Facile Synthesis of Worm-like Micelles by Visible Light Mediated Dispersion Polymerization Using Photoredox Catalyst

Summary

This article describes a process for producing polymeric self-assembled nanoparticles using visible light mediated dispersion polymerization. Using low energy visible light to control the polymerization allows for the reproducible formation of self-assembled worm-like micelles at high solids content.

Abstract

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins with the synthesis of a hydrophilic poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) homopolymer using reversible addition-fragmentation chain-transfer (RAFT) polymerization. Under mild visible light irradiation (λ = 460 nm, 0.7 mW/cm²), this macro-chain transfer agent (macro-CTA) in the presence of a ruthenium based photoredox catalyst, Ru(bpy)₃Cl₂ can be chain extended with a second monomer to form a well-defined block copolymer in a process known as Photoinduced Electron Transfer RAFT (PET-RAFT). When PET-RAFT is used to chain extend POEGMA with benzyl methacrylate (BzMA) in ethanol (EtOH), polymeric nanoparticles with different morphologies are formed in situ according to a polymerization-induced self-assembly (PISA) mechanism. Self-assembly into nanoparticles presenting POEGMA chains at the corona and poly(benzyl methacrylate) (PBzMA) chains in the core occurs in situ due to the growing insolubility of the PBzMA block in ethanol. Interestingly, the formation of highly pure worm-like micelles can be readily monitored by observing the onset of a highly viscous gel in situ due to nanoparticle entanglements occurring during the polymerization. This process thereby allows for a more reproducible synthesis of worm-like micelles simply by monitoring the solution viscosity during the course of the polymerization. In addition, the light stimulus can be intermittently applied in an ON/OFF manner demonstrating temporal control over the nanoparticle morphology.

Introduction

The synthesis of nonspherical (and other) nanoparticle morphologies has traditionally been accomplished using a multistep self-assembly procedure starting with the synthesis and purification of well-defined amphiphilic diblock (or multiblock) copolymers. One of the most common self-assembly techniques was popularized by Eisenberg in the 1990s and involves the dissolution of the amphiphilic block copolymer in a common solvent for both polymer blocks followed by the slow addition of a solvent selective for one of the blocks^1-3. As the selective solvent (typically water) is added, the block copolymer undergoes self-assembly to form polymeric nanoparticles. The final morphology (or mixtures of morphologies) of the nanoparticles are determined by a large number of factors such as the relative lengths of each polymer block, rate of water addition and the nature of the common solvent. However, this approach generally only allows for the production of nanoparticles at relatively low solids content (less than 1 wt%) and so limits its practical scalability⁴. In addition, the reproducible formation of "intermediate" phases such as worm-like micelles can be difficult owing to the narrow range of parameters required to stabilize this nonspherical morphology⁵.

The polymerization-induced self-assembly (PISA) approach partially addresses the drawbacks of the Eisenberg approach by utilizing the polymerization process itself to drive self-assembly in situ allowing for nanoparticle synthesis at much higher solids content (typically 10-30 wt%)^6-8. In a typical PISA approach, a living polymerization process is used to chain extend a solvent soluble macroinitiator (or macro-CTA) with a monomer that is initially soluble in the reaction medium but forms an insoluble polymer. The PISA approach has been used to synthesize worm-like micelles by systematically testing a number of experimental parameters and using detailed phase diagrams as a synthetic "roadmap"^5,9.

Despite their challenging synthesis, there is great interest in worm-like nanoparticles due to their interesting properties relative to their spherical counterparts. For example, we have demonstrated that drug loaded short and long worm-like micelles synthesized using a PISA approach have significantly higher in vitro cytotoxicity compared to spherical micelles or vesicles¹⁰. Others have shown a correlation between nanoparticle aspect ratio and blood circulation time in in vivo models¹¹. Others have shown that the synthesis of worm-like nanoparticles using an appropriate PISA methodology yields a macroscopic gel due to the nanoscale entanglement of the nanoparticle filaments. These gels have demonstrated potential as sterilizable gels owing to their thermoreversible sol-gel behavior¹².

This protocol describes a method allowing for the in situ monitoring of the formation of worm-like micelles by simply observing the solution viscosity during the polymerization. Previous studies of similar worm-like micellar gels have demonstrated that above a critical temperature, these nanoparticles undergo a reversible worm-sphere transition and so form free-flowing dispersions at elevated temperatures. To date, these systems have utilized a thermally sensitive azo compound to initiate the controlled polymerization^13,14 and so gelation may not be readily observed in these systems during the thermal polymerization. From these studies, it was hypothesized that synthesizing PISA derived nanoparticles at lower temperatures may allow for observation of this gelation behavior in situ.

Recently we reported the use of a facile room temperature photopolymerization technique to mediate the PISA process to yield nanoparticles of different morphologies¹⁵. Here, a visualized protocol is presented for the reproducible synthesis of worm-like micelles by observing the solution viscosity behavior during the polymerization. The dispersion polymerization proceeds readily using commercially available light-emitting diodes (LEDs) (λ = 460 nm, 0.7 mW/cm²).

Protocol

1. Synthesis and Characterization of POEGMA Macro-CTA

1. Add oligo(ethylene glycol) methyl ether methacrylate (OEGMA) (12 g, 4 × 10^-2 mol), 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (CPADB) (0.224 g, 8 × 10^-4 mol), 2,2′-azobis(2-methylpropionitrile) (AIBN) (16.4 mg, 0.1 mmol) and 50 ml acetonitrile (MeCN) to a 100 ml round bottom flask.
2. Seal the flask with an appropriately sized rubber septum and steel wire and cool the flask from room temperature to < 4 °C in an ice-water bath.
3. Deoxygenate the flask for 30 min by bubbling nitrogen directly into the reaction mixture through a 21 G needle (0.8 mm x 120 mm) with a second 21 G needle (0.8 mm x 38 mm) acting as a vent.
4. Place the flask in an oil bath at 70 °C for 5.5 hr before quenching the polymerization by immersion in an ice-water bath and exposing the contents to air.
5. Remove the MeCN by agitation under a continuous stream of compressed air and re-dissolve the crude mixture in ~40 ml tetrahydrofuran (THF).
6. Add the contents of the flask dropwise to 400 ml of a rapidly stirred mixture of petroleum spirits (b.p. 40-60 °C) and diethyl ether (70:30, v/v) and continue to stir until the supernatant is no longer cloudy.
   Note: Cooling in an ice-bath may be used to accelerate the precipitation process.
7. Decant the supernatant and re-dissolve the polymer residue in ~40 ml THF.
8. Repeat the precipitation process (steps 1.5-1.7) at least two more times to ensure complete removal of the residual OEGMA monomer. Remove the excess solvent from the purified POEGMA macro-CTA firstly by agitation under a continuous stream of compressed air and drying in a vacuum oven (20 °C, 10 mbar) for 4 hr.
9. Determine the number average molecular weight of the POEGMA macro-CTA by Nuclear Magnetic Resonance (NMR) (M_{n, NMR}) using a previously reported method¹⁵. Using gel permeation chromatography¹⁵ (GPC) (dimethylacetamide as mobile phase and appropriate standards for calibration) calculate the polymer dispersity (Ð).
   Note: Using the above synthesis (steps 1.1-1.8) should yield a POEGMA macro-CTA with M_{n, NMR} = 9,000, and Ð < 1.15. If the molecular weight (and dispersity) of the synthesized POEGMA macro-CTA differs from the synthesis presented here (between 7,000 - 1,000 g/mol), the formation of worm-like micelles (as indicated by in situ gelation) can still occur using the subsequent PISA methodology presented in (section 2) albeit at a slightly altered reaction time.

2. Preparation of POEGMA-b-PBzMA Nanoparticles Using PISA

1. Prepare a 1 mg/ml stock solution of Ru(bpy)₃Cl₂ ^.6H₂O in ethanol (EtOH). Store the stock solution in the refrigerator to minimize solvent evaporation.
2. Plug a Pasteur pipette with a small wad of cotton wool using a second pipette to help pack it tightly. Pour basic aluminum oxide into the pipette with the cotton wool plug to give a column of approximately 5 cm. Remove the monomethyl ether hydroquinone inhibitor in commercial BzMA by passing ~3 ml of BzMA through the column and collecting the deinhibited BzMA eluent.
3. Add POEGMA macroCTA (~9,000 g/mol; 76.9 mg, 8.5 × 10^-6 mol), deinhibited BzMA (0.301 g, 1.71 × 10^-3 mol), Ru(bpy)₃Cl₂^.6H₂O (128 µg, 1.71×10^-7 mol, 128 µl of a 1 mg/ml ethanolic stock solution), 0.383 ml MeCN and 1.402 ml EtOH (1.913 ml total solvent, 80 wt%, 20 v/v% MeCN) to a 4 ml glass vial.
4. Perform the deoxygenation procedure as outlined in steps 1.2-1.3.
5. Place the vial in a 2,000 ml glass beaker (Figure 2) lined with blue LED strips (λ_max = 460 nm, 0.7 mW/cm²) and irradiate at room temperature with magnetic stirring. Monitor the reaction vial routinely after 20 hr and remove it from the reactor when the high viscosity solution forms a free standing gel when the vial is inverted (Figure 3).
   Note: The total time to yield a free-standing gel should be about 24 hr of blue light irradiation using the conditions presented here. Small differences in the light irradiation reactors (physical dimensions, intensity, etc.) may require slightly altered conditions (specifically reaction time) to achieve the in situ formation of worm-like micelles.
6. After removing from the reactor, quench the polymerization by exposing the nanoparticle gel to air for a few minutes and storing the closed vial upright in the dark.

3. Transmission Electron Microscopy (TEM) Imaging of Nanoparticle Morphology

1. Place approximately 40 mg of the crude nanoparticle gel (from section 2) in a 4 ml glass vial.
2. Continuously agitate the nanoparticle gel using a vortex mixer and add 4 ml of EtOH dropwise over a period of at least 5 min. The gel should become a free-flowing solution during the solvent addition.
   Note: If the gel is diluted with EtOH too rapidly or not agitated adequately, some precipitation of the nanoparticles may occur. See step 3.3.
3. Remove any macroscopic aggregates from the diluted nanoparticles by filtering through glass wool.
4. Perform TEM imaging (with uranyl acetate staining) of the diluted sample according to a previously reported procedure.¹⁵

Results

In this study, two-step polymerization protocol is used for the synthesis of worm-like micelles using a PISA approach (Figure 1). In the first step, the polymerization of OEGMA is performed yielding a POEGMA macro-CTA which can be used as a stabilizer in the subsequent polymerization step. The PET-RAFT polymerization proceeds under dispersion conditions owing to the insolubility of PBzMA in ethanol which ultimately leads to nanoparticle formation. During the polymerizatio...

Discussion

This visualized protocol demonstrates the ability to monitor the formation of worm-like micelles simply by observing the onset of gel-like behavior. The utility of this approach lies in the ability to monitor worm formation during the polymerization in comparison to other methods. This procedure can be performed using a two-step polymerization of two commercially available monomers (OEGMA and BzMA) to yield self-assembled POEGMA-b-PBzMA amphiphilic diblock copolymers.

It should be not...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4-Cyano-4- (phenylcarbonothioylthio)pentanoic acid (CPADB)	Sigma-Aldrich	722995-5G
Oligo(ethylene glycol) methyl ether methacrylate (OEGMA)	Sigma-Aldrich	447935-500ML	Average Mn 300, contains 100 ppm MEHQ as inhibitor, 300 ppm BHT as inhibitor
2,2′-Azobis(2-methylpropionitrile) (AIBN)	Sigma-Aldrich
Ru(bpy)3Cl2.6H2O	Sigma-Aldrich	544981-1G
Benzyl methacrylate (BzMA)	Sigma-Aldrich	409448-1L	Contains monomethyl ether hydroquinone as inhibitor
Aluminium oxide (basic)	Chem-Supply Pty Ltd Australia	AL08371000
95% Ethanol (EtOH)	Sucrogen Bio Ethanol	80889
Acetonitrile (MeCN)	Chem-Supply Pty Ltd Australia	RP1005-G2.5L
Tetrahydrofuran (THF)	Chem-Supply Pty Ltd Australia	TA011-2.5L
Petroleum Spirits (40-60 °C)	Chem-Supply Pty Ltd Australia	PA044-2.5L
Diethyl Ether	Chem-Supply Pty Ltd Australia	EA0362.5L
Dimethylacetamide (DMAc)	VWR International Australia	ALFA22916.M1	For GPC analysis
Pasteur pipettes (230 mm)	Labtek	355.050.503
Glass beakers	Labtek	025.01.902 (2L)/ 2110654 (1L)	2 L beaker is for attaching LED strips to form the circular reactor
Commercial LED strip	EcoLab	n/a	λ = 460 nm, 4.8 W/m
4 ml Glass Vials	Labtek	APC502214B
0.9 ml Quartz Cuvette	Starna Scientific Ltd	21/Q/2
Needle (0.8 mm x 38 mm)	Beckton Dickson	302017	For deoxygenating reactions
Needle (0.8 mm x 120 mm)	B Braun Australia	4665643	For deoxygenating reactions
Sleeve stopper septa (rubber septum)	Sigma-Aldrich	z564680/z564702
Stirring hotplates	VWR International Australia/In Vitro Technologies	97018-488/RADRR91200
Vortex mixer	VWR International Australia	412-0098
Vacuum oven	In Vitro Technologies	MEMVO200