Anionic Polymerization of an Amphiphilic Copolymer for Preparation of Block Copolymer Micelles Stabilized by π-π Stacking Interactions

Summary

The key steps of living anionic polymerization of phenyl glycidyl ether (PheGE) on methoxy-polyethylene glycol (mPEG-b-PPheGE) are described. The resulting block copolymer micelles (BCMs) were loaded with doxorubicin 14% (wt%) and sustained release of drug over 4 days under physiologically relevant conditions was obtained.

Abstract

In this study, an amphiphilic copolymer that includes a core-forming block with phenyl groups was synthesized by living anionic polymerization of phenyl glycidyl ether (PheGE) on methoxy-polyethylene glycol (mPEG-b-PPheGE). Characterization of the copolymer revealed a narrow molecular distribution (PDI < 1.03) and confirmed the degree of polymerization of mPEG₁₂₂-b-(PheGE)₁₅. The critical micelle concentration of the copolymer was evaluated using an established fluorescence method with the aggregation behavior evaluated by dynamic light scattering and transmission electronic microscopy. The potential of the copolymer for use in drug delivery applications was evaluated in a preliminary manner including in vitro biocompatibility, loading and release of the hydrophobic anti-cancer drug doxorubicin (DOX). A stable micelle formulation of DOX was prepared with drug loading levels up to 14% (wt%), drug loading efficiencies > 60% (w/w) and sustained release of drug over 4 days under physiologically relevant conditions (acidic and neutral pH, presence of albumin). The high drug loading level and sustained release is attributed to stabilizing π-π interactions between DOX and the core-forming block of the micelles.

Introduction

In aqueous media, amphiphilic block copolymers assemble to form nano-sized block copolymer micelles (BCMs) that consist of a hydrophobic core surrounded by a hydrophilic shell or corona. The micelle core can serve as a reservoir for the incorporation of hydrophobic drugs; while, the hydrophilic corona provides an interface between the core and the external medium. Poly(ethylene glycol) (PEG) and its derivatives are one of the most important classes of polymers and one of the most widely used in drug formulation.^1-3 BCMs have proven to be a worthy drug delivery platform with several formulations relying on this technology now in late stage clinical developme....

Protocol

Figure 1. Schematic showing the nine key steps in the preparation of the mPEG-b-PPheGE copolymer. Please click here to view a larger version of this figure.

1. Preparation of the Reagents under Dry Conditions

1. Preparation of the reagents.
   1. Weigh 15 g of mPEG-5K (M_n=5,400 g/mol, PDI 1.03) and place at 50 °C in an oven under vacuum for 48 hr prior to use.
   ....

Results

Figure 3. Illustration of the anionic polymerization of phenyl glycidyl ether on mPEG macroinitiator to produce mPEG-b-(PheGE)₁₅ for preparation of block copolymer micelles for loading of doxorubicin. The schematic illustrates the deprotonation of the hydroxyl group of mPEG using naphthalene potassium as a radical-anion, followed.......

Discussion

Due to the good control that anionic polymerization provides over molecular weight it is one of the most applied processes in the industry for the preparation of polymers based on oxirane monomers (PEG and PPG). Optimal and stringent conditions must be used for successful polymerization to be achieved. Rigorous purification of all reagents and appropriate apparatus are essential for the living character of the synthesis. Limitations of the current setup are mostly associated with the transfer technique that relies on can.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM/HAMF12	Gibco, Life Technologies	12500	Supplemented with 10% FBS. Warm in 37 °C water bath.
Trypsin-EDTA (0.25%)	Sigma-Aldrich	T4049	Warm in 37 °C water bath
Fetal bovine serum (FBS)	Sigma-Aldrich	F1051	Canada origin
MDA-MB-468 cell line	ATCC	HTB-132
MTS tetrazolium reagent	PROMEGA	G111B
Phenazine ethosulfate (PES)	Sigma-Aldrich	P4544	>95%
mPEG5K (Mn 5,400 g/mol)	Sigma-Aldrich	81323	PDI=1.02
Dimethylsolfoxide (DMSO)	Sigma-Aldrich	D4540	>99.5%
Naphthalene	Sigma-Aldrich	147141	>99%
Phenyl glycidyl ether	Sigma-Aldrich	A32608	>85%
Benzophenone	Sigma-Aldrich	427551	>99%
Potassium	Sigma-Aldrich	451096	>98%
Tetrahydrofuran	Caledon Laboratory Chemicals	8900 1	ACS
Hexane	Caledon Laboratory Chemicals	5500 1	ACS
Calcium hydride (CaH2)	ACP	C-0460	>99.5%
Diethyl Ether	Caledon Laboratory Chemicals	1/10/4800	ACS
Microplate reader	BioTek Instruments
Differential scanning calorimetry (DSC)	TA Instruments Inc	DSC Q100
Gel permeation chromatography (GPC)	Waters	2695 separation moldule / 2414 detector	2 Columns: Agilent Plgel 5 µm Mixed-D
NMR spectroscopy	Varian Mercury 400MHz
Chloroform-d	Sigma-Aldrich	151858	99.96%
DMSO-d	Sigma-Aldrich	156914	99.96%
Vaccum pump	Gardner Denver Welch Vacuum Tech, Inc.		Ultimate pressure 1x10-4 torr
Drierit with indicator, 8 mesh	Sigma-Aldrich	238988	Regenerated at 230 °C for 2 hr