(557g) Development and Characterization of Novel, Micelle-Based Parthenolide Delivery Systems

INTRODUCTION:

Parthenolide
(PTL) is effective at eradicating a number of cancer cells, including
leukemia stem cells (LSCs) but its low solubility in blood limits its use
therapeutically^1,2.  One way to circumvent this problem is
through the use of a polymeric delivery system³.  We are utilizing core-shell micelles to
enable robust PTL loading within the micelles' hydrophobic interior, and we aim
to increase PTL's solubility and circulation time in vivo with a
protective hydrophilic corona.  Using reversible addition-fragmentation chain transfer (RAFT)
polymerization, amphiphilic diblock copolymers of poly(styrene-alt-maleic
anhydride)-b-poly(styrene) (PSMA-b-PS) and poly(styrene-alt-maleic
anhydride)-b-poly(butyl acrylate) (PSMA-b-PBA) were synthesized, and
subsequently assembled into core-shell micelles for PTL delivery.  A variety of micelles made from PSMA-b-PS
and PSMA-b-PBA diblocks of different overall molecular weights and relative hydrophobic:hydrophilic
chain lengths have been characterized with respect to size, morphology, PTL
loading capabilities, stability in aqueous solution, and in vitro cytotoxicity.   

MATERIALS AND METHODS:

Diblock
copolymers of PSMA-b-PS were synthesized in a one-step polymerization by
providing an excess of styrene monomer.  Various molar ratios (18:1, 9:1, 4:1) of styrene:maleic
anhydride monomers were combined in dioxane with
4-cyano-4-(dodecylsulfanyltrithiocarbonyl) sulfanylpentanoic acid (DCT) chain
transfer agent and azobisisobutyronitrile (AIBN) thermal initiator.  The same RAFT chain transfer agent and
thermal initiators were used to synthesize diblocks of PSMA-b-PBA in two-step
polymerizations.  ¹H NMR
was used to confirm conversion of monomer and formation of polymer, and gel permeation
chromatography (GPC) was used to measure polymer molecular weight and
polydispersity index (PDI). 

Diblock
copolymers were self-assembled into micelle nanoparticles by adding water
drop-wise to well-mixed polymer solutions in dimethylformamide.  After self-assembly, micelle size and
morphology was analyzed by dynamic light scattering (DLS) and transmission
electron microscopy (TEM). 
Micelles were then loaded with PTL using solvent replacement techniques,
and the PTL loading efficiency and capacity was determined using high performance
liquid chromatography. 

MV4-11
leukemia cells were cultured in untreated 24-well styrene plates at 5*10⁵
cells/mL with Iscove's Modified Dulbecco's Media (IMDM) containing 10%
heat-inactivated fetal bovine serum, and 1% penicillin/streptomycin.  Cells were dosed with free PTL at 2.5,
5, and 10 μM, and cell counts were taken 24 hours after the dose.  The cytotoxicity profile of free PTL
was compared untreated control groups, as well as PTL-loaded micelles at the
same PTL concentrations. 

RESULTS AND DISCUSSION:

PSMA-b-PS
and PSMA-b-PBA polymers with molecular weights ranging from 18 to 48 kDa and
estimated hydrophobic:hydrophilic molecular weight ratios between 0.5 –
1.2 were synthesized.  All polymers
self-assembled into spherical micelles with diameters between 20 - 200 nm.  Interestingly, distinct morphological
differences can be observed between micelles formed from diblocks of different
hydrophobic:hydrophilic ratios (shown in Figure 1).  PSMA-b-PS micelles successfully loaded PTL with efficiencies
ranging between 20% and 40%.  These
PTL-loaded micelles produced a 50% reduction in MV4-11 leukemia cell viability
at PTL doses of 5 μM. 
Importantly, micelles in the absence of PTL exhibited no significant
cytotoxicity towards these cells, when delivered at the same concentration as
PTL-loaded micelles.

Figure 1:  Representative
TEM Images of Polymer Micelles.  From left to right: PSMA₁₈₆-b-PBA₁₀₇
(brush-like structures, 50 nm in diameter), PSMA₅₉-b-PS₆₃
(spherical micelles, 200 nm in diameter), PSMA₁₀₀-b-PS₂₅₀
(dense spherical micelles, 20 nm in diameter).   

CONCLUSIONS AND FUTURE DIRECTIONS:

PSMA-b-PS and PSMA-b-PBA diblock
copolymers were made using RAFT polymerization, and subsequently self-assembled
into core-shell polymer micelles for drug delivery applications.  A potent leukemia therapeutic, PTL, was
successfully loaded into these micelles and exhibited robust LC cytotoxicity in vitro when delivered via polymer
micelles, where unloaded micelles were non-toxic.  Ongoing experiments are examining PTL release rate in vitro, as well as in vivo circulation time and
biodistribution of different micelle architectures.

REFERENCES:

1.     Guzman,
M.L. et al. The sesquiterpene lactone parthenolide induces apoptosis of human
acute myelogenous leukemia stem and progenitor cells.  Blood 105,
4163-4169, 2005.

2.     Jordan,
C.T.  The leukemic stem cell.  Best
Pract Res Clin Hematology 20, 13-18, 2007.

3.     Torchilin,
V.P. Structure and design of polymeric surfactant-based drug delivery
systems.  J Control Release 73, 137-172, 2001.