**The body of the text was never fully completed, but there is a complete Power Point and poster presentation attached at the bottom.**
My research experience in the summer of 2010 was in Professor Dennis Discher's biophysical engineering and nanobiopolymers lab. This lab is located at University of Pennsylvania in room 129 of the Towne Building. My lab mentor was Abdullah Al Mahmud, postdoctoral researcher in nanobiopolymers. Another RET Nano participant, Myra-Lynn Doughty, also worked in the same lab on the same project.
The overall goal of the research experience was to determine the effectiveness of loading the insecticide bifenthrin into worm micelles formed by diblock copolymers and how the loading affects the overall stability of the worm micelles.
The key molecular component of the research was a diblock copolymer. These feature long sequences, or blocks, of the same monomer unit covalently bound to other long sequences of a different monomer unit (1 Princeton website). When one of these blocks is hydrophilic and the other is hydrophobic, a diblock amphiphile is formed that can self-assemble into one or more of the three main forms: sphere, worm, and/or vesicle. Sphere join together to form worms. Both have a single layer membrane that is hydrophilic on the outside and hydrophobic on the inside, with the inner part of the micelle being hollow. Vesicles feature a bilayer similar to a lipid bilayer, with a hydrophilic core (???).
The hydrophilic block used in this research was poly(ethylene oxide), PEO. It is used due to its biocompatibility (2 Discher, Visualization of degradable). Two hydrophobic blocks were used in the research: polycaprolactone, made with and without the presence of lactic acid, and polybutadiene. Polycaprolactone was chosen due to its biodegradability, which is key to delivery of materials to an organism (3). Polybutadiene's inert nature allows for simple control over vesicle degradation and the release times of contents (5???). In addition, the molecular weight of both the hydrophillic and hydrophobic chains as well as the polydispersity affect the ability to produce the various micelle shapes (???).
Micelle production through addition of chloroform and water....
Multiple studies on delivering cancer drugs via micelles provide a sound model for the loading of bifenthrin into diblock copolymer micelles. The compact inner cores of the micelles serve as containers for drug encapsulation and as the method for controlled release of hydrophobic drugs. The hydrophillic outer layer of the micelles serve as a barrier between the drug and tissues, making it much easier for the body to take in drugs. Worm micelles in particular can last tens of hours or days in vivo. (4??). Due to the circulation time, passive acculmulation of the micelle and drug combo occurs, and a shrinking of tumors has been shown with such drugs as DOX and TAX (6???). Furthermore, the biodegradable nature of polymers like polycaprolactone make them a benign carrier of therapeutics. (4??)
The insecticide bifenthrin is a member of the synthetic pyrethroid pesticide family. It affects the central and peripheral nervous systems of insects, causing paralysis (7???). It is used as a pesticide with a wide variety of applications, from being used to kill a wide variety of organisms, as featured in Ortho Home Defense MAX Perimeter and Indoor Insect Killer, to being used to specifically kill single organisms, like the fire ant, as featured in Scotts Bonus S Max Southern Weed and Feed and Ant Killer. Bifenthrin can be absorbed into plants (tea paper). It is moderately toxic to mammals (cornell webiste #10) and birds (cornell 1), while having low water solubility and a high affinity for soil (cornell 2,3, 7???xxx). The most toxic nature of bifenthrin comes from its ability to be biologically magnificatied as a result of high toxicity in aquatic organisms, including fish, like the bluegill that has a bioaccumulation factor of 6000x (??? or cornell 1,2). The copolymer coating on bifenthrin should make it too large to enter plant pores while also making it soluble in water. Other surfactants have been tested as coatings for pesticides like bifenthrin, but they tend to be toxic and require great amounts to solubilize the compound (XXXXXX).
The lab work involved the usage and mastering of many lab tools were sometimes familiar and sometimes unfamiliar. There was a refining of previously learned techniques in the area of lab equipment use, specifically the stir plate, digital balance, micropipetters, and microscope slide preparation. There was the acquisition of lab preparation techniques, specifically in how to prepare copolymer solutions from stock raw copolymers to be assessed by the methods of solvent evaporation and film rehydration. In order to examine the resulting products from these techniques, there was a training on how to use the Nikon Eclipse TE300 microscope for fluorescent microscopy purposes. Two computer programs were learned as well: Image Pro Plus for acquiring both still and video images from the microscope camera Roper Scientific PVCAM and Image J for analyzing data from microscope, such as measuring contour length of worm micelles in samples. When working with bifenthrin, distilliation and dialysis methods were utilized. There was also necessity to learn how to use a vortex mixer (Fisher Scientific model) and a centrifuge (Labnet's Spectrafuge 24D) because all samples needed to be vortexed, while preparation for high performace liquid chromatography (HPLC) required the centrifuge. To examine the results of the bifenthrin loading based on concentration of bifenthrin remaining within worm micelles, HPLC was performed, with the results analyzed via Shimadzu LC Solution program.
Research Methods and Results
In order to discover how well bifenthrin loaded into worm micelles as well as the micelle stability in bifenthrin's presence, the methods for producing long and flexible worm micelles had to be determined.
First, a comparison of solvent evaporation (both with and without a heating step) and film rehydration methods for producing worm micelles was done on five different copolymers (from here on out, any PEO and polycaprolactone copolymers will be listed as OCL or OCLA, with the latter used in presence of lactic acid; any PEO and polybutadiene will be listed as OB; the numbers next to the copolymers represent the range of molecular weights in the thousands): OCL 2-3, OCL 2-9, OCL 2-12, OCL 2-14, OB 18, and OB 4-6. Analysis of data, as shown in table 1, revealed that solvent evaporation produced more worms, with worms being most prevalent in OCL 2,9, OCL 2,12, OCL 2,14, and OB 4,6.
The second battery of experiments centered around answering two questions:
1. How does the molecular weight of OCL copolymers affect the contour length of worm micelles produced by the copolymers?
2. How is rigidity of OCL copolymers affected by (a) worm micelle length and (b) copolymer molecular weight?
The diblock copolymers used were OCL 2-6, OCL 2-9, OCL 2-12, OCLA 2-9, and OCLA 2-12. OCL was used exclusively here due to its biodegradability. OCL 2-14 was not tested because of its high percentage rigidity and lack of movement already observed. As seen in graph 1, OCL 2-12 had the longest contour length, at 16.63 micrometers. Graph 2 reveals that XXXX had XXXX.
The final experiments answered the following:
1. How well does bifenthrin load into worm micelles?
2. How stable are the worm micelles after loading of bifenthrin?
For these experiments, two separate concentrations of diblock copolymers were used to show differences in numbers of worm micelle production: 1.25 mg/mL and 4 mg/mL. The three diblock copolymers tested were OCL 2-12, OCLA 2-12, and OB 4-6.
1 Block Copolymers http://www.princeton.edu/~polymer/block.html
2 Y. Geng and D.E. Discher. Visualization of degradable worm micelle breakdown in relation to drug release. Polymer 47 (2006): 2519-2525.
3 A. Lavasanifar, J. Samuel, and G. Kwon. Poly(ethylene oxide)-block-poly(L-amino acid) micelles for drug delivery. Advanced Drug Delivery Reviews 54 (2002):169-190.
4?? ....Nanopolymeric therapeutics
5?? Discher Emerging Applications...
6???nano thera note 119
7???mOKRET HOAGLAND 1989.... (bif pdf)