Since the target of our project is to synthesize the Iron Oxide Nanoparticles using biological method and achieve specific targeting towards the breast cancer cell, we designed four different plasmid expressing the carefully selected scFv domain of anti-HER2 antibody and the specific functional domain to enable the purification and conjugation of the antibody, specific bacteria strain was selected to ensure the proper folding of the antibody.

For the future work, when the coating composition of the iron oxide nanoparticles is better determined and the synthetic mechanism of IONPs is better understood, designs of the plasmid can be further optimized to enabling the expression termial of antibody to be the same site as IONPs, along with addition of functional groups in the plasmid design, self assembly of the antibody and IONPs may become possible, which will greatly improve the efficiency for large scale prodection.

E. coli Strain Adoption

Antibodies were required to achieve our goal to target cancer cells. We managed to synthesize antibodies in E. coli by constructing, inducing external plasmids to express desired antibodies and took advantage of its low cost, simplicity, and high yield. However, it was not easy to validly express an eukaryotic gene in a prokaryotic cell due to its simpler inner cell structure, lacking of endoplasmic reticulum and Golgi apparatus, which were crucial to processing protein. These deficiencies might lead to incorrect structural features of antibodies. Besides, in wild type strain of E. coli, the reducing cytoplasm would cause the formation of inclusion bodies, a kind of abnormal structure. Considering that oxidizing environment was important to form disulfide bond, which was indispensable for the biological activity of antibodies. To ensure antibody validity, we therefore adopted SHuffle strain of E. coli, whose cytoplasm was semi-oxidizing, facilitating successful expression of biologically active antibodies in E. coli.

General Idea for Plasmid Construction

To fulfill successful expression of antibodies, plasmid construction was a crucial part. Focusing on breast cancer, we selected HER2 as our targeting site. The pGLO plasmid we used had been verified to be able to express protein since the expressed green-fluorescent protein (GFP) was visible to naked human eyes.
It was plausible that our antibodies could also be expressed since we only altered the protein-coding region. Therefore, we replaced the original protein coding gene with anti-HER2 scFv coding gene. The coding gene was derived by protein-DNA sequence conversion optimization, in frame with 6x His-tag to detect and also purify the expressed protein.

Specific Design in Plasmid Construction for Streptavidin-Biotin Conjugation

As the Streptavidin-Biotin linkage is a very strong interaction in nature, we also had a vector design trying to utilize this binding effect.
By combining the scFv, streptavidin, and His tag into a single plasmid, we can leverage the advantages of each component to enhance our research and development efforts. The scFv provides us with a highly specific and versatile antibody fragment, capable of recognizing various targets of interest. Streptavidin, on the other hand, offers a strong and stable interaction with biotinylated nanoparticles, allowing for efficient targeting and delivery. Lastly, the His tag serves as a well-established purification tag, enabling rapid and reliable protein purification using affinity chromatography.

Test Results of Our Plasmids

We introduced our plasmids into E. coli respectively and induced them to express the protein. The results showed that except for the scFV-streptavidin-his protein was found to be in the inclusion body, all other types of the protein was successfully expressed and purified and gave a satisfying concentration around 1-5 mg/ml after solution displacement to storage buffer.
Then, We conducted the experiment of linking the antibody to the chemically synthesized Iron Oxide Nanoparticles, comparing to the chemically synthesized Iron Oxide Nanoparticles without antibody addition, the cytotoxicity of the group with antibody linkage treatment was higher than the group without the linkage process, indirectly indicating partial bioactivity of the scFv protein.
And the detailed result of our experiment could be found in our result page, especially the cytotoxicity test result and protein purification result.

Future from Our Tests and Other Prospects

For the results of our tests, one thing we could do for the alternative of the scFv-streptavidin-his design is to remove the streptavidin in the design but using chemical method to link the biotin to the antibody, and then link the biotinylated antibody to the avidin linked iron oxide nanoparticles.
One promising direction for future work is to optimize the design of plasmids to enable the expression terminal of antibodies to be located at the same site as the IONPs. This strategic modification would facilitate the self-assembly of antibodies and IONPs if the process is made possible in the future, potentially leading to more efficient and targeted delivery systems. By incorporating functional groups into the plasmid design, we could enhance the binding affinity between the antibodies and IONPs, thereby improving the overall performance of the nanocomplex.
The successful self-assembly of antibodies and IONPs holds great potential for large-scale production. With this advancement, we could streamline the manufacturing process and significantly increase the efficiency of nanoparticle production. Additionally, the ability to self-assemble the components may offer new opportunities for tailoring the properties of the nanocomplex, such as controlling the release kinetics of drugs or enabling specific interactions with biological targets.
The present development in biologically synthesized IONPs offers promising avenues for future research. By further refining the coating composition, understanding the synthetic mechanism, and optimizing plasmid design, scientists are paving the way for improved efficiency, stability, and functionality of these nanoparticles. Ultimately, these advancements hold great potential for revolutionizing various fields, including targeted drug delivery, biomedical imaging, and therapeutics.