TITLE: Preparation of Polymeric Implants for the Sustained Release of Polychlorinated Biphenyls (PCBs) and Their Derivatives
AUTHORS: Amanda J. Bullert, Hui Wang, Hansjoachim Lehmler

[DESCRIPTION]
Polymeric implants are a drug delivery system that can be grafted under the skin to allow for the continuous release of a particular substance or compound, such as polychlorinated biphenyls (PCBs) or their derivatives. In this protocol, we describe the preparation of polymeric polycaprolactone implants loaded with a hydroxylated polychlorinated biphenyl (PCB) metabolite or a related compound (i.e., biphenyl-4-ol). These implants can be grafted subcutaneously in rats to release the test compound continuously. 

[BEFORE_START]
Always wear proper Personal Protective Equipment and work in a fume hood when working with dichloromethane or PCBs. 

[STEPS]
SECTION: 1.	Formulation of polymeric implant
1. Working in a hood, add 7 g of P-80 and 0.7 g of F127 in 20 mL of dichloromethane in a 125 mL glass beaker.
SECTION: 1.	Formulation of polymeric implant
2. Add the test compound (i.e., biphenyl-4-ol or a hydroxylated PCB metabolite) to the P-80/F127 mixture in dichloromethane. 

Note: The amount of the test compound can be based on the percent of the total weight of the polymer (e.g., 1%, 5%, or 10% of the total weight, including the test compound, or 0.08, 0.41, or 0.86 g of the test compound, respectively). Alternatively, proceed directly to Step 3 to prepare the control implants with 0 % of the test compound.
6. Place the beaker in an oil bath at 70 °C to evaporate the solvent by stirring occasionally. Stirring will help the solvent evaporate faster and more evenly.

Note: An oil bath is used to prevent water condensation from getting into the solution. 
7. 
The polymer will return to its original whitish color (Picture 1) and should still be pliable; this will take about 20-30 min in the oil bath, depending on the amount of solvent added.

 
SECTION: 2.	Extrusion of the polymeric implants
8. Load the pliable polymer mixture with the desired amount of the test compound (or not test compound for the control implants) into a 5 mL disposable plastic syringe.
SECTION: 2.	Extrusion of the polymeric implants
9. Attach silicone tubing with the desired internal diameter to the syringe. The diameter of the silicone tubing will determine the final diameter of the implants.
SECTION: 2.	Extrusion of the polymeric implants
11. Force the polymer mixture into the silicone tubing until approximately 7 to 8 cm of the tubing is evenly filled with the polymer mixture. 

Note: The polymer mixture is very viscous, so there will be resistance when extruding into silicone tubing. Place the syringe back in the oven as necessary. 
SECTION: 2.	Extrusion of the polymeric implants
13. Let the extruded polymer mixture set for 24 h at room temperature. 
SECTION: 2.	Extrusion of the polymeric implants
14. Once the polymer has hardened, remove the mold by making a small incision along the silicone tubing.
SECTION: 2.	Extrusion of the polymeric implants
15. Let molded polymer sit out overnight in a fume hood to remove any residual dichloromethane.
SECTION: 2.	Extrusion of the polymeric implants
16. Cut implants to the desired length (e.g., 2 cm)
19. Store implants in labeled amber glass vials at -80°C until use.
4. Allow about 45 minutes to dissolve the polymer, resulting in a clear solution (Figure 1). 

Note: Add more dichloromethane if the solvent no longer covers the polymer pellets. The polymer dissolves faster if it forms an even layer in the beaker. 
SECTION: 1.	Formulation of polymeric implant
3. Cover the solution to prevent the dichloromethane from evaporation 
5. After 45 minutes, uncover and gently stir the polymer solution with a glass stir rod until the mixture is smooth and of a viscous consistency (this will be different depending on the amount of solvent added).
SECTION: 2.	Extrusion of the polymeric implants
10. Place the syringe with the attached silicone tubing in an oven at 70°C for several minutes until the solution is clear. 
SECTION: 2.	Extrusion of the polymeric implants
12. Replace the silicone tubing once the polymer mixture in the tubing reaches between 7 and 8 cm. Repeat this step until the syringe is empty.

 
SECTION: 2.	Extrusion of the polymeric implants
17. Place the implants in a vacuum oven for 2 h to remove residual dichloromethane (PiPiccture 3).
 
18. Weigh and measure each implant individually (Figure 3). The average diameter of implant is 2.3 ± 0.1 mm