Synthesis of PEGylated brush-type copolymers for a plurality of plug-and-play functions

Abstract

There is a great need for functional polymers in various applications. This study aims to develop a well-defined brush-type copolymer P(PEGMA_-N3-co-PEGMEMA)-b-PMAA via the technique of atom transfer radical polymerization (ATRP), and further to demonstrate a plurality of plug-and-play functions of this copolymer.

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Brush-type copolymers represent a unique branching topology of macromolecules in which multiple polymer side chains are grafted to a polymer backbone.^1–4 Varieties of brush-type copolymers have been designed and synthesized with different properties by tailoring the composition and ratios of the polymer side chains.^5,6 For instance, copolymers containing PEG analogue brushes have been proposed for tunable thermosensitivity by controllable radical polymerization.^6–8 Different aggregation behaviors of brush-type amphiphilic diblock copolymers have been investigated using “living”/controlled radical polymerization.⁹ Many functional brush-type copolymers have been utilized for emerging applications such as regulation of surface properties of substrate materials,^10,11 coating of novel biological sensors,¹² and development of environmental stimuli-responsive drug delivery.^13,14 In order to integrate different functions into a single molecular structure, it is critical to perform living polymerization during the synthesis so that the living chains can be employed to initiate the propagation of the side chains or graft distinct polymers in a controllable manner. Among the techniques of “living” polymerization, atom transfer radical polymerization (ATRP) has increasingly drawn attentions of researchers,^15–22 due to many advantages of this method including a broad range of suitable monomers, high tolerance to reagent impurities, and excellent controllability of synthetic conditions.^15–17,19 Copolymers with intriguing molecular structures have been reported via the technique of ATRP, such as star-like,^23,24 cylindrical,^25,26 or other hierarchical configurations,²⁷ demonstrating the powerfulness of ATRP in the synthesis of polymers. However, there is still a rising need in synthesizing new types of copolymers with flexible applications. For example, an ideal copolymer template would require multiple distinct reactive sites for different on-demand reactions subsequently, thus offering a matrix of functions derived from the side chains or backbone. The copolymer should possess an appropriate or tunable hydrophobic/hydrophilic property, so that it can meet the requirements for efficient drug loading and delivery, or effective modification of substrate surface. It remains a challenge to simultaneously achieve these features in the design and synthesis of copolymers.

Previously, we have synthesized a brush-type diblock copolymer and demonstrated its usefulness in assembling composite nanocarriers for gene delivery.²⁸ But the types of the reactive sites integrated in that copolymer were few, thus limiting its application in actively targeted drug delivery. Herein, we report our new efforts in developing a PEGylated brush-type copolymer synthesized with ATRP for a plurality of plug-and-play functions (Scheme 1). The structure of this copolymer includes a carbon backbone and blocks of functionalized side chains for distinct chemical reactions or assembling purposes. One block of this copolymer is composed of PEGylated side chains, offering tunable hyrdrophilic/hydrophobic property and prolonged circulation time in vivo potentially.^5,10,28,29 A controllable percentage of these PEGylated brushes contain active terminals, for example, azide groups, which allows for efficient and specific coupling with alkynylated compounds via CuAAC “Click” chemistry. Carboxylated side chains are enriched in the other block of this copolymer. Therefore, distinct functions can be realized using the carboxylated side chains, including the chemical reactions involved with the formation of amide bonds, or assembling of the copolymer on the surface of nanomaterials through electrostatic interactions. In this manuscript, we have demonstrated the versatile capabilities of this copolymer by taking advantage of multiple active sites for subsequent on-demand reactions or assembling. As the proof of concept, we utilize cyanine 3 alkyne (Cy3), 5-aminofluorescein, and dopamine (DA) modified Fe₃O₄ nanoparticles in our experiments. The functionalized fluorophores are important probes to track cellular activities.³⁰ Dopamine modified Fe₃O₄ nanoparticles have been widely used for bioimaging or constructing drug delivery systems.³¹ These reactions or assembly are efficient and robust, mimicking the concept of plug-and-play in the development of electronic devices. We believe that synthesis of the brush-type copolymers with multiple active (plug-and-play) sites represents an important strategy in the development of functional materials for various applications.

Scheme 1 Illustration of the synthesis of PEGylated brush-type copolymer I–IV.

In this work, we design a robust route to synthesize a brush-type copolymer template with multiple on-demand reactive sites. As shown in Scheme 1, the final copolymer (IV) was synthesized through a series of intermediates (copolymer I, II, IIIa, and IIIb). We chose two different monomers (PEGMA and PEGMEMA) for the PEGylated side chains of the copolymer on purpose. The hydroxyl groups of PEGMA allowed for subsequent reaction with 2-bromoisobutyryl bromide. Copolymerization of methyl terminated monomers (PEGMEMA) separated the side chains of PEGMA and diluted the concentration of hydroxyl groups, thus minimizing the undesired crosslinking reactions. The lengths of side chains derived from these two monomers were optimized to reduce the viscosity of reactants and avoid the undesired embroil of living chains. We developed a protocol of ATRP featured with two steps in one pot in order to synthesize P(PEGMA-co-PEGMEMA) and P(PEGMA-co-PEGMEMA)-b-PtBMA with high yields. Briefly, copolymer I, P(PEGMA-co-PEGMEMA), was synthesized via ATRP reaction using methyl-2-bromopropionate (MBP) as the initiator under nitrogen atmosphere. It maintained an active bromide end as the living site, which served as a macroinitiator for subsequent co-polymerization with tBMA monomers in order to prepare copolymer II, P(PEGMA-co-PEGMEMA)-b-PtBMA.

The chemical structure, composition, and molecular weight of copolymer I and II were verified with ¹H NMR (Fig. S1A and B†) and GPC measurements (Fig. S2†). The characteristic chemical shifts were identified as follows: δ ∼ 0.9–1.1 ppm for signal c and e, H of –CH₃ from P(PEGMA-co-PEGMEMA); δ ∼ 1.4 ppm for signal k, H of –C(CH₃)₃ from PtBMA; δ ∼ 1.8 ppm for signal d, H of –CH₂– from P(PEGMA-co-PEGMEMA); δ ∼ 3.4 ppm for signal j, H of –CH₃– from PEGMEMA side chains; δ ∼ 3.7 ppm for signal g(a), H of –CH₂– from PtBMA; δ ∼ 4.1 ppm for signal f and b, H of –CH₂– from main carbon chains, H of –CH₂CH₂–O– from side chains; δ ∼ 4.3 ppm for signal i, H of –OH at the end of PEGMA. Comparison of P(PEGMA-co-PEGMEMA) and P(PEGMA-co-PEGMEMA)-b-PtBMA with GPC exhibited an increase of molecular weight, suggesting successful copolymerization of the block of PtBMA by ATRP. We repeated the synthesis by ATRP in multiple times and obtained the molecular weights (M̄_n and M̄_w) and the polydispersity indices (PDIs) of the samples (Table 1). The results suggested reproducible synthesis of the copolymers with relative narrow PDIs via ATRP.

Table 1 Characterization of the molecular weights, and molecular weight distributions of copolymer I and copolymer II

Samples	M̄n (GPC) (g mol^−1)	M̄w (GPC) (g mol^−1)	PDI
P(PEGMA5-co-PEGMEMA24)	8850	11 510	1.30
P(PEGMA4-co-PEGMEMA24)	8730	12 310	1.41
P(PEGMA8-co-PEGMEMA24)	9920	11 520	1.16
P(PEGMA5-co-PEGMEMA22)	8480	12 040	1.42
P(PEGMA8-co-PEGMEMA30)-b-PtBMA40	17 580	26 370	1.50
P(PEGMA10-co-PEGMEMA32)-b-PtBMA40	17 690	27 370	1.54
P(PEGMA14-co-PEGMEMA38)-b-PtBMA60	25 180	31 330	1.24
P(PEGMA8-co-PEGMEMA28)-b-PtBMA40	17 880	28 790	1.61

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Copolymer IIIa, P(PEGMA_-Br-co-PEGMEMA)-b-PtBMA, was prepared by a substitution reaction of the hydroxyl groups at the end of PEGMA side chains.^32–34 During this reaction with 2-bromoisobutyryl bromide, the tert-butyl group in the block of PtBMA was kept as a protection of the carboxylic groups. As shown in Fig. 1A, the structure of copolymer IIIa was characterized via ¹H NMR (DMSO-d₆, TMS). The signals at δ ∼ 0.9–1.1 ppm, δ ∼ 1.4 ppm, δ ∼ 3.4 ppm, δ ∼ 3.7 ppm, δ ∼ 4.1 ppm were just described in the case of Fig. S1B.† Only the changed signals at δ ∼ 1.9 ppm and δ ∼ 2.0 ppm were described as follows: δ ∼ 1.9 ppm for signal l, H of –C(CH₃)₂Br from PEGMA side chains; δ ∼ 2.0 ppm for signal d, H of –CH₂– from P(PEGMA-co-PEGMEMA).

Fig. 1 ¹H NMR spectra of copolymer IIIa (A), copolymer IIIb (B) and copolymer IV (C).

Copolymer IIIb, P(PEGMA_-Br-co-PEGMEMA)-b-PMAA, was obtained by hydrolysis of the copolymer IIIa with trifluoroacetic acid (TFA). The reaction of hydrolysis was performed in dichloromethane with excessive TFA at room temperature for 72 h so as to remove the tert-butyl groups in the PtBMA segment. In the ¹H NMR spectra (Fig. 1B), the chemical shift of the protons in tert-butyl groups at δ ∼ 1.4 ppm completely disappeared. A new peak of the chemical shift of the protons at δ ∼ 12.4 ppm was clearly detected, attributed to the carboxyl groups in PMAA block. The other chemical shifts of the protons did not change in the comparison of the ¹H NMR spectra (Fig. 1A and B). These results confirmed that P(PEGMA_-Br-co-PEGMEMA)-b-PMAA copolymer was successfully synthesized by the reaction of hydrolysis. The presence of the carboxyl groups in the copolymer IIIb was also supported by the FT-IR spectra (see discussion of Fig. 2).

Fig. 2 (a) FT-IR spectra of copolymer IV (black) and Cy3-copolymer IV (red). (b) Fluorescent spectra of Cy3 (black) and Cy3-copolymer IV (red). (c) FT-IR spectra of copolymer IV (black) and 5-aminofluorescein-copolymer IV (green). (d) Fluorescent spectra of 5-aminofluorescein (black) and 5-aminofluorescein-copolymer IV (green). Insets: fluorescent images of Cy3-copolymer IV and 5-aminofluorescein-copolymer IV in micro-channels (50 μm).

Copolymer IV, P(PEGMA_-N3-co-PEGMEMA)-b-PMAA, was synthesized via a nucleophilic substitution reaction to replace the bromide groups with the azide groups in the PEGMA side chains. ¹H NMR analysis was employed to verify the structure of the copolymer (Fig. 1C). Most of ¹H NMR signals of the final product were similar to those of copolymer IIIb (Fig. 1B). A new peak of the chemical shift of the protons at δ ∼ 1.4 ppm was detected, attributed to the methyl groups adjacent to the azide groups after the nucleophilic substitution reaction.³⁵ Originally the chemical shift of the protons in the methyl groups adjacent to the bromide group was nearly at δ ∼ 1.9 ppm in the copolymer IIIa or copolymer IIIb. The substitution of the bromide groups with the azide groups made a change in the local induced magnetic fields, leading to a decrease of the chemical shift. The phenomenon of reduced chemical shift that we observed was consistent with the report in the literature.^35,36

We demonstrated that copolymer IV simultaneously enabled a panel of different reactions or assembling on the demands. The reaction of “Click” chemistry was performed between the alkynyl-terminated fluorophore molecules (Cy3) and P(PEGMA_-N3-co-PEGMEMA)-b-PMAA, as a demonstration of facile fluorescent labeling with this brush-type copolymer. We compared the FT-IR spectra of the samples before and after the reaction of “Click” chemistry. As shown in Fig. 2a (black curve), the characteristic absorbance peak at 2100 cm⁻¹ clearly suggested the presence of the azide groups in the copolymer IV. The peak at 1730 cm⁻¹ derived from the vibration of C=O of the carboxyl groups. The broad and intensive absorbance peak ranging from 2800 cm⁻¹ to 3600 cm⁻¹ was mainly contributed by the characteristic stretching vibration of –OH in carboxylic acid groups. After the reaction with alkynyl-terminated Cy3 (red curve in Fig. 2a), the characteristic peak at 2100 cm⁻¹ disappeared totally, suggesting a complete consumption of the azide groups in the reaction of “Click” chemistry. This reaction did not affect the carboxyl groups. After the purification by dialysis for 72 h, the fluorescent spectrum of Cy3 labeled copolymer exhibited an emission peak around 570 nm, matching the original fluorophore consistently (Fig. 2b). Intensive fluorescent luminescence was detected with an inverted fluorescent microscope when the solution of Cy3 labeled copolymer was used to fill the PDMS micro-channels (inset of Fig. 2b). Besides the fluorescent labeling, integration of the azide groups in the side chains makes the copolymer a very powerful model for many kinds of different functions on the demands via “Click” chemistry. For instance, alkyl-terminated peptides with tumor-homing capabilities can efficiently be coupled to the side chains of copolymers for targeted drug delivery. As a matter of fact, the bromide group of copolymer IIIb can also serve as a reactive site for introducing various end-functionalized grafts through nucleophilic addition.³⁷ These possibilities of introducing different chemical groups at the side chains definitely enlarge the range of applications using the template copolymers.

The carboxyl groups in the PMAA block of the copolymer IV were able to form covalent bonds with various amine-modified molecules via amidation reaction. In this work, 5-aminofluorescein molecules were conjugated to copolymer IV, as another demonstration of versatility of the brush-type copolymer. As shown in Fig. 2c (green curve), after the amidation reaction, the new peak at 1645 cm⁻¹ was primarily contributed by the characteristic absorption of the amide groups. The peaks in the range of 1100 cm⁻¹ and 1250 cm⁻¹ were possibly derived from the conjugate benzene rings of 5-aminofluorescein molecules. These pieces of evidence indicated successful conjugation of the copolymer IV with 5-aminofluorescein. There might be some carboxyl groups (with peak at 1730 cm⁻¹) left, depending on the molar ratio of the reagents and the reaction time. The characteristic absorbance peak at 2100 cm⁻¹ contributed by the azide groups was present for both samples before and after the amidation reaction (Fig. 2c, black curve and green curve, respectively). It indicated that the reaction for the carboxyl groups was independent, without influence on the azide groups at other side chains. As shown in Fig. 2d, an emission peak around 530 nm was present in the fluorescent spectrum of 5-aminofluorescein conjugated copolymer IV. The inset of Fig. 2d was a fluorescent image of the PDMS micro-channels filled by the solution of 5-aminofluorescein conjugated copolymer IV. The results supported a successful conjugation between copolymer IV and 5-aminofluorescein via the amidation reaction.

The carboxyl groups in the block of PMMA also provided an alternative function by enabling the assembling of copolymer IV on the positive charged surface via electrostatic interactions. We demonstrated that copolymer IV can be assembled onto the surface of dopamine modified Fe₃O₄ nanoparticles (Fe₃O₄-DA). The TEM images and zeta potentials of the assembled complexes (Fe₃O₄-DA@polymer IV) were acquired, in comparison to Fe₃O₄-DA nanoparticles (Fig. 3). The diameter of Fe₃O₄-DA nanoparticles was approximately 100 nm (Fig. 3a). After the assembling by electrostatic interactions, there was a thin layer of polymer (6–8 nm thickness) on the surface of the nanoparticles (white arrows in the Fig. 3b). The nanoparticles were slightly aggregated to each other after the assembling. In addition, there was a significant change in the zeta-potential from 15.4 mV to −8.8 mV (Fig. 3c and d), due to the assembling effect of copolymer IV on the surface of nanoparticles. It was straightforward to perform electrostatic interaction-based assembling, which could be conveniently applied on the demands.

Fig. 3 TEM images of (a) Fe₃O₄-DA NPs and (b) Fe₃O₄-DA@copolymer IV complexes, respectively. The white arrow indicates copolymer assembled on the surface by electrostatic interactions. Size distribution and zeta potentials of (c) Fe₃O₄-DA NPs and (d) Fe₃O₄-DA@copolymer IV complexes.

Conclusions

In summary, a unique brush-type copolymer P(PEGMA_-N3-co-PEGMEMA)-b-PMAA was successfully synthesized via ATRP reaction, exhibiting a plurality of plug-and-play functions. The chemical structures of the intermediate and final products during the synthesis were carefully characterized. The brush-type copolymer was grafted with a large number of PEGylated side chains, a tunable percentage of which were terminated with the reactive sites such as the azide groups for “Click” chemistry. The other block of the copolymer was rich in carboxylated side chains, which could be employed for on-demand functions such as the amidation reaction or electrostatic interaction-based assembling. In this work, we demonstrated the versatile capabilities of this copolymer with several examples. It is very important to synthesize the brush-type copolymers with multiple active (plug-and-play) sites in the development of functional materials, which shall find a variety of applications such as targeted drug delivery and smart surface materials of substrates.

Acknowledgements

This work was supported by the Major State Basic Research Development Program (2013CB932702, 2012CB932601), and by the National Natural Science Foundation of China (21275106, 21374066); a project supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions, and Doctoral Fund of Ministry of Education of China (20123201120025).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra06484h

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