Douglas S. Lewis and Dr. William G. Pitt, Chemical Engineering

Introduction

In the past decade, many efforts have been made to improve chemotherapeutic treatments for cancer patients. One of the specific efforts seeks to actively deliver anti-cancer drugs to the target tissue rendering the chemotherapy more efficient and less damaging to nearby uninfected cells. The lab of Dr. William Pitt is currently investigating the site-directed delivery of drugs using polymeric nanocarriers that release their contents upon irradiation with ultrasound (US).1-4 In a typical application without active targeting, drug sequestered in a carrier is infused into the circulatory system and the US is focused on the tumor site, such that as the polymeric carriers flow through the insonated volume, they release their contents to the tumor tissue. The downside to this current approach is only 10% of the drug content is released per insonation event. This observation led to the hypothesis that if it were possible to localize all of the polymeric carriers in the insonated volume, the efficiency of drug release could be greatly improved through multiple insonation events. This current project seeks to build on a previous ORCA project in which attachment of antibody (Fab’) fragments to poly(ethylene glycol) (PEG) chains was demonstrated.5 We have since worked to attach Fab’ fragments to Pluronic-105 chains, the principal component of our drug delivery vehicle. This report addresses the progress we have made in this direction.

Synthesis

The synthesis of the antibody-conjugated P-105 micelles can be divided into three phases: 1) Micelle Stabilization 2) P-105 Activation and 3) Fab’ Preparation and Attachment. A summary of this procedure follows below.

Micelle Stabilization – Pluronic micelles are stabilized by adding 0.5 wt% N,N-diethyl acrylamide (NNDEA), N,N-Bis(acryloyl)cystamine (BAC) crosslinker in a 1:20 molar ratio of BAC:NNDEA, and AIBN initiator to a 10 wt% Pluronic solution, and polymerizing at 65°C under nitrogen for 24 hours.

P-105 Activation – The stabilized micelles are then freeze-dried and dissolved in dry dichloromethane together with a 10-fold excess of acryloyl chloride and 2-fold excess of triethylamine and stirred under nitrogen overnight at 25°C. The resulting P-105-acrylate was dried with anhydrous MgSO4 and subsequently precipitated in ether. A small portion was characterized by H-NMR while the rest was stored at -20°C.6

Fab’ Preparation and Attachment – Anti-Human Albumin Antibody isolated from a goat was purchased from Sigma. The Fab’ fragments were prepared using an ImmunoPure Fab’ Preparation Kit (Pierce, Rockford, IL). After pooling collected fractions, the fragments were immediately added to the P-105-acrylate solution with stirring for 12 hours at 25°C. The relative ratio of Fab’ fragments per micelle was determined by performing wavelength scans with a UV-vis spectrophotometer.

Fab’ Binding Assay

Due to the many steps involved in the anti-albumin Fab’ preparation and attachment, it is necessary to ensure that their binding capacity had not been compromised. A simple way of calculating the binding efficiency involves mixing the P-105-anti albumin micelles in the presence of a known concentration of human albumin solution. After gently mixing for 1 hour, the resulting solution is spun at 10,000 rpm in a centrifuge for 30 minutes to allow the micelle-Fab’-antigen complexes to form a pellet at the bottom of the centrifuge tube. The resulting albumin concentration in the supernatant was then determined with a spectrophotometer and compared to the original concentration of human albumin to determine the binding capacity of the anti-albumin.

Results and Discussion

The synthesis of P-105-Fab’ drug carriers described above presented more difficulties than simply attaching Fab’ fragments to PEG chains as previously demonstrated. Several attempts were made to attach the albumin Fab’ fragments without success. While there may be several contributing factors, steric hindrance between the Fab’s free sulfhydryls and the acrylate-capped P-105 chains is believed to be the main reason that there was no covalent attachment. The correct question to then ask is whether the three main steps of synthesis were performed in the correct order. Theoretically, it may make more sense to first cap the P-105 chains with acrylates, attach the Fab’ fragments to the acrylate-capped chains, and then polymerize the inner-penetrating network after the Fab’ fragments are already attached. While this approach may solve the problem of steric hindrance, it also exposes the Fab’ fragments to a greater chance of denaturation considering that the polymerization normally takes place at 65 °C.

Conclusion

The synthetic pathway described in this report does not produce the results that were originally expected. There may be a way to still produce the same end result by changing the order of the synthetic steps to avoid problems of steric hindrance. Future work on this project will involve tweaking these steps in order to identify the best avenue for synthesis. If future attempts prove successful, in vitro and in vivo tests will surely follow.

References

• Zeng, Y et al. J Biomat Sci Polym Edn 17, 591-604 (2006).
• Pruitt, JD et al. Macromolecules 33, 9306-9 (2000).
• Husseini, GA et al. J Control Rel 69, 43-52 (2000).
• Husseini, GA et al. J Control Rel 107, 253-61 (2005).
• Lewis, DS et al, 2006 BYU Undergraduate Student Journal
• Hahn, MS et al. Biomaterials 27, 2519-24 (2006).