1. Block Copolymer Construction by SPPS Nano-Engineering Block Copolymer Micelles as Novel Drug Delivery Vehicles Gary H. Van Domselaar †, Lena C. Andrew, David S. Wishart ‡ Departments of Computing Science and Biological Sciences University of Alberta Unimer Composition Abstract Acknowledgments and References Amphipathic block copolymers are linear polymeric molecules (called unimers) consisting of alternating hydrophobic and hydrophilic segments. Under the proper conditions of temperature, concentration, solvent selectivity, and polymer composition, the block copolymer unimers can self- assemble into extraordinarily stable micellar suspensions. This phenomenon has prompted researchers to investigate the use of biocompatible block copolymers as delivery systems for hydrophobic small molecule drugs. Solid Phase Peptide Synthesis (SPPS) has been used to construct block copolymer unimers with a poly(ethylene oxide) (PEO) hydrophilic shell and a hydrophobic peptide core. This approach is advantageous as it allows for fine control over the construction of the block copolymer hydrophobic section. The size and composition of the peptide segment can modulate block copolymer aggregation and stability, as well as impart specific drug affinity to the micelles. To demonstrate the utility of SPPS in the construction and analysis of block copolymer micelles, two sets (one of constant composition and variable core-length, the other of constant length but of variable composition) of novel unimers were assembled and their micellization properties investigated.  The amino acid core is assembled on an insoluble polymer bead ( ).  The backbone ( ) and side-chain ( )-protected amino acid ( ) is attached to the bead.  The backbone is deprotected to accommodate the next amino acid.  The “incoming” amino acid is coupled to the resin boundamino acid.  The cycle of backbone deprotection and coupling is continued until the PAA (poly(amino acid)) core is complete.  The PAA core is cleaved from the resin and the side chain protecting groups removed.  Activated PEO (poly(ethylene oxide)) is condensed to the PAA core to form the block copolymer unimer. When exposed to aqueous solvents, block copolymers can spontaneously assemble into micellles. These micelles are characterized by a dilute, highly hydrated shell surrounding an essentially solvent-excluded core. Micelles of this nature range in size from approximately 15 nm up to about 100 nm, which is on the same order of size as the smaller viruses, such as poliovirus. Amphipathic block copolymers consist of a linear arrangement of alternating hydrophilic and hydrophobic segments. The composition of these segments is a critical factor in the design of pharmaceutically-useful block copolymers for drug delivery. Specifically: - each block should be biocompatible, and preferably biodegradable, - the shell-forming block should be “stealthy”, allowing it to circulate in vivo for long periods of time, and - the core-forming block must have good drug loading and release properties. Typical block copolymers for drug delivery incorporate PEO (poly(ethylene oxide)) as the shell- forming block, due to its inertness in vivo. PEO is distinguished as being FDA-approved for in vivo use. A wider range of core-forming block compositions are being investigated, but most research activity is focussed on amino acid cores and their derivatives. B A Hydrophobic Block Hydrophilic Block - Forms insoluble core - Stores drug - Forms soluble shell - Interacts with the body Poly(ethylene oxide)-block-Poly(amino acid) (PEO-PAA) = 100  m H 2 N-PEO 5000 -GFLYWFLY Micelle Architecture Investigation: Core Length and Composition The first set of block copolymers were of constant composition and variable core-length, PEO 5000 -GY n, (n=7,9,12,15). The scattering data clearly shows that for the polytyrosine cores studied, critical micelle concentrations decreased with increasing core length. The other set of block copolymer constructs consisted of cores of constant length but of variable composition, PEO 5000 -G(X) 15. As shown in the above figure, PEO 5000 -G(FLYW) 3 displayed the highest CMC value (0.20 mg/ml), whereas the block copolymer unimer PEO 5000 - G(L) 15 displayed the lowest minimum concentration for micellar assembly, 0.014 mg/ml. These results indicate that for the amino acid cores investigated, critical micelle concentrations were sensitive to variations in core composition. Micelle Formation and Drug Loading Micellization is achieved by dialyzing the block copolymers from a non-selective solvent (dimethylformamide or dimethylacetamide) into a selective solvent (aqueous buffer). Drug loading of the micelles is achieved by placing the hydrophobic drug in solution with the block copolymers prior to dialysis. Upon micellization, the drug becomes embedded within the cores of the micelles. A drug release rate study was performed on all the block copolymer constructs loaded with doxorubicin. The drug loaded micelles were incubated at 37 C at a concentration below their CMC. Drug release was measured by high performance size exclusion chromatagraphy over time. Only the structures depicted above showed long term stability. One construct in particular, PEO 5000 -GY 15 showed excellent long term stability below its CMC. This study shows that PEO 5000 -GY 15 has suitable properties as a drug delivery vehicle for doxorubicin. Two sets of block copolymer constructs were assembled in order to study the impact of core length and composition on micellization and block copolymer micelle forming capability and stability. When exposed to water, all constructs spontaneously assembled into micelles, as evidenced by transmission electron micrographs. CMCs (critical micelle concentrations, the smallest concentration of block copolymer required for micellization to occur) were determined by light scattering experiments in distilled water (  =600nm). The CMC values for spontaneous micellization of the block copolymer constructs studied were found to compare favorably with the CMC values of other block copolymer micelles used for drug delivery. We would like to thank PENCE and the MRC (Rx&D-HRF and PMAC-HRF graduate scholarships) for financial support. We would also like to thank Dr. G. Kwon and Dr. D. Jette for their invaluable input. Van Domselaar G.H., Kwon G.S., Andrew L.C., and Wishart D.S. (2003) Surfaces and Colloids B: Biointerfaces 30:323- 334. G. Kwon, M. Naito, Y. Sakurai, K. Kataoka, J. Control. Rel. 1994, 32, 269-277. K. Kataoka, G. Kwon, M. Yokoyama, T. Okano, Y. Sakurai, J. Control. Rel. 1993, 24, 119-132. Block Copolymer Unimer CMCs (Constant Core Length) Doxorubicin Counts Per Minute [ Block Copolymer Unimer (mg/ml) ] PEO 5000 -GY 15 PEO 5000 -GL 15 Block Copolymer CMCs (Constant Core Composition) Scattering Intensity (Arbitrary Units) [ Block Copolymer Unimer (mg/ml) ] PEO 5000 -GY 12 PEO 5000 -GY 7 PEO 5000 -GY 9 † gary.vandomselaar@ualberta.ca ‡ david.wishart@ualberta.ca PEO 5000 -GY 15 ConstructCMC (mg/ml) 0.05 0.08 0.19 0.46 ConstructCMC (mg/ml) PEO 5000 -G(FLYW) 3 FLY PEO 5000 -GF 15 0.20 0.05 0.019 0.014 PEO 5000 -GY 15 Investigation: Drug Release Rate PEO 5000 -GY 15 PEO 5000 -GY 12 PEO 5000 -GF 15