Project Description



Background

Current Status of Cancer, Focus on Breast Cancer
Cancer continues to be a global health concern, with breast cancer ranking among the most prevalent and life-threatening malignancies worldwide. The increasing incidence of breast cancer underscores the urgent need for innovative treatment strategies. In the public data from World Health Organization (WHO), breast cancer tops the list as the most commonly diagnosed cancer worldwide and claims a significant number of lives.

Estimated age-standardized incidence and mortality rates (World) in 2020, World, both sexes, all ages (excl.NMSC)

Figure 1. Estimated age-standardized incidence and mortality rates (World) in 2020, World, both sexes, all ages (excl.NMSC).



Breast cancer is a heterogeneous disease characterized by the uncontrolled growth of cells in the breast tissue. It is the most common cancer among women, with a significant impact on their health and quality of life. Advances in early detection and treatment have improved survival rates, but challenges persist, including late-stage diagnoses and resistance to conventional therapies.

The global burden of breast cancer is substantial, with millions of new cases diagnosed annually. Geographical disparities in incidence and mortality rates emphasize the importance of tailored approaches to prevention, diagnosis, and treatment. Breast cancer awareness, research, and healthcare infrastructure play pivotal roles in addressing this global challenge.
Current Problems in Treating Breast Cancer
Current Treatment towards Breast Cancer

Figure 2. Current Treatment towards Breast Cancer.

Treating breast cancer remains a complex and multifaceted challenge, despite significant advancements in the field of oncology. Several current problems and challenges persist in the treatment of breast cancer:

Late-stage Diagonsis
One of the most significant challenges is the late-stage diagnosis of breast cancer. Many patients are diagnosed when the disease has already reached an advanced stage, reducing treatment options and overall survival rates. Enhancing early detection methods and improving breast cancer screening programs are essential to address this issue.

Tumor Heterogeneity
Breast cancer is not a single disease but a heterogeneous group of diseases with varying molecular subtypes. Each subtype may require different treatment strategies. Tailoring treatments to the specific subtype of breast cancer is crucial for optimizing outcomes, but this can complicate treatment decision-making.

Resistance to Therapy
Resistance to therapy, whether it's chemotherapy, hormonal therapy, or targeted therapy, remains a significant concern. Breast cancer cells can develop resistance to treatment over time, leading to disease progression. Researchers are actively studying the mechanisms of resistance to develop more effective therapies.

Toxicity and Side Effects
Toxicity and Side Effects: Breast cancer treatments, including chemotherapy and radiation therapy, can cause severe side effects and toxicity. These side effects can impact a patient's quality of life and sometimes result in treatment interruptions or discontinuations. Finding ways to minimize side effects while maintaining treatment efficacy is an ongoing challenge.

Cost of Treatment
Breast cancer treatment can be expensive, and the financial burden can be overwhelming for patients and their families. Access to innovative therapies and adequate insurance coverage remains a concern, and addressing the cost of cancer care is crucial for improving patient outcomes and reducing disparities in care.
Current Status of Iron Oxide Nanoparticle Synthesis
Iron oxide nanoparticles have emerged as versatile materials with promising applications in the medical field,. The synthesis of these nanoparticles can be categorized into three main approaches: physical synthesis, chemical synthesis, and biological synthesis, each with its own set of advantages and disadvantages.


Physical Synthesis
Historically, physical synthesis methods have been employed to produce iron oxide nanoparticles. These methods involve high-temperature processes such as thermal decomposition and laser ablation. While physical synthesis techniques yield nanoparticles with excellent crystallinity and controlled size distribution, they often suffer from limited scalability and can require harsh reaction conditions, which may not be ideal for medical applications.

Chemical Synthesis
Chemical synthesis techniques, such as co-precipitation and solvothermal methods, have become more prevalent due to their scalability and relative simplicity. These methods enable the fine-tuning of nanoparticle properties like size, shape, and surface functionalization. However, they may introduce impurities and require the use of toxic reagents, raising concerns about biocompatibility for medical use. Also the chemical synthesis process requires toxic or harmful reagents, which may augment the environmental concerns.

Biological Synthesis
The advent of biological synthesis methods has garnered considerable interest in recent years. Utilizing microorganisms, plants, or biomolecules, this approach offers eco-friendly and biocompatible routes to produce iron oxide nanoparticles. Biologically synthesized nanoparticles often exhibit excellent biocompatibility and can be tailored for specific medical applications. However, the control over size and shape can be more challenging compared to chemical methods.
Application of Iron Oxide Nanoparticles in Medical Field Currently
In the medical field, iron oxide nanoparticles have found several potential applications, including but not limited to:

Magnetic Resonance Imaging (MRI)
Iron oxide nanoparticles are utilized as contrast agents in MRI scans due to their magnetic properties, aiding in the detection and characterization of cancerous lesions.

Drug Delivery
Their surface properties and unique ability for agent design allow for the attachment of therapeutic agents, facilitating targeted drug delivery to cancer cells while minimizing systemic side effects.

Hyperthermia Treatment
Iron oxide nanoparticles can generate heat when exposed to an alternating magnetic field, making them suitable for hyperthermia therapy, where cancer cells are selectively heated and destroyed.

Our Solution

Since the treatment of breast cancer nowadays is still facing many challenges like significant side effects, low specificity and cannot deal with the subgroup of breast cancer well, we are trying to develop a new method for the treatment of breast cancer. Our method is based on the iron oxide nanoparticles, which could be linked with the antibody targeting overexpressed protein on the surface of some breast cancer subgroup cells.

We choose to use iron oxide nanoparticles as the carrier backbond mainly because of its relative high biocompatibility in different types of nanoparticles, especially the biologically synthesized iron oxide nanoparticles. And the iron oxide nanoparticles could be synthesized by the E. coli dircetly with ferric ions as the inducer, which is also a green way to synthesize nanoparticles.

Though our final outlook is to have the IONPs directly linked to the antibody in a self-assembly manner, the coating composition of the IONPs is still a problem to be solved. In this way, bringing forward a self-assembly design for the IONPs could be hard and unpractical at this stage.

So, instead of the self-assembly design, we choose to synthesize the IONPs and the antibody separately, and then link them together with the help of the NHS-PEG-Maleimide bridge or through EDC-NHS linkage. This way, we could still have the IONPs and the antibody linked together for specific targeting.

For the antibody, we choose to use the scFv domain of the anti HER2 antibody, who had been proved to be properly expressed in the SHuffle strain E. coli. So we integrate the scFv domain of the anti HER2 into a vector with the functional groups for linkage and separation. Then, the vector is transformed into the SHuffle strain E. coli for protein expression and folding. After purification, the antibody was linked to the IONPs. And finally we tested the cytotoxicity of the IONPs with or without the antibody linked to it, indirectly tested the affinity of the antibody to the HER2 receptor.

Beyond that, we also designed the fluorescent staining method to test the affinity of the antibody to the HER2 receptor. And doxorubicin junction method was also designed to test the cytotoxicity of the IONPs, though this part of experiment haven't been conducted by us due to the limited time.

Our Experiment Flow Chart

  • Genetically edited E. coli is used to synthesize Iron Oxide Nanoparticles (IONPs) by adding Fe3+ for induction.
    After leaving the system in shaker for 1-2 days, the IONPs are separated from the culture by untrasonification and filtration method designed and tested by us own.
    The success of the synthetic process were verified by TEM and DLS analysis.
  • Here, several vectors contain the scFv domain of anti HER2 antibody and the functional groups for linkage and separation are designed by us. Then, the vectors are transformed into SHuffle strain E. coli for protein expression and folding.
    After the expression, the protein is purified by His-trap column and the success of the purification is verified by SDS-PAGE.
  • Since now we had the IONPs and the scFv domain of anti HER2 antibody, we need to link them together.
    In total two methods are optimized and used by us, one is the direct linkage between the carboxyl group on the nanoparticle and the amine group on the antibody, which is done with the help of EDC/NHS to have a milder reaction condition.
    For the other one, the carboxyl group on the nanoparticle and the thiol group on the antibody are used for linkage through NHS-PEG-Maleimide. For better result, in our vector design, one group with a cystine added before and after the polyhis tag is added to the antibody. This way, the antibody can be linked to the nanoparticle without affecting its binding domain too much.
    To better verify the linking method, positive control using chemically synthesized IONPs with citric acid coating were used for the linkage.
  • To this stage, we had the IONPs with the antibody linked to it. Multiple controlled experiment were conducted to verify the affinity of the antibody to the HER2 receptor and the cytotoxicity of the IONPs. Confocal analysis and flow cytometry were used for visualization of the antibody affinity. And CCK-8 assay was used for the cytotoxicity examination.

Software Design

We designed two softwares in order to achieve high throughput data analysis.

The first one is Particle Size Distribution Counter for Transmission Electron Microscope image analysis. This software could automatically generate the plot for the size distribution of the nanoparticles in the TEM images. By "fine-tuning" image processing parameters, this software could be used for different types of materials observed under microscopes beyond just nanoparticles under TEM. To utilize this software effectively, users only need to upload image files into the software and set a scale bar for software to process. It helps to significantly decline great amount of repetitive work. Furthermore, the software processes the graph, extracting particle size information and generating a statistical distribution graph, both the statistical distribution graph and the schematic diagram indicating the particles included in the statistics would be shown as the output.

For example, the following image is the output from this software when a TEM image of the IONPs is uploaded, and the size distribution figure of the IONPs is shown below:

Output of the software for one sample TEM figure

Figure 1.Output of the software for one sample TEM figure


For the second one, we have developed a script specifically designed for high-throughput analysis of confocal image fluorescence intensity, inspired by tedious data processing in wet lab experiments. This script provides the high throughput processing of TIF images using macro language in Fiji. With our program, we can directly perform automatic normalization and statistical analysis of fluorescence intensity for all images in a given folder, significantly reducing repetitive workload and streamlining the process. Actually, you can change the core codes with different functions in this high-throughput frame. Besides, figures with specific nomenclature could be picked out to be analyzed by this program, which is efficient and time-saving when only a subgroup of the data is needed for analysis.

Size Distribution Output Figure

Figure 2. Size Distribution Data Output Figure


Beyond Experiment

As our project focuses on developing a bio-synthesis method for iron oxide nanoparticles for targeted cancer therapy. We have collaborated with medical professionals in oncology, radiotherapy, and surgery, as well as experts in nanotechnology and synthetic biology. Safety is a central theme, addressing the limitations and side effects of existing therapies like chemotherapy. By utilizing iron oxide nanoparticles as drug carriers, we aim to enhance specificity and minimize impact on healthy cells. Rigorous testing protocols and in vitro studies assess biocompatibility and toxicity. Stakeholder engagement with patient advocacy groups, healthcare providers, and pharmaceutical companies helps shape our research and development process, prioritizing safety. Overall, our human practices ensure the safety, efficacy, and accessibility of our proposed solution for cancer therapy.



Reference


Biosynthesis of IONPs:

[1]. Crespo, K. A., Baronetti, J. L., Quinteros, M. A., Páez, P. L., & Paraje, M. G. (2017). Intra- and Extracellular Biosynthesis and Characterization of Iron Nanoparticles from Prokaryotic Microorganisms with Anticoagulant Activity. Pharmaceutical research, 34(3), 591–598. https://doi.org/10.1007/s11095-016-2084-0
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[3]. Fatemi, M., Mollania, N., Momeni-Moghaddam, M., & Sadeghifar, F. (2018). Extracellular biosynthesis of magnetic iron oxide nanoparticles by Bacillus cereus strain HMH1: Characterization and in vitro cytotoxicity analysis on MCF-7 and 3T3 cell lines. Journal of biotechnology, 270, 1–11. https://doi.org/10.1016/j.jbiotec.2018.01.021

Nanoparticle Separation:

[4]. Muraoka, S., Lin, W., Chen, M., Hersh, S. W., Emili, A., Xia, W., & Ikezu, T. (2020). Assessment of separation methods for extracellular vesicles from human and mouse brain tissues and human cerebrospinal fluids. Methods (San Diego, Calif.), 177, 35–49. https://doi.org/10.1016/j.ymeth.2020.02.002
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[7]. Tian, X., Ruan, L., Zhou, S., Wu, L., Cao, J., Qi, X., Zhang, X., & Shen, S. (2022). Appropriate Size of Fe3O4 Nanoparticles for Cancer Therapy by Ferroptosis. ACS applied bio materials, 5(4), 1692–1699. https://doi.org/10.1021/acsabm.2c00068

Vector Design and Bacteria Strain Selection for Protein Expression:

[8]. Ahmadzadeh, M., Farshdari, F., Nematollahi, L., Behdani, M., & Mohit, E. (2020). Anti-HER2 scFv Expression in Escherichia coli SHuffle®T7 Express Cells: Effects on Solubility and Biological Activity. Molecular biotechnology, 62(1), 18–30. https://doi.org/10.1007/s12033-019-00221-2
[9]. Braakman, I., & Bulleid, N. J. (2011). Protein folding and modification in the mammalian endoplasmic reticulum. Annual review of biochemistry, 80, 71–99. https://doi.org/10.1146/annurev-biochem-062209-093836
[10]. Rashid M. H. (2022). Full-length recombinant antibodies from Escherichia coli: production, characterization, effector function (Fc) engineering, and clinical evaluation. mAbs, 14(1), 2111748. https://doi.org/10.1080/19420862.2022.2111748
[11]. Robinson, M. P., Ke, N., Lobstein, J., Peterson, C., Szkodny, A., Mansell, T. J., Tuckey, C., Riggs, P. D., Colussi, P. A., Noren, C. J., Taron, C. H., DeLisa, M. P., & Berkmen, M. (2015). Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria. Nature communications, 6, 8072. https://doi.org/10.1038/ncomms9072
[12]. Schöttler, S., Becker, G., Winzen, S., Steinbach, T., Mohr, K., Landfester, K., Mailänder, V., & Wurm, F. R. (2016). Protein adsorption is required for stealth effect of poly(ethylene glycol)- and poly(phosphoester)-coated nanocarriers. Nature nanotechnology, 11(4), 372–377. https://doi.org/10.1038/nnano.2015.330

Application of IONPs:

[13]. Omura T. (1998). Mitochondria-targeting sequence, a multi-role sorting sequence recognized at all steps of protein import into mitochondria. Journal of biochemistry, 123(6), 1010–1016. https://doi.org/10.1093/oxfordjournals.jbchem.a022036
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[16]. Ali, A., Zafar, H., Zia, M., Ul Haq, I., Phull, A. R., Ali, J. S., & Hussain, A. (2016). Synthesis, characterization, applications, and challenges of iron oxide nanoparticles. Nanotechnology, science and applications, 9, 49–67. https://doi.org/10.2147/NSA.S99986

NHS-PEG-Maleimide Linkage:

[17]. Zhou, H., Fan, Z., Lemons, P. K., & Cheng, H. (2016). A Facile Approach to Functionalize Cell Membrane-Coated Nanoparticles. Theranostics, 6(7), 1012–1022. https://doi.org/10.7150/thno.15095

EDC/NHS Linkage:

[18]. Saei, A., Asfia, S., Kouchakzadeh, H., & Rahmandoust, M. (2020). Antibody-modified magnetic nanoparticles as specific high-efficient cell-separation agents. Journal of biomedical materials research. Part B, Applied biomaterials, 108(6), 2633–2642. https://doi.org/10.1002/jbm.b.34595
[19]. Hu, F., Chen, K., Xu, H., & Gu, H. (2018). Design and preparation of bi-functionalized short-chain modified zwitterionic nanoparticles. Acta biomaterialia, 72, 239–247. https://doi.org/10.1016/j.actbio.2018.03.038

IONPs Antibody Conjugation Method beyond EDC/NHS and NHS-PEG-Maleimide:

[20]. Lee, C., & Kang, S. (2021). Development of HER2-Targeting-Ligand-Modified Albumin Nanoparticles Based on the SpyTag/SpyCatcher System for Photothermal Therapy. Biomacromolecules, 22(6), 2649–2658. https://doi.org/10.1021/acs.biomac.1c00336
[21]. D'Agata, R., Palladino, P., & Spoto, G. (2017). Streptavidin-coated gold nanoparticles: critical role of oligonucleotides on stability and fractal aggregation. Beilstein journal of nanotechnology, 8, 1–11. https://doi.org/10.3762/bjnano.8.1
[22]. Ren, W. X., Han, J., Uhm, S., Jang, Y. J., Kang, C., Kim, J. H., & Kim, J. S. (2015). Recent development of biotin conjugation in biological imaging, sensing, and target delivery. Chemical communications (Cambridge, England), 51(52), 10403–10418. https://doi.org/10.1039/c5cc03075g
[23]. Hersch, N., Wolters, B., Ungvari, Z., Gautam, T., Deshpande, D., Merkel, R., Csiszar, A., Hoffmann, B., & Csiszár, A. (2016). Biotin-conjugated fusogenic liposomes for high-quality cell purification. Journal of biomaterials applications, 30(6), 846–856. https://doi.org/10.1177/0885328215603026

Targeting of IONPs:

[24]. Korangath, P., Barnett, J. D., Sharma, A., Henderson, E. T., Stewart, J., Yu, S. H., Kandala, S. K., Yang, C. T., Caserto, J. S., Hedayati, M., Armstrong, T. D., Jaffee, E., Gruettner, C., Zhou, X. C., Fu, W., Hu, C., Sukumar, S., Simons, B. W., & Ivkov, R. (2020). Nanoparticle interactions with immune cells dominate tumor retention and induce T cell-mediated tumor suppression in models of breast cancer. Science advances, 6(13), eaay1601. https://doi.org/10.1126/sciadv.aay1601
[25]. Juan, A., Cimas, F. J., Bravo, I., Pandiella, A., Ocaña, A., & Alonso-Moreno, C. (2020). Antibody Conjugation of Nanoparticles as Therapeutics for Breast Cancer Treatment. International journal of molecular sciences, 21(17), 6018. https://doi.org/10.3390/ijms21176018
[26]. Toporkiewicz, M., Meissner, J., Matusewicz, L., Czogalla, A., & Sikorski, A. F. (2015). Toward a magic or imaginary bullet? Ligands for drug targeting to cancer cells: principles, hopes, and challenges. International journal of nanomedicine, 10, 1399–1414.
[27]. Chen, H., Wang, L., Yu, Q., Qian, W., Tiwari, D., Yi, H., Wang, A. Y., Huang, J., Yang, L., & Mao, H. (2013). Anti-HER2 antibody and ScFvEGFR-conjugated antifouling magnetic iron oxide nanoparticles for targeting and magnetic resonance imaging of breast cancer. International journal of nanomedicine, 8, 3781–3794. https://doi.org/10.2147/IJN.S49069

Antibody Affinity Assessment:

[28]. Ren, W. X., Han, J., Uhm, S., Jang, Y. J., Kang, C., Kim, J. H., & Kim, J. S. (2015). Recent development of biotin conjugation in biological imaging, sensing, and target delivery. Chemical communications (Cambridge, England), 51(52), 10403–10418. https://doi.org/10.1039/c5cc03075g

Drug Delivery:

[29]. Chen, H., Wang, L., Yu, Q., Qian, W., Tiwari, D., Yi, H., Wang, A. Y., Huang, J., Yang, L., & Mao, H. (2013). Anti-HER2 antibody and ScFvEGFR-conjugated antifouling magnetic iron oxide nanoparticles for targeting and magnetic resonance imaging of breast cancer. International journal of nanomedicine, 8, 3781–3794. https://doi.org/10.2147/IJN.S49069
[30]. Kovach, A. K., Gambino, J. M., Nguyen, V., Nelson, Z., Szasz, T., Liao, J., Williams, L., Bulla, S., & Prabhu, R. (2016). Prospective Preliminary In Vitro Investigation of a Magnetic Iron Oxide Nanoparticle Conjugated with Ligand CD80 and VEGF Antibody As a Targeted Drug Delivery System for the Induction of Cell Death in Rodent Osteosarcoma Cells. BioResearch open access, 5(1), 299–307. https://doi.org/10.1089/biores.2016.0028