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Abstract: In Acute Myeloid Leukemia (AML), immature white blood cells, called myeloblasts, are formed from blood stem cells and build up in the bone marrow, blocking out white blood cells, red blood cells and platelets.1 Current treatments for AML are aimed at eliminating proliferative blast populations with limited success in targeting the leukemia stem cell population, often resulting in the use of chemotherapeutic agents that have potent effects on patients.2 The creation of drug-infused nanoparticles in targeted drug delivery as a therapeutic option for cancer patients has allowed selective delivery of potent drugs to tumor and cancerous cells, decreasing the drug’s effects on healthy cells.3 The most recent research regarding nanoparticles and AML includes antagomiR-126 nanoparticles that target miR-126 expression in AML blasts and the targeted delivery of microRNA-29b by transferrin-conjugated anionic lipopolyplex nanoparticles.4 Melittin, a toxin found in bee venom, has the ability to poke holes in cell membranes, as confirmed by research on the drug’s antiviral effects on HIV and most recently, by work with melittin fusion proteins and gastric cancer. 5 While nanoparticles are currently being researched to target AML blasts, melittin nanoparticles have not yet been used to target AML blasts. Using similar experimental models to the in vivo research and proposals on miR-126 and microRNA-29b, a transferrin-conjugated, melittin perfluorocarbon nanoparticle is proposed as a novel therapeutic strategy in AML.
Background:
Acute myeloid leukemia (AML) is a hematopoietic stem cell disorder characterized by a block in hematopoiesis that results in growth of a clonal population of neoplastic cells or blasts. 7 Thirty-three percent of people diagnosed with leukemia have AML. 8 Common treatments for leukemia currently include chemotherapy and bone marrow transplantation; however, side-effects such as infection and accumulation of kidney stones still exist. As a result, therapy that minimizes the effects of potent drugs is urgently needed. 7
Nanoparticles, which are particles between 1 and 100 nm in size, can help to deliver drugs to target cells. 9 By using nanoparticles, potent drugs can be transported directly to target cells to limit side effects on healthy cells.10 Most nanoparticles are composed of lipids so that they are non-toxic and can be delivered intravenously to the patient.11 In previous years, nanoparticles have had their successes. Doxil was the first nanoparticle drug to be approved by the FDA.12 Doxil is used to treat Kaposi’s sarcoma, ovarian cancer, breast cancer, and other tumors. 12 Melittin, a cytolytic peptide component of bee venom, contains a C-k and an N-terminus. The C-terminus locates the cytotoxicity functional domain of melittin and is hydrophilic. The N-terminus is the hydrophobic region.13 Melittin can exemplify cationic 26-residue peptides that manifest membrane-disrupting activity when incorporated into traditional bilayer delivery systems.13 It is a nonspecific cytolytic process that attacks all lipid membranes, which leads to significant toxicity. A large amount of free melittin has harmful side effects and symptoms within humans.14 Therefore, using nanoparticles to deliver the toxin to attack a viral envelope or lipid membrane prevents the need to systematically pump high doses of the toxin through the bloodstream.15 After purification, melittin has previously been encapsulated in nanoparticles to successfully rupture HIV cell envelopes,14 which gives researchers the potential to target leukemia with a similar method.
Perfluorocarbon nanoparticles are chosen for this proposal. They contain lipid and Perfluorooctyl-bromide.16 Egg lecithin, PEG-DSPE, and scFv will be used to create the liposome structure of nanoparticles. Egg lecithin contains phospholipids.17 It has emulsification and lubricant properties, which can help to lower the surface tension and reduce friction between surfaces. 17 PEG-DSPE is a kind of amphiphilic lipid that can help to reduce the clearance of liposomal formulations in vivo so that nanoparticles can circulate for a longer time. 18 A single-chain fragment variable (scFv) can be generated through recombinant antibody technology from OKT9, a monoclonal anti-TfR antibody. 19 Its function is to ensure that the nanoparticles are able to bind to acute myeloid leukemia blasts. 19 Acute myeloid leukemia blasts have a high transferrin receptor expression partly due to their proliferative potential, and scFV can bind with those transferrin receptors.20 Researchers have already been successful in targeting AML blasts with transferrin-conjugated anionic lipopolyplex nanoparticles. 20 The research, in vivo, results tested in mice with AML blasts confirmed that transferrin –conjugated nanoparticles were twice as efficient in the delivery of microRNA-29b to AML blasts compared to non transferrin-conjugated nanoparticles or free microRNA-29b drug delivery. 21 Perfluorooctyl-bromide is added to the liposome structure to compose a complete nanoparticle. Perfluorooctyl-bromide, a kind of perfluorocarbon compound, makes liposomes resistant to melittin so that the drug does not destroy its vesicle. 22 The system will contain a phospholipid bilayer membrane composed of egg lecithin, PEG DSPE, and an scFv. Perfluorooctyl-bromide will be added in between the two monolayer membranes at the hydrophobic tails of the lipids (Figure 1). 22 The core of the perfluorocarbon nanoparticle will be composed of water in order to keep the hydrophilic heads faced toward the inside of the nanoparticle. Because melittin has both hydrophobic and hydrophilic regions, its hydrophobic region will face the hydrophobic interior while the hydrophilic will face the water interface. 22 The infusion of melittin into the nanoparticle is assisted by an α-peptide, which links the N-terminus of the melittin and helps to bury the melittin in the lipid layer of the nanoparticle.13 The melittin has a tryptophan side chain (TrP) on its hydrophilic region, which will form a hydrogen bond with perfluorooctyl-bromide. 23 This hydrogen bond affects the lytic activity of melittin on the lipid layer so that the nanoparticle will not be destroyed by the melittin. 23 Once the nanoparticle is completed, the melittin nanoparticles will be injected into the bloodstream and circulate until the scFV binds to the high transferrin receptor surface on an acute myeloid leukemia blast. The melittin will be released into the target cell using contact facilitated drug delivery. In this process, a hemifusion will occur between the vesicle and the target cell lipid layers to achieve lipid mixing.24 Then the melittin will diffuse out of the vesicle into the target cell lipid membrane. The melittin will destroy the integrity of the cancer cell membrane and induce apoptosis.
Figure 1. Perfluorocarbon Nanoparticles. A perfluorooctyl bromide shell (light blue) is formed between each layer of the lipid bilayer. Melittin (red) lies inside the outer lipid layer. The hydrophobic region of melittin binds to the hydrophobic tail of lipid, while the hydrophilic region of melittin binds to the hydrophilic head of lipid.
Objective:
The goal of this research proposal is to create melittin-loaded perfluorocarbon nanoparticles to provide a novel therapeutic strategy in the targeted treatment of AML, ultimately limiting the side effects and increasing the specificity of current treatments.
Approach:
Construction of perfluorocarbon nanoparticles:
The liposomes designed in this experiment will include 99% of egg lecithin, 0.9% of PEG-DSPE, and 0.1% of the scFv.17 To conjugate the scFV on the surface of the PEG DSPE, the scFv will be engineered with a C-terminal cysteine by CANTAB 5E Cys vectors for site-directed conjugation.25 After mixing egg lecithin, PEG DSPE and scFv, this lipid mixture will be dissolved into chloroform, evaporated under reduced pressure and dried in a 50℃ vacuum oven to form a thin polymer layer. 17 Water will be added to the resulting thin layer and stirred under heat to form liposomes. The liposomes will be combined with perfluorooctyl-bromide, one of the perfluorocarbon compounds, by processing them at 20,000 lbf/in2 for 4 minutes with an S110 Microfluidics emulsifier. 17 Nanoparticles with perfluorooctyl-bromide within the liposome are then resistant to melittin. After the liposome has been constructed, melittin will be added to the nanoparticles. 17 The nanoparticles will be incubated and stirred at a concentration of 1mM melittin in water for 72 hours.17 Lastly, the perfluorocarbon nanoparticles will be isolated from solution that contains free melittin by low speed centrifugation and washed with PBS. 17 In Vitro Experiment:
To start the experiment, the nanoparticle will be tested in vitro. The in vitro experiment is designed to examine if the melittin actually affects the cells. A sample of AML cell line, CRL-2740, will be cultivated on four identical tissue culture plates, with a concentration of 1 × 106 cells/ml (Figure 2). 26 Each plate will be labelled A, B, C and D. Plate A and plate B will be negative controls because they should not inhibit cell growth. Plate A will have only saline added to the cells. Nanoparticles without melittin will be added to plate B. Plates C and D will be positive controls because these plates should be affected by the drug. Melittin in its free drug form will be added to plate C. Plate D will be added with the nano-carrier with melittin (Table 1). The four plates will then be incubated for 48 hours. The total number of AML cells and the number of apoptotic cells in each culture plate will be observed and recorded by flow cytometry.26 It is expected that the number of AML cells will grow rapidly, and the number of apoptotic cells will be relatively small in both plate A and B. In contrast, there will be a relatively large amount of apoptotic AML cells in both plate C and D, since many of them will be killed by the nanoparticle-free melittin and nanoparticle-carried melittin.
Figure 2. In Vitro Experiment. Culture plates will contain saline (A), nanoparticles (B), melittin (C), and nanoparticle-carried melittin (D).
Table 1. Controls of In Vitro Experiment. Culture plate A is added with saline, which is a negative control. Culture plate B is added with only nanoparticles, which is a negative control. Culture plate C is added with melittin, which is a positive control. Culture plate D was added with both nanoparticle and melittin.
In Vivo Experiment:
After in vitro testing and necessary modifications, the melittin-infused nanoparticles will be tested in vivo in leukemic non-obese diabetic/severe combined immunodeficient mouse models. 6 The purpose of undergoing the in vivo experiment it to determine that, if the drug works, how the drug and the nanoparticle affects bodily systems. Twelve mice transplanted with AML will be divided into four groups (Figure 3). Group A and B will be negative controls. Mice in group A will only be injected with saline. Group B will be injected with nanoparticles lacking the drug. Mice in group C will be positive controls, being injected with only melittin. Group D will be injected with 0.2mg/kg nano-carried melittin (Table 2).27 The injection will last for 2 weeks. Throughout the two weeks, the weight of mice in each group will be recorded and compared. After two weeks, early pharmacodynamic data including spleen weights, blood smears, and blast differentiation will be collected 27. It is expected that the weights of mice in groups A, B and C will continue to decrease for various reasons. The decrease in weight in group A and B will be caused by further development of AML, while group C might be affected by the side-effect of melittin. The weight of mice in group D with drug-carried nanoparticles will most likely keep in constant. These experiments will be executed in accordance with OSU institutional guidelines for animal care and under protocols approved by the OSU Institutional Animal Care and Use Committee.6 Figure 3. In vivo experiment. Three mice in group A are provided with saline. Three mice in group B are injected with nanoparticles. Three mice in group C are injected with melittin. Three mice in group D are injected with both nanoparticles and melittin.
Table 2. Controls of In Vivo Experiment. Culture plate A is provided with saline, which is a negative control. Culture plate B is injected with only nanoparticles, which is a negative control. Culture plate C is injected with melittin, which is a positive control. Culture plate D was added with both nanoparticle and melittin.
Impact:
This drug-delivery system will provide targeted therapy for patients with acute myeloid leukemia and will eliminate the effects of chemotherapeutic agents by only targeting the cancerous cells. Scientific research has already been done on the targeted delivery of microRNA-29b to AML blasts and researchers have successfully used melittin to target HIV in in vivo experiments. Further research has also confirmed the use of OKT9 to target transferrin receptors in the targeted delivery of therapeutic agents against cancer. However, there is no current research combining these techniques and using OKT9-conjugated perfluorocarbon nanoparticles to deliver melittin to Acute Myeloid Leukemia Blasts. Melittin has the potential to kill cancer cells due to its ability to penetrate the cell membrane. Nanoparticles have been successful in delivering drugs to cancerous cells, but researchers often struggle to deliver a drug with a potent enough concentration to kill the cancerous cells. Because perfluorocarbon nanoparticles have a phospholipid bilayer on the outside of the capsule, melittin can be delivered to AML blasts through these nanoparticles. By combining these techniques, this approach could provide a novel treatment opportunity for the 70% of AML patients who experience a relapse or do not reach complete remission with this disease. 2
Summary:
This proposal offers a method to more effectively treat AML. The combination of perfluorocarbon nanoparticles and melittin can create a treatment that is much more selective and less detrimental than chemotherapy. We intend to incorporate Perfluorooctyl-bromide, egg lecithin, scFv, and PEG DSPE to create the nanoparticle. This combination of molecules should be effective in the treatment of AML and potentially other cancers with specific ligands.
Acknowledgements:
We would like to thank Praful Nair, Dr. Wattenbarger, and Brianna Karpowicz for their assistance in this research proposal. Their help aided in researching critical information for the methods and details of this proposal.■ References
[1] What you need to know about Leukemia. Los Angeles, CA: National Cancer Institute, 2013.
[2] National Cancer Institue. "Facts and Statistics." Leukemia and Lymphoma Society. Last modified 2016. Accessed July 7, 2016. https://www.lls.org/http%3A/ llsorg.prod.acquia-sites.com/facts-and-statistics/facts-and-statistics-overvie w/ facts-and-statistics.
[3] Angle, Annie. "Treatment for Leukaemia." Cancer Council. Last modified April 30, 2013. Accessed July 7, 2016. http://www.cancervic.org.au/about-cancer/ cancer_types/leukaemia/treatment_for_leukaemia.htm.
[4] Tracy R. Daniels, Ezequiel Bernabeu, José A. Rodríguez. “Transferrin receptors and the targeted delivery of therapeutic agents against cancer.” Vol 1820: 291-317. 2011 August 5 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3500658/
[5] Strait, Julia Evangelou. "Nanoparticles loaded with bee venom kill HIV." The Source. Last modified March 7, 2013. Accessed July 6, 2016. https://source.wustl.edu/2013/03/nanoparticles-loaded-with-bee-venom-kill-hiv/.
[6] “Targeted Delivery of microRNA-29b by Transferrin-Conjugated Anionic Lipopolyplex Nanoparticles: A Novel Therapeutic Strategy in Acute Myeloid Leukemia.”http://clincancerres.aacrjournals.org/content/19/9/2355.full.
[7] Shipley, Joshua L., and James N. Butera. "Acute Myelogenous Leukemia." Science Direct, May 20, 2009, 649-58.
[8] National Cancer Institute. "SEER Stat Fact Sheets: Leukemia." Surveillance, Epidemiology and End Results Program. Accessed July 7, 2016. http://seer.cancer.gov/statfacts/html/leuks.html#incidence-mortality.
[9] Davis, Mark E., Zhuo Georgia Chen, and Dong M. Shin. "Nanoparticle therapeutics: an emerging treatment modality for cancer." Reviews 7 (September 2008): 771-82.
[10] Wang, Andrew Z., Omid C. Farokhzad, and Robert Langer. "Nanoparticle Delivery of Cancer Drugs." Annual Review of Medicine, no. 63 (2012): 185-99.
[11] Brannon-Peppas, Lisa, and James O. Blanchette. "Nanoparticle and targeted systems for cancer therapy." Advanced Drug Delivery Reviews, 2012, 206-12.
[12] Y, Barenholz. "The first FDA-approved nano-drug: lessons learned." PubMed 160, no. 2 (March 29, 2012). http://www.ncbi.nlm.nih.gov/ pubmed/22484195. "Doxorubicin Liposomal." Chemocare. Accessed July 14, 2016. http://chemocare.com/ chemotherapy/drug-info/doxorubicin-liposomal.aspx.
[13] Huang, Chuan, Honglin Jin, and Yuan Qian. "Hybrid Melittin Cytolytic PeptideDriven Ultrasmall Lipid Nanoparticles Block Melanoma Growth in Vivo." ACS Nano 7, no. 7 (2013): 5791-800. Accessed July 21, 2016. http://pubs.acs.org/doi/pdf/10.1021/nn400683s. [14] Kim, Yong-Wan, Pankaj Kumar Chaturvedi, Sung Nam Chun, Yang Gu Lee, and Woong Shick Ahn. "Honeybee venom possesses anticancer and anviviral effects by differential inhibition of HPV E6 and E7 expression on cervical cancer cell line." Oncology Reports, January 28, 2015, 1675-82. Accessed July 6, 2016. doi:2015.3760.
[15] Racaniello, Vincent, Dr. "Virus Structure." TWIV. Last modified July 7, 2012. Accessed July 6, 2016. http://www.twiv.tv/virus-structure/.
[16] “Water-soluble perfluorooctyl bromide-liposome nanosphere and preparation method.” GooglePatent. April 30, 2014. Web. Accessed July 14, 2016. http://www.google.com/patents/CN103751106A?cl=en.
[17] Andrew P. Jallouk, Kelle H. Moley, Kenan Omurtag “Nanoparticle incorporation of melittin reduces sperm and vaginal epithelium cytotoxicity.” Accessed July 14, 2016. http://digitalcommons.wustl.edu/cgi/viewcontent.cgiarticle=3821&context=open_access_pubs.
[18] Alric, Christophe. "Covalent conjugation of cysteine-engineered scFv to PEGylated magnetic nano probes for immunotargeting of breast cancer cells." Royal Society of Chemistry 6 (March 7, 2016): 37099-109.
[19] Daniels, Tracy R. "Transferrin receptors and the targeted delivery of therapeautic agents against cancer." PubMed 3 (March 2012): 291-317.
[20] Morishita, Yoshihisa. "Up-Regulation of Transferrin Receptor Gene Expression by Granulocyte Colony-stimulating Factor in Human Myeloid Leukemia Cells." Cancer Research 50 (December 15, 1990): 7955-61.
[21] “Targeted Delivery of microRNA-29b by Transferrin-Conjugated Anionic Lipopolyplex Nanoparticles: A Novel Therapeutic Strategy in Acute Myeloid Leukemia.” http://clincancerres.aacrjournals.org/content/19/9/2355.full.
[22] Lee, Sun-Joo, Paul H. Schlesinger, and Samuel A. Wickline. "Interaction of Melittin Peptides with Perfluorocarbon Nanoemulsion Particles." National Insitutes of Health, December 29, 2012. Accessed December 6, 2011. jp209543c.
[23] Lee, Sun-Joo, Paul H. Schlesinger, and Samuel A. Wickline. "Interaction of Melittin Peptides with Perfluorocarbon Nanoemulsion Particles." National Insitutes of Health, December 29, 2012. Accessed December 6, 2011. jp209543c.
[24] Jallouk, Andrew P. "Delivery of Protease-Activated Cytolytic Peptide Prodrug by Perfluorocarbon Nanoparticles." Bioconjugate Chemistry.
[25] no. 8 (June 2015). Accessed June 21, 2016. doi:10.1021.
[26] H, Albrecht. "Production of soluble ScFvs with C-terminal-free thiol for site-specific conjugation or stable dimeric ScFvs on demand." PubMed 1(February 15, 2004): 16-26.
[27] "Leukemia Cell Lines." The Essentials of Life Science Research, 2013.
[28] Bernot, K. M. "Toward personalized therapy in AML: in vivo benefit of targeting aberrant epigenetic in MLL-PTD-associated AML." Leukemia 7 (June 23, 2013).
[29] “Transferrin receptor expression on AML blasts is related to their proliferative potential.” http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2141.1988.tb07633.x/abstract
Background:
Acute myeloid leukemia (AML) is a hematopoietic stem cell disorder characterized by a block in hematopoiesis that results in growth of a clonal population of neoplastic cells or blasts. 7 Thirty-three percent of people diagnosed with leukemia have AML. 8 Common treatments for leukemia currently include chemotherapy and bone marrow transplantation; however, side-effects such as infection and accumulation of kidney stones still exist. As a result, therapy that minimizes the effects of potent drugs is urgently needed. 7
Nanoparticles, which are particles between 1 and 100 nm in size, can help to deliver drugs to target cells. 9 By using nanoparticles, potent drugs can be transported directly to target cells to limit side effects on healthy cells.10 Most nanoparticles are composed of lipids so that they are non-toxic and can be delivered intravenously to the patient.11 In previous years, nanoparticles have had their successes. Doxil was the first nanoparticle drug to be approved by the FDA.12 Doxil is used to treat Kaposi’s sarcoma, ovarian cancer, breast cancer, and other tumors. 12 Melittin, a cytolytic peptide component of bee venom, contains a C-k and an N-terminus. The C-terminus locates the cytotoxicity functional domain of melittin and is hydrophilic. The N-terminus is the hydrophobic region.13 Melittin can exemplify cationic 26-residue peptides that manifest membrane-disrupting activity when incorporated into traditional bilayer delivery systems.13 It is a nonspecific cytolytic process that attacks all lipid membranes, which leads to significant toxicity. A large amount of free melittin has harmful side effects and symptoms within humans.14 Therefore, using nanoparticles to deliver the toxin to attack a viral envelope or lipid membrane prevents the need to systematically pump high doses of the toxin through the bloodstream.15 After purification, melittin has previously been encapsulated in nanoparticles to successfully rupture HIV cell envelopes,14 which gives researchers the potential to target leukemia with a similar method.
Perfluorocarbon nanoparticles are chosen for this proposal. They contain lipid and Perfluorooctyl-bromide.16 Egg lecithin, PEG-DSPE, and scFv will be used to create the liposome structure of nanoparticles. Egg lecithin contains phospholipids.17 It has emulsification and lubricant properties, which can help to lower the surface tension and reduce friction between surfaces. 17 PEG-DSPE is a kind of amphiphilic lipid that can help to reduce the clearance of liposomal formulations in vivo so that nanoparticles can circulate for a longer time. 18 A single-chain fragment variable (scFv) can be generated through recombinant antibody technology from OKT9, a monoclonal anti-TfR antibody. 19 Its function is to ensure that the nanoparticles are able to bind to acute myeloid leukemia blasts. 19 Acute myeloid leukemia blasts have a high transferrin receptor expression partly due to their proliferative potential, and scFV can bind with those transferrin receptors.20 Researchers have already been successful in targeting AML blasts with transferrin-conjugated anionic lipopolyplex nanoparticles. 20 The research, in vivo, results tested in mice with AML blasts confirmed that transferrin –conjugated nanoparticles were twice as efficient in the delivery of microRNA-29b to AML blasts compared to non transferrin-conjugated nanoparticles or free microRNA-29b drug delivery. 21 Perfluorooctyl-bromide is added to the liposome structure to compose a complete nanoparticle. Perfluorooctyl-bromide, a kind of perfluorocarbon compound, makes liposomes resistant to melittin so that the drug does not destroy its vesicle. 22 The system will contain a phospholipid bilayer membrane composed of egg lecithin, PEG DSPE, and an scFv. Perfluorooctyl-bromide will be added in between the two monolayer membranes at the hydrophobic tails of the lipids (Figure 1). 22 The core of the perfluorocarbon nanoparticle will be composed of water in order to keep the hydrophilic heads faced toward the inside of the nanoparticle. Because melittin has both hydrophobic and hydrophilic regions, its hydrophobic region will face the hydrophobic interior while the hydrophilic will face the water interface. 22 The infusion of melittin into the nanoparticle is assisted by an α-peptide, which links the N-terminus of the melittin and helps to bury the melittin in the lipid layer of the nanoparticle.13 The melittin has a tryptophan side chain (TrP) on its hydrophilic region, which will form a hydrogen bond with perfluorooctyl-bromide. 23 This hydrogen bond affects the lytic activity of melittin on the lipid layer so that the nanoparticle will not be destroyed by the melittin. 23 Once the nanoparticle is completed, the melittin nanoparticles will be injected into the bloodstream and circulate until the scFV binds to the high transferrin receptor surface on an acute myeloid leukemia blast. The melittin will be released into the target cell using contact facilitated drug delivery. In this process, a hemifusion will occur between the vesicle and the target cell lipid layers to achieve lipid mixing.24 Then the melittin will diffuse out of the vesicle into the target cell lipid membrane. The melittin will destroy the integrity of the cancer cell membrane and induce apoptosis.
Figure 1. Perfluorocarbon Nanoparticles. A perfluorooctyl bromide shell (light blue) is formed between each layer of the lipid bilayer. Melittin (red) lies inside the outer lipid layer. The hydrophobic region of melittin binds to the hydrophobic tail of lipid, while the hydrophilic region of melittin binds to the hydrophilic head of lipid.
Objective:
The goal of this research proposal is to create melittin-loaded perfluorocarbon nanoparticles to provide a novel therapeutic strategy in the targeted treatment of AML, ultimately limiting the side effects and increasing the specificity of current treatments.
Approach:
Construction of perfluorocarbon nanoparticles:
The liposomes designed in this experiment will include 99% of egg lecithin, 0.9% of PEG-DSPE, and 0.1% of the scFv.17 To conjugate the scFV on the surface of the PEG DSPE, the scFv will be engineered with a C-terminal cysteine by CANTAB 5E Cys vectors for site-directed conjugation.25 After mixing egg lecithin, PEG DSPE and scFv, this lipid mixture will be dissolved into chloroform, evaporated under reduced pressure and dried in a 50℃ vacuum oven to form a thin polymer layer. 17 Water will be added to the resulting thin layer and stirred under heat to form liposomes. The liposomes will be combined with perfluorooctyl-bromide, one of the perfluorocarbon compounds, by processing them at 20,000 lbf/in2 for 4 minutes with an S110 Microfluidics emulsifier. 17 Nanoparticles with perfluorooctyl-bromide within the liposome are then resistant to melittin. After the liposome has been constructed, melittin will be added to the nanoparticles. 17 The nanoparticles will be incubated and stirred at a concentration of 1mM melittin in water for 72 hours.17 Lastly, the perfluorocarbon nanoparticles will be isolated from solution that contains free melittin by low speed centrifugation and washed with PBS. 17 In Vitro Experiment:
To start the experiment, the nanoparticle will be tested in vitro. The in vitro experiment is designed to examine if the melittin actually affects the cells. A sample of AML cell line, CRL-2740, will be cultivated on four identical tissue culture plates, with a concentration of 1 × 106 cells/ml (Figure 2). 26 Each plate will be labelled A, B, C and D. Plate A and plate B will be negative controls because they should not inhibit cell growth. Plate A will have only saline added to the cells. Nanoparticles without melittin will be added to plate B. Plates C and D will be positive controls because these plates should be affected by the drug. Melittin in its free drug form will be added to plate C. Plate D will be added with the nano-carrier with melittin (Table 1). The four plates will then be incubated for 48 hours. The total number of AML cells and the number of apoptotic cells in each culture plate will be observed and recorded by flow cytometry.26 It is expected that the number of AML cells will grow rapidly, and the number of apoptotic cells will be relatively small in both plate A and B. In contrast, there will be a relatively large amount of apoptotic AML cells in both plate C and D, since many of them will be killed by the nanoparticle-free melittin and nanoparticle-carried melittin.
Figure 2. In Vitro Experiment. Culture plates will contain saline (A), nanoparticles (B), melittin (C), and nanoparticle-carried melittin (D).
Table 1. Controls of In Vitro Experiment. Culture plate A is added with saline, which is a negative control. Culture plate B is added with only nanoparticles, which is a negative control. Culture plate C is added with melittin, which is a positive control. Culture plate D was added with both nanoparticle and melittin.
In Vivo Experiment:
After in vitro testing and necessary modifications, the melittin-infused nanoparticles will be tested in vivo in leukemic non-obese diabetic/severe combined immunodeficient mouse models. 6 The purpose of undergoing the in vivo experiment it to determine that, if the drug works, how the drug and the nanoparticle affects bodily systems. Twelve mice transplanted with AML will be divided into four groups (Figure 3). Group A and B will be negative controls. Mice in group A will only be injected with saline. Group B will be injected with nanoparticles lacking the drug. Mice in group C will be positive controls, being injected with only melittin. Group D will be injected with 0.2mg/kg nano-carried melittin (Table 2).27 The injection will last for 2 weeks. Throughout the two weeks, the weight of mice in each group will be recorded and compared. After two weeks, early pharmacodynamic data including spleen weights, blood smears, and blast differentiation will be collected 27. It is expected that the weights of mice in groups A, B and C will continue to decrease for various reasons. The decrease in weight in group A and B will be caused by further development of AML, while group C might be affected by the side-effect of melittin. The weight of mice in group D with drug-carried nanoparticles will most likely keep in constant. These experiments will be executed in accordance with OSU institutional guidelines for animal care and under protocols approved by the OSU Institutional Animal Care and Use Committee.6 Figure 3. In vivo experiment. Three mice in group A are provided with saline. Three mice in group B are injected with nanoparticles. Three mice in group C are injected with melittin. Three mice in group D are injected with both nanoparticles and melittin.
Table 2. Controls of In Vivo Experiment. Culture plate A is provided with saline, which is a negative control. Culture plate B is injected with only nanoparticles, which is a negative control. Culture plate C is injected with melittin, which is a positive control. Culture plate D was added with both nanoparticle and melittin.
Impact:
This drug-delivery system will provide targeted therapy for patients with acute myeloid leukemia and will eliminate the effects of chemotherapeutic agents by only targeting the cancerous cells. Scientific research has already been done on the targeted delivery of microRNA-29b to AML blasts and researchers have successfully used melittin to target HIV in in vivo experiments. Further research has also confirmed the use of OKT9 to target transferrin receptors in the targeted delivery of therapeutic agents against cancer. However, there is no current research combining these techniques and using OKT9-conjugated perfluorocarbon nanoparticles to deliver melittin to Acute Myeloid Leukemia Blasts. Melittin has the potential to kill cancer cells due to its ability to penetrate the cell membrane. Nanoparticles have been successful in delivering drugs to cancerous cells, but researchers often struggle to deliver a drug with a potent enough concentration to kill the cancerous cells. Because perfluorocarbon nanoparticles have a phospholipid bilayer on the outside of the capsule, melittin can be delivered to AML blasts through these nanoparticles. By combining these techniques, this approach could provide a novel treatment opportunity for the 70% of AML patients who experience a relapse or do not reach complete remission with this disease. 2
Summary:
This proposal offers a method to more effectively treat AML. The combination of perfluorocarbon nanoparticles and melittin can create a treatment that is much more selective and less detrimental than chemotherapy. We intend to incorporate Perfluorooctyl-bromide, egg lecithin, scFv, and PEG DSPE to create the nanoparticle. This combination of molecules should be effective in the treatment of AML and potentially other cancers with specific ligands.
Acknowledgements:
We would like to thank Praful Nair, Dr. Wattenbarger, and Brianna Karpowicz for their assistance in this research proposal. Their help aided in researching critical information for the methods and details of this proposal.■ References
[1] What you need to know about Leukemia. Los Angeles, CA: National Cancer Institute, 2013.
[2] National Cancer Institue. "Facts and Statistics." Leukemia and Lymphoma Society. Last modified 2016. Accessed July 7, 2016. https://www.lls.org/http%3A/ llsorg.prod.acquia-sites.com/facts-and-statistics/facts-and-statistics-overvie w/ facts-and-statistics.
[3] Angle, Annie. "Treatment for Leukaemia." Cancer Council. Last modified April 30, 2013. Accessed July 7, 2016. http://www.cancervic.org.au/about-cancer/ cancer_types/leukaemia/treatment_for_leukaemia.htm.
[4] Tracy R. Daniels, Ezequiel Bernabeu, José A. Rodríguez. “Transferrin receptors and the targeted delivery of therapeutic agents against cancer.” Vol 1820: 291-317. 2011 August 5 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3500658/
[5] Strait, Julia Evangelou. "Nanoparticles loaded with bee venom kill HIV." The Source. Last modified March 7, 2013. Accessed July 6, 2016. https://source.wustl.edu/2013/03/nanoparticles-loaded-with-bee-venom-kill-hiv/.
[6] “Targeted Delivery of microRNA-29b by Transferrin-Conjugated Anionic Lipopolyplex Nanoparticles: A Novel Therapeutic Strategy in Acute Myeloid Leukemia.”http://clincancerres.aacrjournals.org/content/19/9/2355.full.
[7] Shipley, Joshua L., and James N. Butera. "Acute Myelogenous Leukemia." Science Direct, May 20, 2009, 649-58.
[8] National Cancer Institute. "SEER Stat Fact Sheets: Leukemia." Surveillance, Epidemiology and End Results Program. Accessed July 7, 2016. http://seer.cancer.gov/statfacts/html/leuks.html#incidence-mortality.
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