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A collection of reports, essays, and presentations I've worked on throughout the years. Some recent, and some from when I first made this website back in middle school! Enjoy :)
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A mock grant proposal written for my Biochemistry of Cancer course. Specific Aims
The goal of this proposal is to investigate ways to increase gold nanoparticle efficiency in targeting and treatment of leukemia. Gold nanoparticles (AuNPs) have been used in diagnostics and therapeutics involving many diseases due to their ability to target cells via antibody labeling while remaining biologically inert. It has been concluded that changes in the size of the nanoparticles can alter cell uptake of AuNPs (Chithrani, 2006), but not how to manipulate this property to enhance therapeutic efficacy in leukemia. We have chosen to use 6-Mercaptopurine (6-MP) because it is a popular anti-leukemia drug that has been demonstrated to be compatible with gold nanoparticles in targeting cancerous cell lines at an increased efficacy (Faid, 2023). The consensus seems to be that smaller nanoparticles are more successfully uptaken by cells, enabling them to more effectively target them, however, especially in therapeutic applications, the nanoparticles must be large enough to carry those molecules responsible for treatment. We hypothesize that spherical gold nanoparticles approximately 50 nm in diameter will be more effective at inducing apoptosis in leukemia cells than other sizes of nanoparticles or 6-MP alone, as it will maximize cellular uptake and circulation of the treatment throughout the body while effectively extending the short half-life of 6-MP which limits therapeutic applications. Data indicates that 6-MP decreases the survival of a breast cancer cell line when delivered using gold nanoparticles (Faid, 2023). This suggests that gold nanoparticles carrying 6-MP can be designed to similarly selectively target leukemia cells and deliver treatment to them. We have chosen to study the effect of cellular uptake as a means of inducing apoptosis using two representative leukemia cell lines in mice as a model system. Therefore, the specific aims of this proposal are:
Introduction In most instances, leukemia starts with the formation of abnormal white blood cells in the bone marrow (Leukemia - Cancer Stat Facts, n.d.). These cells grow inside the bone marrow in a rapid and uncontrolled fashion, quickly crowding out cells that would otherwise develop into healthy red and white blood cells or platelets (Professional, n.d.). Decrease in synthesis of new cells to replenish the blood results in less healthy cells being released into the bloodstream, decreasing oxygen transport efficiency throughout the body (red blood cells), and the body’s immune response (white blood cells and platelets) (Professional, n.d.). Unlike many other cancers, leukemia does not commonly form solid tumors, meaning surgical interventions cannot usually be utilized in treatment plans. Additionally, leukemia does not follow the stage system used to categorize other cancers (Stage I through IV, indicating severity and the extent to which the cancer has developed). Instead, leukemia is classified into four main types, determined by the type of abnormal cell present and the speed of progression: acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myelogenous leukemia (CML) (Professional, n.d.). 6-Mercaptopurine (6-MP) is a popular anti-leukemic medication which possesses a multitude of adverse effects and has a short biological half-life (Faid, 2023), resulting in its infrequent use. 6-MP is a purine antagonist, structurally analogous to adenine (Chong & Werth, 2019). If integrated into DNA, upon its activation it can cause apoptosis or DNA damage, in addition to its ability to inhibit purine synthesis or synthesis of ATP and GTP (Chong & Werth, 2019), all of which are necessary for cellular proliferation. Theoretically 6-MP would be very effective against cancerous cells because of this activity, however it also targets healthy rapidly dividing cells, such as those found in the bone marrow, which leukemia overpowers. Gold nanoparticles (AuNPs) loaded with 6-MP have been previously been demonstrated to decrease survivability of a breast cancer line (Faid, 2023). This supports research which suggests AuNPs have the ability to enhance peptide treatment efficacy and effectiveness (Navidi-Moghadam-Foumani et al., 2023). AuNPs have been used in many applications, including diagnostic and therapeutic methods focusing on a variety of diseases. Since their initial discovery, multiple synthesis methods have been hypothesized and developed: Faraday’s two-phase method, the Brust–Schiffrin method, Turkevich method, Frens method (Xia et al., 2016), and the Martin method (Hammami et al., 2021), each with their own impediments stemming from inconsistent size or shape of the AuNPs, or small experimental yields. The basic chemistry surrounding AuNP synthesis involves the reduction of AuCl4- ions to Au+ ions via citrate and the simultaneous oxidation of citrate into sodium acetone dicarboxylate, with the gradual increase in Au+ concentration eventually triggering the nucleation and growth of the AuNPs (Xia et al., 2016). Synthesis of uniform spherical AuNPs has proved the most experimentally challenging–especially when compared with AuNR, or gold nanorods–at sizes larger than 40 nm (Xia et al., 2016). This is of particular interest because spherical AuNPs have many advantageous characteristics (such as a high surface-to-volume ratio, high biocompatibility, and minimal toxicity) (Hammami et al., 2021), and studies have concluded maximum cellular uptake of AuNPs occurs at sizes of about 50 nm (Chithrani et al., 2006). New methodology based on the Frens method initially put forth in 1973 has been demonstrated to successfully synthesize spherical AuNps at sizes from 2 to 330 nm, with the size of the nanoparticles resulting from careful manipulation of reaction conditions (Xia et al., 2016). AuNPs can accumulate in solid tumors, so AuNP synthesis is often followed by therapeutic loading. Since leukemia does not form solid tumors, antibodies that recognize antigens present on leukemia cells must be conjugated to the AuNPs so that they can properly target the cells (He et al., 2021). There are many recognized leukemia-specific antigens–as well as antigens which are highly associated with, but not specific to leukemia–that appropriate antibodies can be designed to interact with, depending on the specific cell line (Anguille et al., 2012). Experimental Design Aim 1: Testing varying sizes of nanoparticles in their ability to deliver 6-MP and preserve its biological half-life. Preparing Nanoparticles Before the AuNPs can be tested, various uniformly-sized batches of spherical AuNPs must be synthesized. Following the modified Frens method put forth by Xia et al. (2016), gold nanoparticles should be synthesized in at least 15 sizes across the spectrum of possibility. The sizes of fragments will be representative of the entire range of possible sizes, however there will be a higher concentration of AuNPs in the immediate region surrounding 50 nm, because this size of AuNP has been previously demonstrated to experience the highest cellular uptake (Chithrani et al., 2006). Additional experimentation will be performed on a smaller subset of the range if AuNPs substantially smaller or larger than 50 nm (<20 nm or >80 nm) perform similarly or better than the 50 nm AuNPs in 6-MP binding or delivery, which is not anticipated based on current literature. Such specificity in the size of the nanoparticles across the entire range of possibility is due to the improved methodology utilized by Xia et al. to produce uniform batches of AuNPs even <5 nm and >40 nm, which was previously impossible (2016). After synthesis of the AuNPs, they will be characterized for size and shape using transmission electron microscopy (TEM) and dynamic light scattering (DLS) as a secondary control measure, to ensure the nanoparticles are of uniform and expected size as well as being free from aggregation (Chithrani et al., 2006), which can effect the movement of the AuNPs. Alternatively, an analytical ultracentrifuge outfitted with a multiwavelength extinction detector can simultaneously record optical and sedimentation data on AuNP samples, determining all means of dimensional characteristics, as well as aspect ratio and volume values as demonstrated by Wawra et al. (2018). Before the nanoparticles can be loaded with 6-MP, their surface must be properly prepared with antibodies targeting antigens present on the leukemia cells they will be targeting. This would be accomplished by experimental best practices to attach antibodies that target leukemia-specific antigens, likely using thiolated ligands to facilitate the binding (Anguille et al., 2012). Loading Nanoparticles with 6-MP and Testing Drug Release AuNP 6-MP can be prepared simply by coating the synthesized AuNPs under standard reaction conditions used by researchers because the chemical characteristics of the two components are amenable to binding to each other. Drug loading efficiency will be determined by X-ray photoelectron spectroscopy (XPS), as it has been shown to measure the bonding between AuNPs and antibodies (Mukherjee et al., 2007), and should be able to do the same for 6-MP. Microtiter plates have been established as appropriate vessels to conduct incubation of cancer cells with loaded AuNPs (Faid et al., 2023). Following this precedent, each of the synthesized AuNP sizes loaded with 6-MP will be incubated with a sample of leukemia cells from a well studied cell line–such as K562, considered an ideal model (Phelan & Szabo, 2019)–with proper controls. After an appropriate incubation time, optical density of the treated and untreated cells can be compared to determine the survival rate of the leukemia cells. This will demonstrate the ability of the AuNPs to decrease survivability of the cancer. Additional in vitro testing would then be performed utilizing a dialysis membrane, allowing for the measurement of release kinetics of 6-MP (Yu et al., 2019). Determining Biological Half-Life of 6-MP A murine animal model will be utilized for in vivo study of the 6-MP loaded AuNPs. After inoculation of the animal with the human K562 line and an appropriate incubation period, 6-MP loaded AuNPs can be introduced into the blood stream. The 6-MP concentration in blood serum can then be determined by taking samples at regular intervals determined by the results of the release kinetics experimentation, including proper controls. High performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS) can be used to measure the concentration, and the half-life can be mathematically modeled by pharmacokinetic modelling. Expected Results There should be a clear size-dependent effect on both drug loading and release properties. Small nanoparticles are believed to have higher drug loading efficiencies because of their larger surface-area-to-volume ratios, so it is likely the smaller sizes (~50 nm) will be demonstrated to have the best drug loading efficiencies. Incredibly small nanoparticles (<10 nm) have small extinction (or absorption) cross sections, which is understood to reduce their ability to function as drug carriers (Sibuyi et al., 2021). Larger nanoparticles may have slower drug release kinetics, which would effectively increase the biological half-life of 6-MP. If this is the case, it is possible an intermediary size of AuNP will have the best performance overall, even if it shows neither the highest drug loading efficiency nor the longest biological half-life of 6-MP. Anticipated Complications Issues with AuNP synthesis, such as inconsistent size distribution and batches that are nonuniform in terms of size and/or shape are possible, because of how delicate the process is. Care should be taken to optimize synthesis parameters and multiple controls for quality should be implemented. If nanoparticles of uniform size cannot be synthesized, methods to filter outliers should be implemented. Nanoparticle aggregation or non-specific drug interactions can alter drug release kinetics, however these adverse effects should be avoidable by implementing multiple replicates to the experiments performed. If AuNPs cannot be loaded with 6-MP, reconsideration of the protocol with consultation of existing literature should be performed. A different drug can be experimented with in place of 6-MP, such as Rituximab or another drug approved for leukemia treatment (Drugs Approved for Leukemia, 2024). Aim 2: Test the ability of gold nanoparticles to target leukemia cells by inducing apoptosis. Preparing Nanoparticles Nanoparticles should be prepared as outlined in Aim 1 above, focusing on those sizes of nanoparticles deemed most successful as a result of the first round of experimentation. A handful of sizes of nanoparticles will be prepared, to maintain size as a possible variable affecting the ability of the AuNPs to induce apoptosis. Multiple leukemia cell lines should be used, in addition to a noncanceous line as control. A good sampling of well-studied leukemia lines representing all four types of leukemia is present in the “Leukemia Cell Line Panel” (American Type Culture Collection, 2022). The AuNPs should be prepared with appropriate antibodies for each of the cell lines that will be utilized. Cell Culturing, Treatment, and Assessment of Apoptosis The leukemia cell lines will be cultured under standard conditions and given an incubation period before AuNP treatment is applied. The cells should be exposed to the varied sizes of AuNPs with appropriate controls in place. If deemed appropriate, additional experimentation can be performed exposing the cells to different concentrations of AuNPs of the same size, determined by the results of the size-varied experiment. After an established treatment period, multiple analyses should be performed on the treated cells to measure the level of apoptotic induction. Immunoblot analysis will be performed to confirm apoptosis (Mukherjee et al., 2007) by screening for key apoptotic markers–such as CD261, Bcl-2, BAX, and Caspases 3, 8, and 9 (Bio-Rad, n.d.)–and then quantified to determine any significance. The treated cells will also be analyzed by flow cytometry, using Annexin V to stain apoptotic cells. Expected Results As a result of treatment with AuNPs, there should be an increase in apoptosis visible in both the immunoblot and flow cytometry. The extent of apoptotic induction should be consistent between the two measurements, with specificity towards the various leukemia cell lines. There should be little, if any, cytotoxicity to the healthy cell control. Anticipated Complications In addition to the complications stated in Aim 1, non-specific binding of the AuNPs to off-target cells or insufficient targeting efficiency can occur. This can be avoided by validating targeting specificity of the antibody/antigen pairs and optimizing nanoparticle surface functionalization. Variation in different leukemia cell lines is also likely, which is why testing multiple lines and performing replicates of the experimentation is necessary. If one (or more) of the cell lines outlined in the “Leukemia Cell Line Panel” (American Type Culture Collection, 2022) does not respond to treatment, different well-studied leukemia cell lines can be tested instead. Aim 3: Demonstrating the efficacy of gold nanoparticle delivery of 6-MP to cancer cells of various leukemia cell lines. Preparing Nanoparticles and Cell Culturing AuNPs should be prepared as outlined in Aims 1 and 2, with batches of uniform shape and size being synthesized and prepared with antibodies for the cancer lines that will be utilized. A variety of cell lines should be used, ideally the same representative lines used in Aim 2. AuNPs should be loaded with 6-MP for eventual treatment of the cell lines. The cell lines should be cultured under standard conditions with an incubation period before treatment is applied. 6-MP AuNP should be applied to the cell lines and given enough time for drug release to occur (as determined by the experimentation described under Aim 1) before results are measured. Assessing Treatment Efficacy Flow cytometry using annexin V staining and immuloblot analysis should be performed as outlined in Aim 2 to measure apoptosis induction. This can be compared to the performance of the AuNPs without 6-MP used in Aim 2, and any differences should be noted. In vivo Experimentation A murine leukemia xenograft model should be prepared to study efficacy in vivo. After proper inoculation and incubation of the murine model, the prepared 6-MP loaded AuNPs can be introduced. The animals should be monitored, and upon their death, histologic studies can be performed by utilizing hematoxylin and eosin, anti-huCD45, or anti-huCD33 staining, to better visualize the cancerous cells present (Antonelli et al., 2016). The cancerous tissues can also be analyzed for drug uptake. Expected Results The efficacy of drug delivery and apoptosis induction is expected to be higher in cell lines known to have higher drug sensistivity. They should also be higher than the apoptosis levels seen in the experiments testing Aim 2, because of the addition of 6-MP in this experiment. The in vivo studies should show regression of the cancer and improved survival in those models treated with the 6-MP loaded nanoparticles when compared to the untreated models. Both in vivo and in vitro results should show more efficacy in 6-MP AuNPs than the untreated cells, with the AuNPs not loaded with 6-MP somewhere in between. Anticipated Complications The different cell lines will likely show variability in drug uptake and metabolism, which can result in inconsistent responses to treatment. Performing replicate experiments should assist in providing validation to the demonstrated results. The in vivo studies might demonstrate heterogeneity in the cancer cells and subsequent immune response. Using the appropriate animal model should avoid this issue. Literature Cited American Type Culture Collection. (2022). LEUKEMIA CELL LINE PANEL. https://www.atcc.org/-/media/product-assets/documents/panels/cell-biology/leukemia-cell-panel.pdf?rev=f054d18d0c5543a5a398a17117149a5c Anguille, S., Van Tendeloo, V. F., & Berneman, Z. (2012). Leukemia-associated antigens and their relevance to the immunotherapy of acute myeloid leukemia. Leukemia, 26(10), 2186–2196. https://doi.org/10.1038/leu.2012.145 Antonelli, A., Noort, W. A., Jaques, J., De Boer, B., De Jong-Korlaar, R., Brouwers-Vos, A. Z., Lubbers-Aalders, L., Van Velzen, J. F., Bloem, A. C., Yuan, H., De Bruijn, J., Ossenkoppele, G. J., Martens, A. C., Vellenga, E., Groen, R. W., & Schuringa, J. J. (2016). Establishing human leukemia xenograft mouse models by implanting human bone marrow–like scaffold-based niches. Blood, 128(25), 2949–2959. https://doi.org/10.1182/blood-2016-05-719021 Bio-Rad. (n.d.). Apoptosis Analysis by Western Blotting | Bio-Rad. Bio-Rad. https://www.bio-rad-antibodies.com/apoptosis-westernblot.html Chithrani, D. B., Ghazani, A. A., & Chan, W. C. W. (2006). Determining the Size and Shape Dependence of Gold Nanoparticle Uptake into Mammalian Cells. Nano Letters, 6(4), 662–668. https://doi.org/10.1021/nl052396o Chong, B. F., & Werth, V. P. (2019). Management of cutaneous lupus erythematosus. In Elsevier eBooks (pp. 719–726). https://doi.org/10.1016/b978-0-323-47927-1.00058-x Connor, D. M., & Broome, A. (2018). Gold nanoparticles for the delivery of cancer therapeutics. In Advances in cancer research (pp. 163–184). https://doi.org/10.1016/bs.acr.2018.05.001 Drugs approved for leukemia. (2024, April 1). Cancer.gov. https://www.cancer.gov/about-cancer/treatment/drugs/leukemia Faid, A. H., Hussein, F., Mostafa, E. M., Shouman, S. A., Badr, Y., & Sliem, M. A. (2023). Hybrid chitosan gold nanoparticles for photothermal therapy and enhanced cytotoxic action of 6-mercaptopurine on breast cancer cell line. Beni-Seuf University Journal of Basic and Applied Sciences /Beni-Suef University Journal of Basic and Applied Sciences, 12(1). https://doi.org/10.1186/s43088-023-00419-z Hammami, I., Alabdallah, N. M., Jomaa, A. A., & Kamoun, M. (2021). Gold nanoparticles: Synthesis properties and applications. Journal of King Saud University. Science/Maǧallaẗ Ǧāmiʹaẗ Al-malik Saʹūd. al-ʹUlūm, 33(7), 101560. https://doi.org/10.1016/j.jksus.2021.101560 He, J., Liu, S., Zhang, Y., Chu, X., Zhou, L., Zhao, Z., Qiu, S., Guo, Y., Ding, H., Pan, Y., & Pan, J. (2021). The application of and strategy for gold nanoparticles in cancer immunotherapy. Frontiers in Pharmacology, 12. https://doi.org/10.3389/fphar.2021.687399 Leukemia - Cancer Stat Facts. (n.d.). SEER. https://seer.cancer.gov/statfacts/html/leuks.html Mukherjee, P., Bhattacharya, R., Bone, N. D., Lee, Y. K., Patra, C. R., Wang, S., Lu, L., Secreto, C., Banerjee, P., Yaszemski, M. J., Kay, N. E., & Mukhopadhyay, D. (2007). Potential therapeutic application of gold nanoparticles in B-chronic lymphocytic leukemia (BCLL): enhancing apoptosis. Journal of Nanobiotechnology, 5(1), 4. https://doi.org/10.1186/1477-3155-5-4 Navidi-Moghadam-Foumani, R., Fazilati, M., Ardestani, M. S., Zanjanchi, P., & Asghari, S. M. (2023). Gold nanoparticle conjugation and tumor accumulation of a VEGF receptor-targeting peptidomimetic. Journal of the Iranian Chemical Society, 21(1), 293–303. https://doi.org/10.1007/s13738-023-02925-4 Phelan, S. A., & Szabo, E. (2019). Undergraduate lab series using the K562 human leukemia cell line: Model for cell growth, death, and differentiation in an advanced cell biology course. Biochemistry and Molecular Biology Education, 47(3), 263–271. https://doi.org/10.1002/bmb.21222 Professional, C. C. M. (n.d.). Leukemia. Cleveland Clinic. https://my.clevelandclinic.org/health/diseases/4365-leukemia Sibuyi, N. R. S., Moabelo, K. L., Fadaka, A. O., Meyer, M., Onani, M. O., Madiehe, A. M., & Meyer, M. (2021). Multifunctional Gold nanoparticles for improved diagnostic and therapeutic applications: A review. Nanoscale Research Letters, 16(1). https://doi.org/10.1186/s11671-021-03632-w Wawra, S. E., Pflug, L., Thajudeen, T., Kryschi, C., Stingl, M., & Peukert, W. (2018). Determination of the two-dimensional distributions of gold nanorods by multiwavelength analytical ultracentrifugation. Nature Communications, 9(1). https://doi.org/10.1038/s41467-018-07366-9 Xia, H., Xiahou, Y., Zhang, P., Ding, W., & Wang, D. (2016). Revitalizing the Frens Method To Synthesize Uniform, Quasi-Spherical Gold Nanoparticles with Deliberately Regulated Sizes from 2 to 330 nm. Langmuir, 32(23), 5870–5880. https://doi.org/10.1021/acs.langmuir.6b01312 Yu, M., Yuan, W., Li, D., Schwendeman, A., & Schwendeman, S. P. (2019). Predicting drug release kinetics from nanocarriers inside dialysis bags. Journal of Controlled Release, 315, 23–30. https://doi.org/10.1016/j.jconrel.2019.09.016 Comments are closed.
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