Device for Converting Elastin-Like Polypeptide Aggregate to Soluble Form October 19th, 2005 Team Members: Eric Lee (Team Leader) John Harrison (Communications) Albert Kwansa (BSAC) Sasha-Cai Lesher-Pérez (BWIG) Client Dr. Darin Y. Furgeson Pharmaceutical Sciences School of Pharmacy University of Wisconsin-Madison Advisor Professor William L. Murphy Department of Biomedical EngineeringIntroduction The goal for our project this semester is to develop a device that will enhance the solubility of Elastin-like polypeptide (ELP) aggregate. The device will incorporate temperature control, salt extraction, and particle size reduction capabilities. In addition, the device will be engineered in ways that it is semi-automatic and requires minimal labor to operate. The motivation of the ELP project lies within our client’s research in cancer treatments. Currently, chemotherapy is one of the most effective cancer treatments available; however, the drug delivery system in chemotherapy is non-specific. Chemotherapy injects toxic chemicals into the blood stream, and the chemicals are carried throughout the body affecting both the cancer cells and normal cells. Chemotherapy patients are in a constant state of nausea, they are sick and listless, and they generally lose his/her hair. Our client, Dr. Furgeson, is researching possible drug delivery system with Elastin-like polypeptide, such that the cancer treatment will be cell specific and non-viral. Elastin-like polypeptides are artificial biopolymers that are thermal responsive. Below a characteristic transition temperature (Tt), ELPs are soluble in isotonic solution, but when the temperature is raised above the Tt, ELP hydrophobically collapses and aggregates. Interestingly, depending on the conditions, the transition of ELP between its soluble and aggregate state is totally reversible. ELP is a promising cancer drug delivery carrier due to its thermal responsive characteristic. Research is being conducted to tag the cancer drugs with the Elastin-like polypeptide such that the drug can be delivered to targeted cells. By raising thetemperature around the targeted cells via heat pad or infrared, the ELP aggregates in regions with temperatures above Tt. The targeted cells engulf the aggregate surrounding it by endocytosis. Once inside the cell, the drug-ELP complex is sent to lysosome (digestive organelle) for proteolytic cleavage of the polypeptide. The cancer treatment is harmless while it is in the drug-ELP complex, however, once the ELP is digested by the lysosome, the drug is administered to the targeted cells and acts to terminate the cellular activities. Current Method of ELP ExtractionThe current process for obtaining elastin-like protein is to first transform the gene which encodes for ELP into a bacteria plasmid. The bacteria would synthesize the specific ELP in large quantities according to the transformed gene. Once ample amount of ELP is grown within the bacterial cells, the bacterial cells are put through sonication. The sonication breaks down the cell membrane and organelles, releasing ELP into the containing solution. Extraction of ELP is done by separating the broken cell organelles from ELP by differential centrifugation. Once finished, the pellets (containing organelles) are discarded. The ELP is rinsed with PBS (phosphate buffered saline) and then placed in protein disulfide isomerase until ELP aggregates. With another cycle of differential centrifugation, further purification is achieved because the insoluble ELP aggregates are contained in the pellet and the supernatant is discarded. After purification, the ELP needs to be resolubilized. The current method is to first extract salts with ITC (inverse transition cycling). Salts lowers the transition temperature so in extracting them the transition temperature will go up and resuspension will be simpler. The next step is to pipette the ELP aggregate with cold PBS baths and continuously do this until the ELP isresuspended. A one liter culture of the bacteria typically creates 50 -100 mg of ELP, resuspending 300-600 mg will take 12 hours with the current method of continuous pipetting. This process is time consuming and ergonomically unsound because of the continuous repetition. Design Specifications The device should be able to completely automate or partially automate the resolubilization of the ELP in cold PBS solution. It should be able to recover 75 – 80% of the ELP synthesized by the bacteria. The product should be able to resolubilize at least 300 – 600 mg of the ELP in 12 hrs, which is the current rate of manual resolubilization. Temperature of the ELP material should be maintained below Tt, which may be 5oC – 75oC depending on the ELP composition and concentration (Urry, Dan W.). Construction should be of durable materials that can withstand the reduced temperatures and allow for heat transfer to cooling surroundings for temperature control. Particle size of the ELP pellet material should be reduced to maximize contact area between the ELP and the PBS solution. The device should not be too heavy and if so desired should be easily transportable by one person. It should be able to fit within the confines of a laboratory bench top or fume hood and should be secured on top of a surface to prevent slippage. Proposed Designs To transform ELP to its soluble form, we will manipulate three principle factors that affect ELP solubility: salt concentration, temperature, and particle size. Salt extraction will increase the transition temperature which will make the temperature control aspect of the mechanism easier to deal with because the PBS bath will not need tobe cooled as much as if there was salt in the solution. Particle size reduction will allow the ELP aggregate’s surface area to increase, this will cause more contact with the chilled PBS solution, increasing solubility in a more efficient form. There are five main forms of particle size reduction; crushing, grinding, pulverizing, homogenizing, and blending. Crushing and grinding are typically done with solids, crushing can be done with crystal-like structures, grinding needs to be in a solid form, typically crystal-like form. Pulverizing is the use of sonication or other such methods where the desired particles that need to be reduced can be broken with vibrations, such as cell organelles. Homogenization is the constant process of mixing and spreading the
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