Unimolecular micelles from graft copolymer with binary side chains

Abstract

A novel amphiphilic binary graft copolymer poly(glycidyl methacrylate)-graft-[poly(2-cinnamoyl-oxyethyl methacrylate)-random-methoxy polyethylene glycol] (PGMA-g-(PCEMA-r-MPEG)) was successfully synthesized by a combination of atom transfer radical polymerization (ATRP) and click reaction, in which alkyne-end-functionalized poly(2-cinnamoyloxyethyl methacrylate) (PCEMA–C≡CH) and poly(ethylene glycol) methyl ether (MPEG–C≡CH) were grafted onto a poly(3-azide-2-hydroxy-propyl methacrylate) (P(GMA-N₃) backbone. This polymer was used to prepare stable unimolecular micelles (UMMs), which could be produced using either high or low polymer concentrations. Since water is a good solvent only for MPEG but a poor solvent for both PGMA and PCEMA, the hydrophobic PGMA and PCEMA segments aggregated together to form a dense core that was surrounded by a corona based on the soluble MPEG segments. PCEMA was photo-crosslinkable, and thus the UMMs could be crosslinked by shining UV light on the system to yield permanent UMMs. The morphologies of the UMMs were characterized by TEM, AFM, and DLS. Both the TEM and AFM observations indicated that the crosslinked UMMs had a diameter of ∼13 nm, while the DLS measurements indicated they had a diameter of ∼34 nm. The unimolecular state of the micelles was confirmed by SEC, as well as a comparison of the theoretical mass per graft copolymer molecule with that of an individual micelle. Moreover, the morphology of the UMMs was unperturbed by the crosslinking reaction although they became more compact and had a slightly smaller diameter.

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1. Introduction

Polymeric micelles possessing a hydrophobic core and a hydrophilic shell have attracted much attention in recent years due to their numerous potential applications in drug delivery,^1–3 cancer imaging,^4,5 photothermal therapy,⁶ and as nanoreactors.^7–9 However, traditional multimolecular micelles assembled from multi-macromolecules are stable only above their critical micelle concentration (CMC)¹⁰ and they tend to undergo disassembly upon alteration of the external conditions such as the concentration, pH, solvent, temperature, ionic strengths, flow stress, or other parameters.¹¹ This instability associated with multimolecular micelles can be overcome by crosslinking the core^12,13 and/or the shell^14,15 of the self-assembled multimolecular micelles or by developing unimolecular micelles.^2,4 The topologies of unimolecular micelles (UMMs) bearing a core–shell architecture¹⁶ resemble those of conventional multimolecular polymeric micelles.¹⁷ In contrast with multimolecular micelles, UMMs possess significant thermodynamic stability in aqueous solutions due to their covalently reinforced structures.¹⁸

To date, UMMs have been successfully prepared from a number of amphiphilic polymers with various topologies (Fig. 1), such as linear copolymers,^19–21 macrocyclic copolymers,²² star-shaped copolymers,^16,23 graft copolymers,^4,24–26 and hyperbranched copolymers.^27,28

Fig. 1 Polymers with various topologies used to prepare UMMs.

Graft copolymers possess unique architectures containing a backbone chain and many side-chains.²⁹ They bear a cylindrical brush conformation if D/R_g < 2, where D is the average distance between the grafted chains and R_g is the radius of gyration of the polymer in solution.³⁰ It is noteworthy that the morphology and the size of UMMs based on amphiphilic graft polymers can be conveniently and precisely tuned by changing the length of the backbone or the side-chains, as well as the grafting density. Moreover, their synthesis is relatively simple. Therefore, graft copolymers are promising precursors for UMMs.

However, in view of documents, while copolymers with other topologies have been widely used to prepare UMMs, research focusing on the preparation of UMMs based on graft copolymer is relatively scarce. There are a few examples on graft copolymers that have been used to investigate the formation of UMMs by the collapse of their backbone or side-chains, i.e., the flower-like unimolecular structures of poly(methyl methacrylate)-graft-polystyrene (PMMA-g-PS)²⁵ formed in selective solvents and the core–shell nanostructures based on PGA-g-(PCL-b-PEO).²⁴ Moreover, all of the graft copolymers that have been used as precursors for UMMs were homograft copolymers (Fig. 1d(1)) or graft copolymers bearing block copolymer side-chains (Fig. 1d(2)). That is, both of these examples possess one type of pendent polymer chain and they are thus described as unitary graft copolymers. However, unitary graft copolymers suffer from difficulty in adjusting the morphology of the UMMs and size of the hydrophobic core simultaneously.

We envisioned that this problem may be solved by using binary graft copolymers as the precursors for UMMs with the topology as shown in Fig. 1d(3). Here, different from unitary graft copolymers, the hydrophobic and hydrophilic segments of binary graft copolymers randomly distribute in the backbone chains of the graft copolymers. This unique structure of binary graft copolymers were expected to make it possible to adjust the morphology of the obtained UMMs and the size of their hydrophobic core simultaneously in one step by controlling the initial molar ratio of the side chains to the backbone chain. However, to the best of our knowledge, there have been no reports on UMMs from binary graft copolymers.

Herein, to confirm our hypothesis, we design a new kind of binary graft polymers and investigated the formation of UMMs in a selective solvent via intramolecular collapse. Specifically, a novel well-defined amphiphilic binary graft copolymer PGMA-g-(PCEMA-r-MPEG), denoted as GP, was synthesized by grafting MPEG–C≡CH and PCEMA–C≡CH as side-chains onto a third polymer backbone P(GMA-N₃) via Cu catalyzed alkyne–azide cycloaddition (CuAAC). The photo-crosslinkable PCEMA component was introduced in this work to help investigate the formation of the unimolecular micelles. The one-pot grafting reactions were quantitative at a [C≡CH] to [N₃] molar ratio of ≤39/260 or when x + y of Scheme 1 was ≤15%. The residual N₃ groups were then deactivated by reacting them with propargyl alcohol.

Scheme 1 Structure of PGMA-g-(PCEMA-r-MPEG).

In order to obtain UMMs, we initially adopted Liu's approach,³¹ which was an effective strategy to inhibit intermolecular association. This strategy involved adding a GP solution in DMF into deionized water under constant stirring and UV irradiation (if photo-crosslinking was desired). Since water is a good solvent only for MPEG but a poor solvent for both PGMA and PCEMA, the hydrophobic PGMA and PCEMA segments aggregated together to form a dense core while the soluble MPEG segments stretched into the water phase and served as a corona. The structure of the obtained UMMs could be locked by shining UV light onto the system in order to crosslink the PCEMA segment, thus yielding permanent crosslinked UMMs. Importantly, we also found that while UMMs could be obtained without subjecting them to crosslinking under UV irradiation, to our surprise, GP also underwent assembly at a relative high polymer concentration of 10 mg mL⁻¹ by directly adding deionized water into a GP solution in DMF and then dialyzing this dispersion again with a large amount of deionized water.

Usually, the high dilution method (i.e., <0.004 mg mL⁻¹) was normally employed to avoid undesired intermolecular association.^32,33 However, this method is very noneconomical in terms of solvent use. This inefficient and costly method can be improved by a continuous addition stategy,³⁴ which was employed by Liu to prepare unimolecular tadpoles.³¹ This strategy ensured that UMMs were prepared at final graft copolymer concentrations that were thousands of times higher than what could have been achieved with the high dilution method. Up to now, UMMs using unitary graft copolymers as the precursors have been prepared at a relative high concentration (∼0.3 mg mL⁻¹) in some published literature.^35,36 For example, Takaya Terashima and coworkers found two of their amphiphilic random copolymer underwent intramolecular folding even at 60 mg mL⁻¹.²⁰ This was because the proper hydrophilic/hydrophobic ratio successfully suppressed intermolecular association. In our case, new binary graft copolymer could also be assembled into UMMs at high polymer concentrations of 10 mg mL⁻¹. This finding could be not attractive in fundamental study on preparation of UMMs based on designed binary graft copolymers, but may be useful for potential application, i.e., drug delivery.

2. Experimental section

2.1 Materials and reagents

2,2′-Dipyridine (bpy, 99%), N,N,N′,N′′,N′′-pentamethyldiethylenetriamine (PMDETA, 99%), poly(ethylene glycol) methyl ether (MPEG, M_n = 5000 g mol⁻¹), dimethylaminopyridine (DMAP, 99.9%), cinnamoyl chloride (CC, 99.5%), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl, 98%), were purchased from Aldrich and used as received. In addition, sodium azide (NaN₃, 99.5%), copper(II) chloride (CuCl₂, 99%), tetrabutylammonium fluoride (TBAF, 98%), copper(II) sulfate pentahydrate (CuSO₄·5H₂O, 99%), sodium ascorbate (SA, 99%), N,N-dimethylformamide (DMF, 99%), dichloromethane (CH₂Cl₂, 99%), methanol (MeOH, 99%), tetrahydrofuran (THF, 99%), hexane (99%), 2-butanone (99%), and ammonium chloride (AlCl₃, 99%) were purchased from Aladdin Reagents of China and used as received.

Glycidyl methacrylate (GMA, Aladdin, 98%) was passed through a basic alumina column to remove the inhibitor. 2-Hydroxyethyl methacrylate (HEMA, Aladdin, 98%) was purified by initially washing an aqueous solution of this monomer (25 vol% HEMA, 100 mL) with hexane (4 × 200 mL). The monomer HEMA was subsequently precipitated from the aqueous phase by the addition of NaCl, and then it was dried over MgSO₄ prior to distillation under reduced pressure.³⁷ 2-Methoxyethyl 2-bromoisobutyrate and 3-(trimethylsilyl)propargyl 2-bromoisobutyrate were synthesized according to a literature procedure.³⁸

Copper(I) bromide (CuBr, Aladdin, 98%) and copper(I) chloride (CuCl, Aladdin, 98%) were rinsed with glacial acetic acid and subsequently washed with methanol before they were dried under vacuum. Propargyl alcohol was dried over anhydrous magnesium sulfate for 12 h and distilled immediately before use. Meanwhile, triethylamine (99%, Aladdin) was refluxed over sodium and distilled before use. Pyridine, diethyl ether, 1,4-dioxane and diphenyl ether were purchased from Aladdin Reagents of China and refluxed over CaH₂ overnight and distilled prior to use.

2.2 Synthesis of PGMA and P(GMA-N₃)

A typical procedure to prepare PGMA₂₆₀ was performed as follows. A round-bottom flask charged with diphenyl ether (20.0 mL), 2-methoxyethyl 2-bromoisobutyrate (45.3 mg, 0.201 mmol), GMA (20.0 g, 0.141 mol), CuBr (50.5 mg, 0.353 mmol) was degassed by three freeze–evacuate–thaw cycles, and the deoxygenated PMDETA (61.2 mg, 0.353 mmol) was then transferred into the flask under the protection of argon. The polymerization was carried out in an oil bath at 30 °C for 3 h. The flask was subsequently immersed into liquid nitrogen and then its contents were exposed to air. The crude product was diluted with 100 mL of dichloromethane and passed through an activated neutral alumina column. The filtrate was concentrated to ∼35 mL and precipitated three times via addition into a large amount of hexane. The precipitate was dried under vacuum for 24 h (yield: 40.1%).

P(GMA-N₃) was prepared by a method described in the literature.³⁹ PGMA₂₆₀ (5.61 g, bearing 39.5 mmol of epoxy groups), NaN₃ (10.3 g, 158 mmol) and AlCl₃ (0.133 g, 1.00 mmol) was dissolved in 100 mL of DMF and stirred for 48 h in a temperature-controlled oil bath at 50 °C. After the reaction, insoluble impurities were removed via filtration. The filtrate was concentrated under reduced pressure and then precipitated in water three times. The product was dried under vacuum to provide 7.18 g of a white solid in a 98.2% yield.

2.3 Synthesis of PHEMA–C≡CH

PHEMA–C≡CH was synthesized via ATRP using 3-(trimethylsilyl)propargyl 2-bromoisobutyrate as the initiator. The initial molar ratio of [HEMA]₀/[I]₀/[CuCl]₀/[CuCl₂]₀/[bpy]₀ was 60/1/1/0.2/2, while [HEMA]₀ = 7.69 M in a solvent mixture of 2-butanone and methanol (3/2 by volume). The mixture was degassed by three freeze–evacuate–thaw cycles and backfilled with argon. The polymerization was conducted at 50 °C for 1.5 h and then quenched by exposing the reaction mixture to air. The crude product was diluted with 100 mL of methanol and passed through an activated neutral alumina column to remove the catalyst. The filtrate was concentrated to ∼45 mL and added into an excess of water to precipitate the polymer. The final product was dried under vacuum at room temperature for 24 h (yield: 58.9%).

To remove the trimethylsilyl protecting group introduced by the initiator, the product obtained above (10.0 g, bearing 1.18 mmol of trimethylsilyl groups), DMF (80 mL) and TBAF (1.23 g, 4.71 mmol) were stirred together at room temperature for 72 h. After most of the solvent was removed under reduced pressure, the polymer solution was added to an excess of deionized water (300 mL) in order to precipitate the polymer. The polymer was redissolved in ∼30 mL of methanol and precipitated into 300 mL of water again. The obtained product was dried under vacuum for 24 h, thus generating 8.0 g of PHEMA–C≡CH as a light yellow solid in an 81% yield.

2.4 Synthesis of PCEMA–C≡CH

In order to prepare PCEMA–C≡CH, PHEMA–C≡CH (5.80 g, bearing 44.6 mmol of hydroxyl groups) was reacted with cinnamoyl chloride (14.8 g, 89.2 mmol) in freshly distilled pyridine (160 mL). The mixture was stirred at room temperature for 12 h and then filtered to remove the pyridine salts. The filtrate was concentrated to ∼30 mL and added to an excess of methanol in order to precipitate PCEMA–C≡CH. This polymer was then dissolved in dichloromethane and then precipitated again from methanol in order to purify it further. The precipitate was then dried at room temperature under vacuum for 24 h. The yield was 9.06 g (78.1%).

2.5 Synthesis of MPEG–C≡CH

MPEG–C≡CH was synthesized in two steps. First, 4-oxo-4-(prop-2-ynyloxy)butanoic acid was synthesized according to a published procedure.⁴⁰ Propargyl alcohol (4.72 g, 84.3 mmol), succinic anhydride (15.8 g, 158 mmol), triethylamine (16.0 g, 158 mmol) and pyridine (12.5 g, 158 mmol) were dissolved in 100 mL of dry 1,4-dioxane. The reaction was performed at room temperature for 24 h and the solvent was subsequently evaporated under vacuum. The crude product was dissolved in 300 mL CH₂Cl₂ and washed six times with cold 1 M HCl (10 mL). The organic phase was dried over MgSO₄, filtered and evaporated to obtain 4-oxo-4-(prop-2-ynyloxy)butanoic acid. The yield was ∼91%.

In the second step, poly(ethylene glycol) methyl ether was reacted with 4-oxo-4-(prop-2-ynyloxy)butanoic acid to yield MPEG–C≡CH. In a typical procedure, MPEG (M_n = 5000 g mol⁻¹, 20.0 g, 4.00 mmol), 4-oxo-4-(prop-2-ynyloxy)butanoic acid (1.87 g, 12.0 mmol), DMAP (1.95 g, 16.0 mmol) and EDC·HCl (4.60 g, 24.0 mmol) were dissolved in 100 mL CH₂Cl₂. The solution was stirred at room temperature for 72 h and then sequentially washed twice with 2 M HCl (5 mL), saturated NaHCO₃ (10 mL) and distilled water (10 mL). The organic phase was collected and dried over anhydrous MgSO₄ for 12 h and filtered. The filtrate was concentrated via rotary evaporation and precipitated from an excess of cold diethyl ether. The precipitate was then dried under vacuum for 12 h, thus yielding 17.5 g of MPEG–C≡CH as a white powder in an 85.1% yield.

2.6 Synthesis of PGMA-g-(PCEMA-r-MPEG)

The general procedure employed to graft the alkyne-terminated side-chains onto the azide-bearing polymer backbone was as follows. P(GMA-N₃)₂₆₀ (97.2 mg, bearing 0.525 mmol of azide groups), PCEMA₆₅–C≡CH (150 mg, bearing 8.88 × 10⁻³ mmol of alkyne groups), MPEG₁₁₄–C≡CH (350 mg, bearing 0.0700 mmol of alkyne groups), CuSO₄·5H₂O (6.45 mg, 0.0258 mmol), sodium ascorbate (10.3 mg, 0.0520 mmol), and DMF (12 mL) were added into a round-bottom flask equipped with a magnetic stirrer. The mixture was purged with argon for 50 min. The reaction mixture was subsequently stirred at room temperature for 48 h. Finally, propargyl alcohol (0.800 g, 14.3 mmol) was injected into the flask to deactivate the residual azide groups. The crude product was diluted with 100 mL of CH₂Cl₂ and passed through an activated neutral alumina column to remove the catalyst. After most of the CH₂Cl₂ was removed via rotary evaporation, the residue was dialyzed against water for 2–3 days to remove the sodium ascorbate. The final product was then collected after it had been freeze dried, at a yield of ∼65%.

2.7 Preparation of the UMMs

Crosslinked UMMs were prepared by pumping 2 mL of the GP solution (in DMF, 5 mg mL⁻¹) into 18 mL of deionized water, which was kept at 25 °C under constant stirring and irradiation. The tip of the GP addition needle was wrapped with a piece of tinfoil in the shape of a trumpet to avoid premature crosslinking of the newly added sample. The irradiation was continued for another 2 h after the addition of the GP. The irradiated samples were then transferred to a rinsed dialysis bag (MWCO 10 K) and dialyzed against a large volume of deionized water for 24 h to remove the DMF.

2.8 Characterization techniques

2.8.1 Size exclusion chromatography (SEC). The number-average molecular weight (M_n) and polydispersity index (M_w/M_n) of each polymer were determined by size exclusion chromatography (SEC). SEC measurements were performed at 30 °C using a Waters 1515 system equipped with a Waters 2414 refractive index (RI) detector. PS standards were used to calibrate the SEC system, and DMF was used as the eluent at a flow rate of 1.00 mL min⁻¹ at 35 °C.

2.8.2 Nuclear magnetic resonance spectroscopy (NMR). Nuclear magnetic resonance spectroscopy (NMR) spectra were recorded using a Bruker Avance-400 spectrometer operated in the Fourier transform mode. Deuterated chloroform (CDCl₃), deuterated water (D₂O) or deuterated dimethyl sulfoxide (DMSO-d₆) were used as the solvents.

2.8.3 Atomic force microscopy (AFM). Samples were prepared for atomic force microscopy (AFM) characterization by drop-casting a dilute solution of the corresponding micelles at a concentration of 0.005 mg mL⁻¹ onto a freshly cleaved mica surface. The samples were then dried under high vacuum to remove any residual solvents. The AFM images were captured by a MultiMode 8 SPM AFM system (Bruker) using a ScanAsyst mode.

2.8.4 Transmission electron microscopy (TEM). Prior to observation via transmission electron microscopy (TEM), the samples were prepared by drop-casting a dilute solution of micelles (∼0.003 mg mL⁻¹) onto nitrocellulose-coated 200 mesh copper grids before they were dried under vacuum. The samples were subsequently stained by placing a drop of an aqueous phosphotungstic acid solution (1.3 wt%) onto the copper grids. After 30 s, the excess aqueous phosphotungstic acid solution was blotted away with filter paper. TEM images were obtained using a JEM-100CX II microscope operated at 80 kV.

2.8.5 Dynamic light scattering (DLS). The hydrodynamic diameters of the micelles in aqueous solutions were determined via DLS using a Malvern Zetasizer Nano S equipped with a 633 nm laser. All light scattering experiments were performed at 25 °C at a detection angle of 90°. All the samples (0.5 mg mL⁻¹) were filtered through filters with a pore size of 0.22 μm prior to the DLS measurements.

2.8.6 UV-vis spectroscopy. UV-vis absorption spectra of the crosslinked micelle sample was recorded on UV-vis spectrophotometer UV-2450 (Shimadzu) using 1 cm-path length quartz cuvettes. Spectra were collected within a range of 200–800 nm. The concentration of the sample was 0.05 mg mL⁻¹.

3. Results and discussion

3.1 Preparation of the binary graft copolymer PGMA-g-(PCEMA-r-MPEG)

The synthetic pathway for the preparation of PGMA-g-(PCEMA-r-MPEG) is outlined in Scheme 2. P(GMA-N₃), MPEG–C≡CH, and PCEMA–C≡CH were initially synthesized, and then the latter two polymers were grafted onto the P(GMA-N₃) backbone via a click reaction that was catalyzed by CuSO₄·5H₂O/sodium ascorbate under mild conditions to yield the binary graft copolymer PGMA-g-(PCEMA-r-MPEG).

Scheme 2 Synthetic pathway toward PGMA-g-(PCEMA-r-MPEG).

3.1.1 Synthesis of PGMA and P(GMA-N₃). According to Scheme 2, the polymerization of 2-methoxyethyl 2-bromoisobutyrate with GMA in the presence of PMDETA and CuBr at 30 °C for 2 h yielded a well-defined linear PGMA chain. A monomer-to-initiator molar ratio [M]₀/[I]₀ of 700 : 1 was used for this polymerization. 2-Methoxyethyl 2-bromoisobutyrate was used as the initiator to facilitate the determination of the repeat unit numbers by ¹H NMR spectroscopy, because the signals corresponding to the initiator's –OCH₃ group did not overlap with the resonances of the PGMA protons. The resultant polymer was analyzed by ¹H NMR in CDCl₃. The ¹H NMR spectrum of PGMA₂₆₀ is shown in Fig. 3b. A repeat unit number of 260 was obtained by comparing the signal integration corresponding to the initiator's –OCH₃ group at 3.35 ppm with that of the epoxide CH protons at 3.21 ppm. The sample was also analyzed by size exclusion chromatography (SEC) using DMF as the eluent. The number-average molecular weight (M_n) was found to be 93.3 × 10³ g mol⁻¹ with a narrow distribution (PDI) of 1.22 (Table 1).

Table 1 Preparation conditions and molecular characteristics of the precursory polymers

Sample	[M0]/[I0]	Yield^a (%)	DP^b NMR	Mn NMR (kg mol^−1)	Mn SEC (kg mol^−1)	Mw/Mn SEC
a Determined by gravimetric analysis.b Determined via ^1H NMR integration.
PGMA260	700 : 1	40.1	260	36.9	93.3	1.22
P(GMA-N3)260		98.2	260	48.1	1610.2	2.15
MPEG–C≡CH		85.1	114	5.0	28.3	1.01
PCEMA–C≡CH	60 : 1	78.1	65	20.8	41.9	1.44

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P(GMA-N₃) was prepared by ring-opening reaction of the epoxy groups with NaN₃ in the presence of AlCl₃. As shown in Fig. 3a, the signals corresponding to the CH (3.23 ppm) and CH₂ (2.64 ppm, 2.84 ppm) protons of the epoxide ring had shifted to 3.87 and 3.38 ppm, respectively. The signals at 3.79 and 4.29 ppm of the methylene (–COOCH₂–) moiety had also shifted to one signal at 3.87 ppm in P(GMA-N₃) (d peaks for P(GMA-N₃) in Fig. 3a). All of these chemical shifts confirmed the 100% conversion of the epoxy groups to azido groups.

The P(GMA-N₃)₂₆₀ sample was also characterized by SEC using DMF as the eluent. A relatively broad peak (PDI = 2.15) was observed, which was probably due to the enhanced interaction between the azide groups or/and the hydroxyl groups of P(GMA-N₃) and the SEC columns according to previous reports.⁴¹ In order to determine whether the azide groups and/or hydroxyl groups of P(GMA-N₃) influenced the SEC trace, the azide groups and hydroxyl groups of P(GMA-N₃) were protected by reacting P(GMA-N₃) with propargyl alcohol and acetic anhydride in sequence. The resultant polymers obtained from each step were then analyzed by SEC. Fig. 2 shows the SEC traces of P(GMA-N₃) prior to protection (a), P(GMA-N₃) after protection with propargyl alcohol (b) and P(GMA-N₃) after protection by both propargyl alcohol and acetic anhydride (c). The peak remained broad after P(GMA-N₃) had reacted with propargyl alcohol, but became narrow (PDI = 1.50) after the reaction with acetic anhydride. This result suggested that the broad distribution was likely caused by the hydroxyl groups.

Fig. 2 SEC traces of P(GMA-N₃) (a), P(GMA-N₃) after protection with propargyl alcohol (b), and P(GMA-N₃) after protection with both propargyl alcohol and acetic anhydride (c).

3.1.2 Synthesis of PCEMA–C≡CH. As shown in Scheme 2, PCEMA–C≡CH was prepared in three steps. First, PHEMA–C≡C–TMS was synthesized via ATRP using 3-(trimethylsilyl)propargyl 2-bromoisobutyrate as an initiator. Second, PHEMA–C≡C–TMS was activated with TBAF to remove the TMS protecting group, thus yielding PHEMA–C≡CH. Third, the resultant PHEMA–C≡CH was reacted with cinnamoyl chloride to yield PCEMA–C≡CH.

PHEMA–C≡C–TMS was synthesized according to a published literature procedure.⁴² 3-(Trimethylsilyl)propargyl 2-bromoisobutyrate was used as the initiator to prevent possible side reactions between the radicals and the terminal alkyne group during the polymerization.⁴³ The TMS protecting group was readily removed by stirring the polymer in a DMF solution with TBAF. The ¹H NMR spectrum of PHEMA–C≡CH is shown in Fig. 3d. It was apparent that the signal corresponding to the TMS protecting group at 0.14 ppm had disappeared from the spectrum, which demonstrated that this protecting group had been successfully removed.

Fig. 3 ¹H NMR spectra and peak assignments for (PGMA-N₃)₂₆₀ (a), PGMA₂₆₀ (b), PCEMA–C≡CH (c), PHEMA–C≡CH (d), MPEG–C≡CH (e) and 4-oxo-4-(prop-2-ynyloxy)butanoic acid (f).

PCEMA–C≡CH was obtained by reacting PHEMAC≡CH with cinnamoyl chloride in anhydrous pyridine. This cinnamation reaction proceeded quantitatively. Its quantitative occurrence can be appreciated by comparing the ¹H NMR spectra of PHEMA–C≡CH and PCEMA–C≡CH shown in Fig. 3. The peaks appearing at 3.59 and 3.90 ppm in the spectrum of PHEMA–C≡CH (signals c and d, respectively in Fig. 3d) had completely disappeared and were replaced by the signals appearing at 4.08 and 4.21 ppm after the cinnamation reaction as shown in the spectrum of PCEMA–C≡CH (signals c and d, respectively in Fig. 3c). The DP of PCEMA–C≡CH was determined from the integration ratio of the peak at 4.59 ppm (signal e in Fig. 3c) with respect to that of the ethyl groups at 4.08 ppm (signal c in Fig. 3c). The repeat unit number of PCEMA–C≡CH was determined to be 65. SEC characterization revealed that the number-average molecular weight of PCEMA–C≡CH was 4.19 × 10⁴ g mol⁻¹. Meanwhile, the polydispersity index was 1.44.

3.1.3 Synthesis of MPEG–C≡CH. MPEG–C≡CH was obtained by performing an etherification reaction between MPEG and 4-oxo-4-(prop-2-ynyloxy)butanoic acid. Firstly, propargyl alcohol was reacted with succinic anhydride to produce 4-oxo-4-(prop-2-ynyloxy)butanoic acid, which was structurally characterized via ¹H NMR (Fig. 3f). Secondly, 4-oxo-4-(prop-2-ynyloxy)butanoic acid was reacted with the terminal hydroxyl group of a commercial MPEG (M_n = 5000) chain to yield MPEG–C≡CH. The resultant MPEG–C≡CH was carefully characterized by ¹H NMR (Fig. 3e) and SEC (Fig. 5d). A comparison of the proton integrations corresponding to the MPEG chain's terminal CH₃–O– group at 3.27 ppm and that of the two methylene groups of 4-oxo-4-(prop-2-ynyloxy)butanoic acid at 2.64 ppm yielded a ratio of 3.10/4.03, which provided evidence of a complete functionality transformation. As expected, the polydispersity of MPEG–C≡CH was low at 1.01.

3.1.4 Binary graft copolymers. The linear polymer side-chains were grafted onto the backbone via a CuAAC reaction, which was catalyzed by CuSO₄·5H₂O/sodium ascorbate under mild conditions. The residual azide groups were deactivated by reacting them with an excess of propargyl alcohol.

The ¹H NMR spectra of the purified GP is shown in Fig. 4. The PGMA backbone signals were absent because the backbone chain was surrounded by side-chains and tumbled slowly, and thus these signals were too broad to be detected via NMR.³⁸ In contrast, all of the protons of the grafted PCEMA and MPEG chains were visible in the spectrum. A quantitative comparison of the integrations measured at 7.50 and 3.64 ppm yielded a molar ratio of 1.00 : 7.99 for the PCEMA and MPEG repeat units, respectively, while the theoretical value was 1.00 : 7.89. The mass ratio of MPEG–C≡CH to PCEMA–C≡CH was calculated to be 7 : 3.

Fig. 4 ¹H NMR spectrum of the purified GP. This spectrum was recorded at 400 MHz in CDCl₃.

The SEC traces of PCEMA–C≡CH and MPEG–C≡CH and of the crude GP product obtained after the click reactions had been performed are shown in Fig. 5. DMF rather than THF was used as the eluent in this case because GP had poor solubility in THF.

Fig. 5 SEC traces of GP and its three precursors: GP (a), P(GMA-N₃) (b), PCEMA–C≡CH (c), and MPEG–C≡CH (d). DMF was used as the eluent.

As expected, GP had a much shorter retention time than those of its precursors. P(GMA-N₃) had an abnormally short retention time, probably due to the excessive swelling of the P(GMA-N₃) chains in DMF. At these low feed ratios, all of the added polymers underwent quantitative grafting under our reaction conditions, and thus the obtained grafting density matched its targeted value of 15%. The mass fraction of the hydrophilic MPEG chains was 58.6%, and that of hydrophobic PGMA and PCEMA chains combined was 41.4%. The polydispersity index of GP was 1.62 (Table 2).

Table 2 Preparation conditions and molecular characteristics of the binary graft copolymer

Sample	Mass feed ratio^a	Molar feed ratio^b	Mn,SEC (kg mol^−1)	Mn,theor^c (kg mol^−1)	Mw/Mn
a Feed mass ratio between P(GMA-N3), PCEMA–C≡CH, and MPEG–C≡CH.b Feed molar ratio between –N3, PCEMA–C≡CH, and MPEG–C≡CH.c Mn,theor = Mn(P(GMA-N3)) + x × n × 16 900 + y × n × 5000.
GP	1.0 : 1.5 : 3.6	260 : 4.4 : 34.6	2190	294	1.62

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3.2 Graft copolymers vs. cylindrical brushes

Graft copolymers adopt a cylindrical brush conformation and become molecular brushes if the distance between the grafted chains (D) is less than twice the radius of gyration (R_g) of the grafted chains³⁰ and the length of the backbone is significantly longer than that of the side-chains due to intramolecular excluded volume effects.⁴⁴ The conformation of a molecular brush is controlled by steric repulsions between the densely grafted side-chains that enhance the stiffness of the backbone, thus hindering overlapping and entanglement with neighboring polymers.⁴⁵ In this work, the grafting density was ∼15%. In other words, each side-chain was separated by 6.66 monomer units on the polymer backbone. Thus, the distance between the grafted side-chains was 1.7 nm, given by 6.66 × l (length of a monomer unit) = 6.66 × 0.25 nm. To a first approximation, we considered a homograft copolymer PGMA-g-MPEG and calculated the radius of gyration R_g of a MPEG chain in the unperturbed state. The radius of gyration R_g of a MPEG chain with a molar mass of 5000 g mol⁻¹ is ∼3.0 nm according to the literature.⁴⁶ This met the condition D < 2R_g, thus indicating that the grafts in our copolymer were in the brush regime. However, the length of the backbone of P(GMA-N₃)₂₆₀ was less than double that of the side-chains. Therefore we will continue to refer to our copolymers as graft copolymers.

3.3 Preparation of the unimolecular micelles

In order to obtain UMMs, we adopted a clever and innovative flow reactor design in this work to avoid the statistically favored and competing intermolecular reactions.¹⁹ As shown in Scheme 3(b)–(d), this method involved dissolving the GP in DMF, which solubilized both the main chain and the side-chains, and then adding the polymer solution into deionized water via a peristaltic pump under constant stirring and irradiation. The addition rate was kept at ∼0.2 mL min⁻¹, and thus an ultra-low concentration of the GP was retained throughout the self-assembly process. When the GP solution was added into water, the hydrophobic PGMA and PCEMA segments immediately collapsed, while the hydrophilic MPEG chains stretched into water. The GP was then converted into UMMs with hydrophobic PGMA/PCEMA cores that were surrounded by a corona consisting of the hydrophilic MPEG chains. This unimolecular structure was quickly locked by a crosslinking reaction involving the PCEMA. In such a method, intermolecular association was effectively minimized.

Scheme 3 Photographs of vials containing: water (a), GP in DMF (b, 0.5 mg mL⁻¹), crosslinked UMMs in water (c, 0.5 mg mL⁻¹), and non-crosslinked UMMs in water (d, 0.5 mg mL⁻¹). Also shown is a schematic depiction of the formation of the crosslinked and non-crosslinked unimolecular micelles.

As shown in Scheme 3, the prepared crosslinked UMM solution (c) was as transparent and colorless as pure water (a) and the solvated GP solution in DMF (b), but the pathway of a laser beam was not visible in water. The laser beam's pathway in the crosslinked UMM sample was much stronger than that in the GP solution in DMF, suggesting some new sources of scattering were introduced during the above steps. In order to determine the crosslinking degree of the obtained micelle sample, UV-vis spectroscopy was used to monitor the crosslinking process of the non-crosslinked micelle sample by measuring the decrease in the absorbance at 274 nm corresponding to the CEMA double-bond. Its crosslinking degree was determined to be ∼60.1% by comparing the reduced absorbance at 274 nm of the micelle sample with the original absorbance of the non-crosslinked micelle sample.

Size Exclusion Chromatography (SEC) was performed to confirm whether the micelles formed were UMMs or multimolecular micelles. The crosslinked UMMs described above were subjected to dialysis against deionized water to remove DMF in advance. The crosslinked UMM sample was obtained by freeze-drying treatment and redispersal in DMF prior to characterization by SEC. Fig. 6a compares the SEC traces of GP and a micellar GP sample. It was apparent that the SEC peak of the GP micelle sample had shifted to a longer retention time, which was due to a reduction of the hydrodynamic volume V_P.

Fig. 6 SEC traces of the crosslinked UMMs and GP (a), DLS traces (b) of crosslinked (dashed line) and non-crosslinked (solid line) UMMs, TEM image of the crosslinked UMMs (c, negatively stained), TEM image of non-unimolecular micelles (d, negatively stained), AFM image of the crosslinked UMMs (e).

To calculate the hydrodynamic volume V_P of the GP micelle sample at its chromatogram peak, we can correlate its peak PS-equivalent molecular weight M_p with V_P using eqn (1):³¹

Herein, the intrinsic viscosity [η]_P of PS with a molecular weight M_w can be calculated from eqn (2):

[η]_P = 1.10 × 10⁻²M_P^0.725 mL g⁻¹ (2)

The use of both eqn (1) and (2) yielded a hydrodynamic volume V_P reduction of 15.2% for the GP micelle sample relative to its graft copolymer precursor GP. This pronounced decrease in volume suggested that PCEMA and PGMA underwent intramolecular collapse in water. Based on the above analysis, we concluded that GP underwent intramolecular folding in water to form compact UMMs, rather than multimolecular micelles.

The morphology of the crosslinked UMMs was analyzed by TEM. For this measurement, the micelles were aero-sprayed onto nitrocellulose-coated copper grids and then negatively stained with phosphotungstic acid. As shown in Fig. 6c, the path length of the electron beam through the particles increased from the edge to the center because the stain precipitated onto the nitrocellulose-coated grid after deposition of the particles. Thus, the particles appeared white on a dark background. The average diameters of the crosslinked UMMs shown in Fig. 6c were 13 ± 2 nm. AFM measurements were also performed to quantify the structures of the crosslinked UMMs. As shown in Fig. 6e, robust spherical nanoparticles were clearly observed with a mean diameter of 15 nm, which was very close to that determined by TEM. The height of the nanoparticles was ∼6 nm, which closely matched their radius. The size distribution of the crosslinked UMMs was studied by DLS. As shown in Fig. 6b (dashed line), a unimodal peak in the range of 20–60 nm was observed via DLS with a narrow PDI (0.12). The mean hydrodynamic diameter (D_h) of the unimolecular micelles was 34 nm. The D_h value was much larger than that determined by both TEM and AFM because the samples prepared for the TEM and AFM measurements had been dried, but an aqueous solution was employed for the DLS measurements. Consequently, the hydrophilic MPEG chains assumed a stretched conformation in the latter case.

The theoretical mass of a UMM was calculated to be 4.9 × 10⁻¹⁹ g from m_theor = (M(P(GMA-N₃)) + M(PCEMA) × n × x + M(MPEG) × n × y)/NA, where NA is Avogadro's number. Assuming that the crosslinked UMMs measured by AFM was in the shape of a hemisphere with a radius of 6.5 nm, the mass of an individual nanoparticle would be 6.8 × 10⁻¹⁹ g, as given by m_measu = ρ(GP) × (1/2) × (4πr³/3). Herein, the densities of the GP, PCEMA, MPEG, and PGMA were denoted as ρ(GP), ρ(PCEMA) = 1.25 mg mL⁻¹,⁴⁷ ρ(MPEG) = 1.10 mg mL⁻¹,⁴⁸ and ρ(PGMA) = 1.08 mg mL⁻¹.⁴⁹ Assuming that the volume of the GP is the sum of the volumes of individual components, then ρ(GP) was calculated from ρ(GP) = M(GP)/(M(PGMA)/ρ(PGMA) + (M(PCEMA) × 4.4)/ρ(PCEMA) + (M(MPEG) × 34.6)/ρ(MPEG)) to be 1.18 mg mL⁻¹. The values of m_theor and m_measu were very similar, further proving the validity of the conclusion that was reached based on the SEC measurements.

In order to determine whether the same results were obtained without a crosslinking process, a non-crosslinked sample was prepared and observed via TEM. The sample was prepared by adding a GP solution (5 mg mL⁻¹ in DMF) dropwise into deionized water under constant stirring. The DMF was subsequently removed by dialysis against deionized water. The other experimental conditions were the same as those used to prepare the crosslinked UMMs, except that the crosslinking process was not employed in this case. As shown in Fig. 6d, spherical solid particles with a diameter of 15 ± 1 nm were observed via TEM for the non-crosslinked sample. This non-crosslinked micelles was larger than the crosslinked UMMs because the hydrophobic non-crosslinked PCEMA domain was not as compact as the crosslinked PCEMA,⁵⁰ rather than because multimolecular micelles formed in the absence of crosslinking treatment. Therefore, we concluded that stable UMMs with the PCEMA and PGMA components forming the core and the MPEG components forming the corona were prepared through the above non-crosslinking protocol. Therefore, crosslinking did not influence the morphologies of the UMMs, but did cause the crosslinked UMMs to become contracted and exhibit a slightly smaller diameter. For comparison, DLS measurements were also performed on the non-crosslinked UMMs as shown in Fig. 6b (solid line). It can be seen that there was no obvious difference encountered when the micelles were not subjected to crosslinking treatment.

3.4 Factors affecting UMM preparation

The UMMs discussed so far were prepared by the dropwise addition of a GP solution into deionized water under constant stirring (and irradiation). An additional parallel experiment was performed to study the influence of the GP concentration on the formation of the UMMs. Unlike the method described above, in this case 8 mL of deionized water was added dropwise into 1 mL of a GP solution (in DMF, 100 mg mL⁻¹) under constant stirring. The DMF was removed by dialysis against deionized water. The obtained sample was transferred into a quartz round-bottom flask and then irradiated by UV light under magnetic stirring at room temperature for 2 h in order to lock the structure of the micelles.

The micellar solution prepared from the above protocol was added dropwise onto nitrocellulose-coated grids and negatively stained with 1.3 wt% phosphotungstic acid for 30 s prior to TEM observation. Fig. 7a shows a TEM image of the micelles prepared at a high GP concentration. It could be seen that the crosslinked micelles had a spherical morphology and an average diameter of ∼13 nm, which was very similar to that of the crosslinked UMMs obtained in ultra-dilute conditions. This morphology and size of the crosslinked micelles was also confirmed by AFM measurement (Fig. 7b). The hydrodynamic diameters and polydispersity indices of the crosslinked micelles prepared at ultra dilute conditions (dash line) and at a high concentration (solid line) were determined by DLS (Fig. 7c). The polydispersity index and the average hydrodynamic diameter (D_h) of the crosslinked micelles prepared at a high concentration was very close to that of the crosslinked micelles obtained using dilute conditions. Therefore, it was apparent that only UMMs were obtained from the assembly of the binary graft polymer PGMA-g-(PCEMA-r-MPEG), even at a high polymer concentration.

Fig. 7 TEM (a) and AFM (b) images of crosslinked UMMs prepared at a high concentration, DLS traces (c) of crosslinked UMMs prepared at ultra dilute conditions (dashed line) and at a high concentration (solid line).

This phenomenon was unexpected because intermolecular interactions were supposed to become dominant at a high concentration of GP. This may be because the GP had a particular hydrophilic/hydrophobic ratio that caused it to favor the exclusive assembly of UMMs. Takaya Terashima and coworkers have previously investigated the single-chain folding of amphiphilic random copolymers in water.²⁰ They found that with a 20 mol% hydrophobic segment, all of copolymers formed unimolecular micelles in water, regardless of the hydrophobic pendant structures and the degree of polymerization, while those with over 50 mol% hydrophobic segment formed multichain aggregates. More importantly, the compact structure of the copolymers with a 20 mol% hydrophobic segment in water was maintained even at a relatively high concentration of 60 mg mL⁻¹. They concluded this was because both the steric repulsion between multiple hydrophilic chains and the intramolecular hydrophobic interactions successfully isolated the polymer backbone in water to prevent intermolecular aggregation from occurring when the hydrophilic segment reached a certain mol% value. In this study, PGMA-g-(PCEMA-r-MPEG) could serve as an amphiphilic random copolymer bearing longer hydrophilic and hydrophobic pendant chains. The molar fraction of the hydrophobic PCEMA component relative to the side-chains was 11 mol% in this work, which was much smaller than the hydrophobic content of 20 mol% employed by Terashima and coworkers. Therefore, we suspected that the GP's ability to form UMMs even at a high concentration was probably due to the high proportion of its hydrophilic segment. However, it was also worth noting that, in contrast with random copolymers, the hydrophobic main chain of this graft copolymer accounted for a significant portion of its mass. Therefore, not only the molar ratio of PCEMA relative to the total number of side-chains, but also the mass fraction of the hydrophobic segments (PGMA and PCEMA) relative to the total mass of an individual molecule determined whether the final micelles would be unimolecular or multimolecular. In addition, the graft length could also influence the self-assembly behavior. It has been reported that unimolecular spherical micelles were obtained from graft copolymers with longer side-chains, and multimolecular spherical micelles were obtained from those with shorter side-chains.²⁷ From the analysis above, we can conclude that the structure of precursor polymer was critical to control the intra- or intermolecular association through the external factors (concentration,³³ switch rate of solvent quality,³⁶ etc.) would have an effect on it.

4. Conclusion

We have designed and synthesized a novel amphiphilic binary graft copolymer PGMA-g-(PCEMA-r-MPEG) that bore water-soluble “stealthy” MPEG and photo-crosslinkable PCEMA side-chains with a grafting density of ∼15%. The mass ratio of MPEG–C≡CH to PCEMA–C≡CH was 7 : 3. The mass fraction of the hydrophilic MPEG was 58.6%, and that of the combined hydrophobic PGMA and PCEMA components was 41.4%. The degree of polymerization of the PGMA backbone was 260 units, and those of the MPEG–C≡CH and PCEMA–C≡CH side-chains were 114 and 65 units, respectively. This GP readily formed stable UMMs via assembly conditions involving either ultra-dilute or high polymer concentrations. The unimolecular micelles were stable in solution without further crosslinking, but the size of the crosslinked UMMs was slightly smaller than that of the non-crosslinked UMMs. This work offered a facile method based on a new class of binary graft copolymers to prepare unimolecular micelles. In the next step, we will study the effect of hydrophobic/hydrophilic ratio, side/main chain length, and graft density on the morphology and the size of the obtain UMMs.

Acknowledgements

The authors wish to thank the National Natural Science Foundation of China (No. 51173204, 51503124, 21404121, 21404122), the Pearl River Novel Science and Technology Project of Guangzhou, the Development Fund for Special Strategic Emerging Industry in Guangdong Province, the Guangdong Natural Science Foundation (2015A030313799, 2014A030310412, 2015A030313822, 2016A030313163), the Science and Technology Program of Guangzhou City, the Science Research Special Project of Guangzhou City and the Production Education Research Project in Guangdong Province (2015B090915004, 2015B010135002) for providing financial support.

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