Block Copolymer Construction by SPPS

Nano-Engineering Block Copolymer Micelles as Novel Drug Delivery VehiclesGary 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 insolublepolymer bead ( ).

The backbone ( ) and side-chain ( )-protectedamino acid ( ) is attached to the bead.

The backbone is deprotected to accommodate thenext amino acid.

The “incoming” amino acid is coupled to the resinbound amino acid.

The cycle of backbone deprotection and coupling iscontinued until the PAA (poly(amino acid)) core iscomplete.

The PAA core is cleaved from the resin and theside chain protecting groups removed.

Activated PEO (poly(ethylene oxide)) is condensedto the PAA core to form the block copolymerunimer.

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.

BA

Hydrophobic BlockHydrophilic Block

- Forms insoluble core- Stores drug

- Forms soluble shell- Interacts with the body

Poly(ethylene oxide)-block-Poly(amino acid)

(PEO-PAA)

=

100 m

H2N-PEO5000-GFLYWFLY

Micelle Architecture

Investigation: Core Length and Composition

The first set of block copolymers were of constant composition and variable core-length, PEO5000-GYn, (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, PEO5000-G(X)15. As shown in the above figure, PEO5000-G(FLYW)3 displayed the highest CMC value (0.20 mg/ml), whereas the block copolymer unimer PEO5000-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.

NH2

OH

O

O OH

OO OH

OH

O

O

OH

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

PEO5000

-GY15

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

Co

un

ts P

er M

inu

te

[ Block Copolymer Unimer (mg/ml) ]

PEO5000-GY15

PEO5000-GL15

Block Copolymer CMCs(Constant Core Composition)

Sca

tter

ing

In

ten

sity

(A

rbit

rary

Un

its)

[ Block Copolymer Unimer (mg/ml) ]

PEO5000-GY12

PEO5000-GY7

PEO5000-GY9

†gary.vandomselaar@ualberta.ca ‡david.wishart@ualberta.ca

PEO5000-GY15

Construct CMC (mg/ml)

0.05

0.08

0.19

0.46

Construct CMC (mg/ml)

PEO5000-G(FLYW)3FLY

PEO5000-GF15

0.20

0.05

0.019

0.014

PEO5000-GY15

Investigation: Drug Release Rate

0 1 2 3 5 6 7101520253035404550556065707580859095

100

Time (day)

Mic

ell

e D

iss

oc

iati

on

(%

)

PEO5000-GY15

PEO5000-GY12PEO5000-GF15