintained at lower levels in those cultured in the absence of the ligands.
Hind III, 215
Sph I, 221
Sal I, 233
Bam HI, 245
Nsi I, 256
Stu I, 5176
Sca I, 4679
Sph I, 1037
Sty I, 2300
Hpn I, 2794
Saa I, 2946
Kpn I, 2948
Saa I, 2936
Following a twenty four hour incubation period in an incubator at 37ËšC and at 5% CO2, part of the cells that served as positive controls were targeted with nanoparticles functionalized with a γ-secretase inhibitor, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylgly cine t-butyl ester (DAPT). The other part serving as a negative control were incubated with nanoparticles conjugated with folate and they served as control particles for this study. Gamma secretase is an important component of the Notch signaling pathway in that it catalysis the last proteolytic cleavage of the Notch receptor, releasing its intracellular domain (NICD) which then translocates to the nucleus and leads to the expression of Notch target genes. Therefore inhibiting gamma secretase will probably lead to a decrease in Notch signaling hence a reduction in cancer metastasies which is often associated with increased Notch activity in breast cancers.
Prior to the targeting process with DAPT and the control particles, 100μl of both nanoparticles were dissolved in 10mL of DMEM supplemented with 50ml of 10% FCS, 200mM of 5mL L-Glutamate, 4000 Units/mL of penicillin and 10,000mg/mL streptomycin antibiotics. The particles were then disaggregated by sonication in a water bath sonicator for fifteen minutes and this was to ensure that the particles were adequately taken up by the target cells. Old growth media was removed from the cell samples and 2mL of the media containing the particles was added to them. They were incubated at 37ËšC at 5% CO2 in a humidified incubator for another 24 hours after which they were lysed and Notch signaling activity was measured using the luciferase reporter assay method.
Measurement of Notch Signaling activity by Luciferase reporter Assays.
In this study, the simultaneous expression of the Notch target genes and the luciferase gene adjacent to it are used as a measure of Notch signaling activity in MDA-MB-231 cells by luciferase reporter assay method. This method is based on the fact that the luciferase gene is inserted upstream to Notch target genes, and under the influence of the same promoter, it is expressed at the same time as Notch target genes when the latter are expressed due to the activation of Notch signaling. The resulting product of the luciferase gene expression, luciferase catalyzes the oxidative carboxylation of luciferin resulting in the emission of light which can be detected using a luminometer.
After transfecting the cells with the 12XCSL and β-gal plasmids and incubating them with DAPT and control particles, the cells were lysed on ice for about 20 minutes using 1X cell lysis buffer. 1X cell lysis buffer was made by diluting 480μl of a stock solution of 5X cell lysis (Promega Corp, USA) buffer in 1920μl of deoinized water to a total volume of 2400μl. 200μl of the above solution was then added to all the wells containing cells and the volume which was used during the lysis process was different from the required volume of 100μl that was recommended for lysis on a 12 well plate. This volume was used in order to obtain substantial amounts of cell lysate substrates for the study.
3.3 Protein absorption
Transfection of MDA-MB-231 cells and Luciferase reporter assays