Contribution

Overview

We designed a method for the separation of the Iron Oxide Nanoparticles by taking the advantage of ultrasonication and ultrafiltration. This method could relatively precisely separate the nanoparticles of desired size from the biological masses, resulting in a satisfying low polydispersity index, which is also an important factor to control for application. What is especially important is that the method designed by us is nicely suitable for preliminary test, as this method largely reduced the interference factors like salt ions and aggregation in the characterization like TEM and DLS. Several repeat experiments conducted by us showed that the method is easily repeatable. It is also a green way which don't involve the usage of hazardous chemicals.

Feasible Nanoparticle Separation Method from Biological Masses

We designed a method for the separation of the Iron Oxide Nanoparticles by taking the advantage of ultrasonication and ultrafiltration. This method could relatively precisely separate the nanoparticles of desired size from the biological masses, resulting in a satisfying low polydispersity index, which is also an important factor to control for application. What is especially important is that the method designed by us is nicely suitable for preliminary test, as this method largely reduced the interference factors like salt ions and aggregation in the characterization like TEM and DLS. Several repeat experiments conducted by us showed that the method is easily repeatable. It is also a green way which doesn't involve the usage of hazardous chemicals.

NHS-PEG-Maleimide Bridge

There is a facile approach used to functionalize cell membrane-coated nanoparticles, which gives us inspiration in our crucial part, namely antibody-nanoparticle conjugation. Since our iron-oxide nanoparticles are formed via intrinsically protective mechanism from E. coli, and based on the fact that the iron-oxide nanoparticles are membrane-bounded, we speculate that the composition of this membrane is probably highly similar to biologically lipid bilayer, with a vast amount of membrane proteins inlaid. This facile approach utilized amine groups on membrane proteins and thiol groups in certain thiolated enzymes, adopting NHS-PEG-Maleimide as an intermediate molecule, fulfilling conjugation process via amine-NHS reaction and thiol-maleimide reaction, which is also applicable for our situation. However, due to the concern about losing the biological activity of our antibodies, we are not going to thiolate our antibodies by directly using Traut's reagent, but add a cysteine at the end of our antibodies to provide a reactive thiol group, ensuring the smooth completeness of conjugation process. This is a versatile approach that can be applied in various types of conjugation, including ADC (antibody-drug conjugation) and any other kinds of protein-related conjugation.

Mysterious Inclusion Body Formation

pGLO plasmid is able to express green-fluorescent protein (GFP), which can be observed by human naked eyes. Furthermore, we have successfully produced our cysteine-added his-tagged anti-HER2 scFv by replacing GFP-sequencing region with our design scFv sequence.

However, for our back-up conjugation strategy, biotin-streptavidin conjugation, which is a kind of tight conjugation, it is failed to express streptavidin in E. coli, forming intrusion bodies rather than producing normal streptavidin. However, the exact reason is still unknown. Our speculations are that the environment of SHuffle strain of E. coli is not oxidizing enough and pGLO plasmid is somehow not suitable for streptavidin expression. Usually, when the level of expression of protein goes beyond 2% of the total cellular proteins, it leads to inclusion Body formation. What's more, lack of eukaryotic chaperones such as ClpB and post-translational machinery also contribute to the formation of intrusion bodies, and proteins with more highly hydrophobic proteins having more chances to aggregate into inclusion bodies. It reminds us and others that expression environment, including cell host and expressing plasmid, is important for protein production, and different proteins have different requirements. There are several resolutions that might work, including slowing down the expression of proteins by reducing temperature, the concentration of the inducer and the amount of vectors used, adding protein tags which makes it more soluble, and using vectors with weaker promoter. What need to mention is that solubilize and refold the protein of interest from the intrusion bodies is also a possible solution to get target protein.