Polymerization-Induced Self-Assembly of Metallo-Polyelectrolyte Block Copolymers

Abstract

Cobaltocenium-containing polyelectrolyte block copolymer nanoparticles were prepared via polymerization-induced self-assembly (PISA) using aqueous dispersion RAFT polymerization. The cationic steric stabilizer was a macromolecular chain-transfer agent (macro-CTA) based on poly (2-cobaltocenium amidoethyl methacrylate chloride) (PCoAEMACl), and the core-forming block was poly(2-hydroxypropyl methacrylate) (PHPMA). Stable cationic spherical nanoparticles were formed in aqueous solution with low dispersity without adding any salts. The chain extension of macro-CTA with HPMA was efficient and fast. The effects of block copolymer compositions, solid content, charge density, and addition of salts were studied. It was found that the degree of polymerization of both the stabilizer PCoAEMACl and the core-forming PHPMA had a strong influence on the size of nanoparticles.

INTRODUCTION

Self-assembly of amphiphilic block copolymers in water has attracted considerable attention in the recent decades for preparing polymer nanoobjects with a variety of morphologies such as spheres, worms, lamellae, and vesicles, which have applications in drug delivery, reactive vessels, templating, bioimaging, and catalysis.^1–15 Usually, the self-assembly of block copolymers is achieved under low concentrations (e.g. <1 wt%), which would be difficult to implement for large-scale applications.^16–18

Over the past few years, polymerization-induced self-assembly (PISA) has become a highly efficient one-pot approach to producing well-defined block copolymer nanoobjects with different morphologies at high solid contents (10–50% w/w).^19–24 The PISA process typically involves the use of controlled/ living polymerization techniques. RAFT polymerization is the most commonly used technique in PISA due to its “living” character and mild reaction conditions, in addition to low dispersity and good control on molecular weight.²⁵ For PISA, a soluble stabilizer block is chain extended with a second monomer that forms an insoluble block, which induces self-assembly in situ.²⁰ RAFT-mediated PISA can be readily achieved via aqueous emulsion polymerization or dispersion polymerization. In the case of PISA, aqueous dispersion RAFT polymerization is particularly attractive from an environmental and economic perspective and is expected to offer many potential applications.^19,20 For aqueous dispersion polymerization, the stabilizer block or macromolecular chain-transfer agents (macro-CTA) and monomer need to be water-soluble for generating water insoluble diblock copolymers. As a result, there are only a few vinyl monomers that have been used, including N-isopropyl acrylamide (NIPAM),²⁶ 2-methoxyethyl acrylate (MEA),²⁷ diacetone acrylamide (DAAM),²⁸ and 2-hydroxypropyl methacrylate (HPMA).²⁰ The Armes group used HPMA extensively for preparing various morphologies.^19,29,30 A wide range of steric stabilizer macro-CTAs have been employed in aqueous RAFT-mediated PISA, including ionic and nonionic blocks. Water-soluble non-ionic stabilizer blocks are most commonly studied through PISA, compared to ionic stabilizer blocks.^21,30,31

Ionic (e.g.,cationic and anionic) and zwitterionic^32,33 steric stabilizer blocks are used to prepare charged nano-objects.^21,34–37 However, only a few cationic^38–40 steric stabilizer blocks have been reported for aqueous RAFT-mediated PISA, and all of these are based on organo-polyelectrolytes such as poly(quaternized 2-(dimethylamino) ethyl methacrylate) and poly(2-aminoethyl methacrylate).

Herein metallo-polyelectrolyte-based steric stabilizer is chosen for the first time to enable aqueous dispersion RAFT-mediated PISA (Fig. 1). The presence of transition metals allows unique electronic properties such as lipophobicity, ionic binding, and redox chemistry.^41–43 Metallo-polyelectrolytes are highly water soluble, less toxic, and chemically stable,^44–46 which could open up new applications beyond traditional organo-polyelectrolytes.

FIGURE 1.

Schematic representation of polymerization induced self-assembly (PISA) of metallo-polyelectrolyte diblock copolymers.

Specifically we carried out aqueous dispersion RAFT polymerization of HPMA using poly(2-cobaltocenium amidoethyl methacrylate chloride) (PCoAEMACl) as the cationic steric stabilizer macro-CTA. The effects of diblock copolymer composition, charge density, solid contents, and added salts were investigated. The dependence of the size of nanoobjects with the degree of polymerization of PHPMA and PCoAEMACl was also explored.

EXPERIMENTAL SECTION

Materials

Chain transfer agent 2-cyano-2-propyl benzodithioate (97%, CPB) was purchased from Sigma-Aldrich and used directly. Monomer 2-cobaltocenium amidoethyl methacrylate hexafluorophosphate (CoAEMAPF₆) was synthesized by reacting cobaltocenium monocarboxylic acid with 2-aminoethyl methacrylate hydrochloride in the presence of coupling agent N-(3-(dimethylamino)propyl)-N'-ethylcarbodiimide (EDC) according to established methods.^47–49 2-Aminoethyl methacrylate hydrochloride (90%), N-(3-dimethylaminopropyl)-N'-ethylcarbodi imide hydrochloride (EDC-HCl, 98%), 4-(dimethylamino)pyridine and tetrabutylammonium chloride (TBACl) were purchased from Sigma-Aldrich and used as received. HPMA was purchased from Alfa-Aesar, which comprises approximately 75% 2-hydroxypropyl methacrylate and 25% 2-hydroxyisopropyl methacrylate. AIBN and 2,2'-azobis (2-(2-imidazoline-2-yl)propane) dihydrochloride were purchased from VWR. Water was purified using Thermo Scientific Nanopure with ion conductivity at 18.2 MΩ. All other chemicals were from commercial sources and used as received.

Characterization

The purity of monomers and the conversion of polymerization were monitored by 300 MHz ¹H NMR spectroscopy using a Bruker Avance III HD 300 spectrometer. Spectra were recorded in deuterated chloroform, deuterium oxide, or dimethylsulfoxide in ppm (δ) with tetramethylsilane as an internal standard. Molecular weight and conversion of polymers were measured using ¹H NMR spectra.

DLS and Zeta Potential

A Zetasizer Nanoseries ZEN3690 (Malvern Instruments, Malvern, UK) instrument was used to measure the hydrodynamic diameter (Z-average) and Zeta potential of the aggregates. The samples were prepared by dissolving copolymers in filtered (0.2 μm GHP membrane filter) deionized water with a concentration of 0.1 mg/mL. The solutions were at pH 7.0, and the measurements were carried out at 25 °C. The data processing was done using the general-purpose algorithms provided in the Zetasizer Software. Sample measurements were acquired in triplicate and reported as an average and standard error.

Atomic Force Microscopy (AFM)

AFM was accomplished using a Multimode Nanoscope V system (Bruker, Santa Barbara, CA). Tapping mode AFM was used to map the topography by tapping the surface using an oscillating tip. The measurements were achieved using commercial Si cantilevers with a nominal spring constant and resonance frequency at 20–80 Nm⁻¹ and 230–410 kHz, respectively (TESP, Bruker AFM Probes, Santa Barbara, CA). AFM samples were prepared by dropping casting onto oxidized silicon silicon wafer (100 nm thick thermal oxide) and then dried at room temperature before observation.⁵⁰ The silicon wafers were cleaned using acetone-water mixture and then isopropyl alcohol or ethanol. Nanoparticle sample solutions were diluted with deionized water to a concentration of 0.20% w/w.

Transmission Electron Microscopy (TEM)

A JOEL 1400 plus TEM was applied to take images at an operating voltage of 120 kV. Nanoparticle samples were diluted with deionized water to a concentration of 0.2 mg/mL. TEM samples were prepared by dropping solution on carbon-supported copper grids and then dried before observation.

Synthesis of PCoAEMACl

The synthesis of PCoAEMACl macro-CTA was carried out via RAFT polymerization.⁴⁸ Monomer CoAEMAPF₆ (490 mg, 1.0 mmol), initiator AIBN (0.5 mg, 0.003 mmol), and chain transfer agent CPB (3.5 mg, 0.01 mmol) were dissolved in DMF (1.0 mL) in a 10 mL Schlenk flask. The mixture was degassed by purging N₂ for 30 min and then heated at 90 °C for 1–2 h. After the reaction, the homopolymer with PF₆⁻ as a counterion were obtained by precipitation in cold dichloromethane. Finally, the homopolymer showed excellent water-solubility after its counterion exchange from PF₆⁻ to Cl⁻ using TBACl as a phase-transfer ion-exchange reagent. One milliliter of acetonitrile solution of PCoAEMAPF₆ (30 mg mL⁻¹) was slowly dropped into 5 mL of TBACl acetonitrile solution under vigorous stirring. After 5 min, a homopolymer PCoAEMACl with Cl⁻ anion was precipitated and washed three times using acetonitrile to remove excess TBACl and remaining PF₆⁻ ions. The degree of polymerization was determined by ¹H NMR analysis.

Synthesis of Block Copolymer Nanoparticles Via Aqueous Dispersion of RAFT Polymerization

The synthesis of PCoAEMACl-b-PHPMA diblock copolymer nanoparticles with a solid content of 12.5% w/w was conducted via aqueous dispersion RAFT polymerization. Here is an example: macro-CTA PCoAEMACl₂₆ (34 mg, 3.4 μmol), 2,2'-azobis (2-(2-imidazoline-2-yl)propane) dihydrochloride (0.3 mg, 1 μmol) initiator, and HPMA (245 mg, 1.7 mmol) monomer weighed into a 10-mL schlenk flask. Deionized water was added to dissolve all the monomer and initiators producing a 12.5% w/w aqueous solution. The aliquot was subject to freeze-pump-thaw for three times to remove air and filled with nitrogen prior to immersion in an oil bath set at 70 °C. The reaction solution was stirred for 3–4 h to ensure maximum conversion of HPMA monomer (>96% conversion as monitored by ¹H NMR) and quenched by exposure to air. Finally, the nanoparticle solution was purified via dialysis over water (3 × 1 L).

RESULTS AND DISCUSSION

Synthesis of Metallo-Polyelectrolyte Block Copolymers Via Aqueous Dispersion RAFT Polymerization

Cobaltocenium-containing macro-CTA was prepared via RAFT polymerization following an earlier report using CoAEMAPF₆ as a monomer, CPB as a chain transfer agent and AIBN as an initiator (Fig. 2).⁵¹ After the synthesis, an anion exchange process was carried out to prepare cobaltocenium-containing methacrylate polymers with chloride as the counterion.^47,51 The polymerization was terminated at a relatively low conversion (e.g.,55%) to preserve chain-end functionality. These cobaltocenium-containing polymers acted as a stabilizer block in the following PISA process.

FIGURE 2.

Synthesis of diblock copolymers by aqueous dispersion RAFT polymerization.

The cobaltocenium-containing macro-CTA was then chain-extended with HPMA in DI water via aqueous dispersion RAFT polymerization (Fig. 2). The 2,2'-azobis(2-(2-imidazoline-2-yl)propane) dihydrochloride was chosen as a water-soluble initiator. The polymerization was conducted at 70 °C with a series of different degree of polymerization (DP) of HPMA, while the molar ratio of macro-CTA to initiator was fixed. All polymerization proceeded until high conversions of HPMA monomer (>96%) were achieved, as monitored by NMR. Figure 3(a) shows ¹H NMR spectra of macro-CTA and a representative diblock copolymer PCoAEMACl₂₆-PHPMA₅₀₀. From the ¹H NMR spectrum of diblock copolymer, a new peak at 4.6 ppm confirmed the formation of diblock copolymer. The formation of a cloudy/yellowish solution in the reaction mixture indicated the progress of polymerization and the formation of amphiphilic diblock copolymers. The amphiphilicity of diblock copolymer induced the formation of nanoparticles. The solution was further dialyzed against water. Unlike other studies, the molecular weight of these metallocene-containing polymers via RAFT could not be analyzed using GPC, as the cationic cobaltocenium block has a strong interaction with various columns.³⁹

FIGURE 3.

(a) ¹H NMR spectra of macro-CTA and a diblock copolymer; (b) A plot of conversion versus time for aqueous dispersion RAFT polymerization of HPMA using macro-CTA at 70 °C; (c) A plot of particle’s hydrodynamic diameter (D_h) versus time.

A kinetic study was conducted for the polymerization of HPMA using cobaltocenium containing macro-CTA at a solid content of 12.5% w/w and monitored by NMR at different time intervals [Fig. 3(b)]. HPMA conversion was calculated based on the disappearance of the vinyl signal relative to the growing signals at 4.6 ppm. Initially, the reaction mixture was a transparent yellow solution because HPMA and the macro-CTA were both soluble in water, but the turbidity of the solution increased as the polymerization proceeded. This is because the growing PHPMA chains gradually become insoluble in water, leading to form nano-objects in situ and phase separate. The higher monomer conversion (92%) reached within 1 h, confirming the reaction proceeded rapidly. Then the reaction conversion nearly reached a plateau afterward as shown in the Figure 3(b). The progress of micellization of resultant amphiphilic diblock copolymers was also monitored by DLS. DLS study confirmed that the micellization occurred after 10 min of polymerizations, as shown in Figure 3(c). The sizes of nanoparticles increased rapidly with the polymerization time.

A series of PCoAEMACl-b-PHPMA diblock copolymers with various degrees of polymerization of HPMA block with constant DP of PCoAEMACl (e.g.,26) were synthesized with the solid content kept at 12.5% w/w. The DPs of targeting core-forming HPMA block are in the range of 100–650. The resulting nanoparticles are imaged by AFM (Fig. 4 and Supporting Information Fig. S1) and TEM (Fig. 4). All diblock copolymers of PCoAEMACl₂₆-PHPMA_x (x = 100, 200, 250, 350, 450, 500, and 650) formed spherical nanoparticles. Among the nanoparticles, metallocene-based polyelectrolyte block, PCoAEMACl, forms the corona, and the PHPMA forms the core of the stable spherical nanoparticles. The hydrodynamic diameter of spheres was determined by DLS after dilution to 0.10% w/w. The diameter of spheres increased with the increase of the DP of PHPMA [Fig. 5(a)]. DLS study suggested that particles have a relatively narrow distribution with the size ranging from 60 to 235 nm. AFM and TEM images of corresponding diblock copolymers also showed a similar trend of particle size evolution.

FIGURE 4.

Representative AFM (a, b, c) and TEM (d, e, f) images obtained for PCoAEMACl-b-PHPMA prepared by aqueous dispersion RAFT polymerization at 70 °C with a degree of polymerization for core-forming PHPMA block at (a, d) 250; (b, e) 500; and (c, f) 650. All scale bars are 500 nm.

FIGURE 5.

(a) Hydrodynamic diameter versus DP of PHPMA by DLS; (b) Intensity-average particle size distribution curve by DLS; (c) AFM height images of PCoAEMACl₁₃-PHPMA₅₀₀, PCoAEMACl₂₆-PHPMA₅₀₀, and PCoAEMACl₅₂-PHPMA₅₀₀ with 12.5% w/w solid and PCoAEMACl₁₃-PHPMA₅₀₀ with 25% w/w solid content. All scale bars are 500 nm.

To further investigate the metallocene-based PISA, diblock copolymers PCoAEMACl₂₆-PHPMA_x with another solid content (25% w/w) were prepared and different lengths of HPMA block were obtained: x = 250, 500, and 650. These diblock copolymers with higher solid content also formed spherical-shaped nanoparticles, which was confirmed by AFM images (Supporting Information Fig. S2). The hydrodynamic diameters of these nanoparticles are 91.6, 141.2, and 151.0 nm for block copolymers with the DP of PHPMA at 250, 500, and 650 respectively (Table 1). The hydrodynamic diameter of particles with 25% w/w solid content is smaller compared to those with 12.5% w/w solid content. It was postulated that at higher solid contents the higher viscosity could inhibit the motion of polymer chains and thus prevent the formation of larger particles.

TABLE 1.

Summary of Synthesis of Diblock Copolymers: Conversion by ¹H NMR, Solid Contents, Hydrodynamic Diameter (DLS), Polydispersity, and Zeta Potential

PCoAEMACl (DP) : HPMA : Initiator	Conversiona of HPMA (1H NMR)	Solid Content (%, w/w)	Hydrodynamic Diameter, Dh (nm)	Polydispersity	Zeta Potential (mV)
1 (26) : 100 : 0.3	98	12.5	60.2 ± 0.7	0.044	49
1 (26) : 200 : 0.3	98	12.5	76.3 ± 0.5	0.064	49
1 (26) : 250 : 0.3	98	12.5	108.3 ± 0.7	0.032	49
1 (26) : 350 : 0.3	98	12.5	120.6 ± 0.6	0.056	51
1 (26) : 450 : 0.3	96	12.5	160.0 ± 0.5	0.038	51
1 (26) : 500 : 0.3	96	12.5	196.6 ± 0.7	0.024	55
1 (26) : 650 : 0.3	96	12.5	234.9 ± 1.2	0.044	55
1 (26) : 250 : 0.3	97	25	91.6 ± 0.5	0.103	49
1 (26) : 500 : 0.3	96	25	141.2 ± 2.5	0.391	51
1 (26) : 650 : 0.3	96	25	151.0 ± 0.8	0.211	51
1 (13) : 500 : 0.3	96	12.5	143.8 ± 1.8	0.094	52
1 (13) : 650 : 0.3	96	12.5	215.8 ± 0.7	0.057	52
1 (52) : 500 : 0.3	96	12.5	223.7 ± 2.4	0.266	62

DP = degree of polymerization.

^aFinal conversions were measured after 4 h of reaction at 70 °C.

The effect of the length of the stabilizer block was also investigated by varying the DP of macro-CTA PCoAEMACl, while keeping the DP of PHPMA block at 500. The DP of stabilizer block PCoAEMACl was varied as 13, 26, and 52. The observed average hydrodynamic diameters of block copolymer nanoparticles PCoAEMACl₁₃-PHPMA₅₀₀, PCoAEMACl₂₆-PHPMA₅₀₀, and PCoAEMACl₅₂-PHPMA₅₀₀ were around 143.8, 196.6, and 223.7 nm, respectively. The hydrodynamic diameter increases by increasing the DP of stabilizer block PCoAEMACl. The clear shift in the DLS intensity-size distribution curve also indicated the size increase with a higher DP of PCoAEMACl [Fig. 5(b)]. The morphology of nanoparticles did not change with changing the DP of stabilizer block, and AFM imaging observed the formation of spherical nanoparticles. A similar trend of size increase of spherical nanoparticles was also observed in AFM images [Fig. 5(c)]. The larger length of stabilizer block led to higher charge density and higher electrostatic repulsion between the stabilizer chains that resulted in bigger particles. The polydispersity measured by DLS is somehow higher for the longer stabilizer block of PCoAEMACl₅₂-PHPMA₅₀₀.

The Zeta potential values were determined by DLS to measure the colloidal stability and surface charge of particles. The metallocene-based stabilizer block carries cationic charges and is highly ionized in water, leading to the positive zeta potentials ranging from +49 to +65 mV (Table 1). All copolymers showed positive zeta potential values suggesting good colloidal stability of nanoparticles, and the cobaltocenium-containing block located on the outer surface of the aggregates. The zeta potential values are almost similar for the constant DP of cobaltocenium-containing stabilizer block with varying DP of PHPMA. The zeta potential for PCoAEMACl₅₂-PHPMA₅₀₀ was found to be higher at +65 mV, which is due to the higher charge density of longer length of the stabilizer block.

The effect of ionic salts on PISA was investigated. A total of 0.2 M NaCl aqueous solution was used as a solvent to perform the preparation of a block copolymer PCoAEMACl₂₆-PHPMA₄₅₀. However, the morphology of the diblock copolymer solution did not change. Only spherical nanoparticles were formed, which was confirmed by the AFM images [Fig. 6 (b)]. The DLS measurement showed that the hydrodynamic diameter (D_h) of the copolymers significantly decreased, from 160 to 122 nm [Fig. 6(a)], and polydispersity also reduced significantly, which is potentially due to the ion screening effect that reduces the repulsion between the positively charged cobaltocenium blocks, leading to chain contraction. AFM imaging of PCoAEMACl₂₆-PHPMA₄₅₀ in 0.2 M NaCl salt solution also confirmed the formation of smaller spherical nanoparticles [Fig. 6(b)].

FIGURE 6.

(a) DLS intensity-average particle size distribution curve of PCoAEMACl₂₆-PHPMA₄₅₀ in water and 0.2 M NaCl solution; (b) AFM images of PCoAEMACl₂₆-PHPMA₄₅₀ nanoparticles in water and in 0.2 M NaCl solution. All scale bars are 500 nm.

CONCLUSIONS

In summary, the PISA of metallo-polyelectrolyte block copolymers is reported. The nanoparticles were prepared via aqueous dispersion RAFT polymerization of HPMA using cobaltocenium-based macro-CTA. The high conversion (>96%) of HPMA was achieved. Spherical nanoparticles were formed after the PISA process, as confirmed by both AFM and TEM images. A monotonic increase in the size of particles was observed with the increase of the length of the core-forming PHPMA block with a fixed length of the corona block. Particle size also increased with increasing the charge density and the length of the stabilizer block. Overall, the PISA of metallo-polyelectrolyte block copolymers offers a new platform for the preparation of cationic metal-containing nano-objects.