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.