Hydrophilic Hollow Molecularly Imprinted Polymer Microparticles with

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Hydrophilic Hollow Molecularly Imprinted Polymer Microparticles with Photo- and Thermoresponsive Template Binding and Release Properties in Aqueous Media Chenxi Li, Yue Ma, Hui Niu, and Huiqi Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b08868 • Publication Date (Web): 19 Nov 2015 Downloaded from http://pubs.acs.org on November 29, 2015

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ACS Applied Materials & Interfaces

Hydrophilic Hollow Molecularly Imprinted Polymer Microparticles with Photo- and Thermoresponsive Template Binding and Release Properties in Aqueous Media

Chenxi Li, Yue Ma,† Hui Niu,† and Huiqi Zhang* State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Functional Polymer Materials (Ministry of Education), Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), and College of Chemistry, Nankai University, Tianjin 300071, People’s Republic of China

†

These two authors contribute equally to this work.

1

ACS Paragon Plus Environment


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ABSTRACT: A facile, general, and efficient approach to prepare hydrophilic hollow molecularly imprinted polymer (MIP) microparticles with photo- and thermoresponsive template binding and release behaviors in aqueous media is described, which includes the preparation of uniform “living” silica submicrospheres bearing surface atom transfer radical polymerization (ATRP)-initiating groups (i.e., alkyl halide groups) via a one-pot sol-gel method, their subsequent grafting of azobenzene (azo)-containing MIP shell and poly(N-isopropylacrylamide)-block-poly(2-hydroxyethyl

methacrylate)

(PNIPAAm-b-PHEMA) brushes via successive surface-initiated ATRP, and final removal of the silica core. The successful synthesis of such hydrophilic hollow MIP microparticles was confirmed with SEM, FT-IR, water dispersion stability, and static contact angle studies. They proved to show apparently higher template binding capacities than the corresponding solid ones and obvious photo- and thermoresponsive template binding properties in aqueous solutions. Moreover, their pronounced light- and temperature-controlled template release in aqueous

media

was

also

demonstrated.

In

particular,

the

introduction

of

PNIPAAm-b-PHEMA brushes onto hollow MIP microparticles imparted them with high surface hydrophilicity both below and above the lower critical solution temperature of PNIPAAm, which paves the way for their applications in such areas as controlled drug/chemical delivery and smart bioanalysis. KEYWORDS:

molecularly

imprinted

polymers,

hollow

water-compatible, stimuli-responsive, controlled drug delivery

2


polymer

microparticles,

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1. INTRODUCTION As synthetic receptors with tailor-made recognition sites, molecularly imprinted polymers (MIPs) have long been considered to be promising substitutes for biological receptors (e.g., antibodies and enzymes) because of their good molecular affinity and selectivity, easy synthesis, high stability, and low cost.1-10 Although dramatic advances have been made in the field of molecular imprinting, there still exist some challenges to be addressed before broad and routine practical applications of MIPs can be eventually realized. In contrast with biological receptors that show outstanding molecular recognition ability and high stimuli-responsivity (e.g., thermoresponsivity) in aqueous solutions, most of the previously obtained MIPs lose their specific bindings in aqueous solutions (mainly due to their high surface hydrophobicity),1,2,10 and have poor stimuli-responsivity (because of their high crosslinking degrees typically required to stabilize binding sites2). These downsides of the MIPs largely restrict their applications in such areas as smart bioanalysis and controlled drug delivery. Over the past two decades, many efforts have been devoted to developing water-compatible10-12 and/or stimuli-responsive13,14 MIPs. Some stimuli-responsive MIP hydrogels have been designed for such purposes by using certain hydrophilic responsive (co)monomers (e.g., N-isopropylacrylamide (NIPAAm), acrylic acid, or azobenzene (azo) monomer) and cautiously selecting kinds and amounts of the utilized crosslinkers.15-18 However, rather laborious optimization of MIP formulations is usually needed for this theoretically simple approach, thus making it very difficult to obtain dual or multiple stimuli-responsive MIPs. Moreover, the resulting MIP hydrogels usually have nonspherical shapes and large sizes after their backbreaking grinding and sieving (their diameters are 3



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typically tens of micrometers and such relatively large MIP particles have many inaccessible binding sites and thus lower template binding capacities), which make them unsuitable for use in smart binding assays and drug delivery.19,20 Therefore, it is highly desirable to develop useful strategies for the preparation of water-compatible and (dual or multiple) stimuli-responsive MIP particles with small micro- or nanometer sizes and high template loadings. To address the above issues, our group has recently developed some efficient methods to generate water-compatible and single (thermo-), dual (photo- and thermo-) or multiple (photo-, thermo- and pH-) stimuli-responsive MIP microspheres.21-24 “Living” normal or photoresponsive MIP microspheres bearing controlled/“living” radical polymerization (CRP)-initiating groups were first prepared, which were subsequently grafted with hydrophilic polymer brushes of either thermoresponsive (i.e., poly(NIPAAm) or briefly PNIPAAm)

or

both

thermo-

and

pH-responsive

(i.e.,

poly(NIPAAm-co-2-(dimethylamino)ethyl methacrylate)) properties via surface-initiated CRPs.21-24 Although these MIP particles proved highly promising in controlled and sustained chemical or drug delivery/release systems, the use of the above-mentioned polymer brushes with stimuli-responsive collapse and dissolution behaviors as the responsive layer would result in hydrophobic MIP particles upon their collapse with increasing temperatures or pH values of MIP aqueous solutions, which makes them inappropriate for drug delivery application in aqueous media because their hydrophobic surfaces will lead to high nonspecific bindings of organic compounds and proteins present in biological media. In addition, the template loading capacities of such MIPs still need to be improved to achieve their optimal drug delivery effects. 4


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In this paper, we describe a facile, general, and efficient approach to prepare advanced water-compatible and stimuli-responsive MIP microparticles that can overcome the above problems (Scheme 1). Uniform “living” silica submicrospheres bearing alkyl halide groups (i.e., atom transfer radical polymerization (ATRP)-initiating groups) were first prepared via a one-pot sol-gel method, and they were subsequently grafted with an azo-containing MIP shell and

hydrophilic

thermoresponsive

block

copolymer

brushes

(i.e.,

poly(NIPAAm)-block-poly(2-hydroxyethyl methacrylate) or briefly PNIPAAm-b-PHEMA) via successive surface-initiated ATRP. Finally, the desired hydrophilic hollow MIP micropartilces were readily obtained by removal of the silica core. The simple combination of a photoresponsive MIP layer with hydrophilic thermoresponsive polymer brushes readily

Scheme 1. Schematic Illustration for the Preparation of Photo- and Thermoresponsive Hydrophilic Hollow Azo-Containing MIP Microparticles Bearing PNIPAAm-b-PHEMA Brushes. 5



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imparted the resulting MIP particles with obvious dual stimuli-responsive template binding and release capability in aqueous solutions. In addition, the introduction of a hollow structure into such MIP particles significantly improved their template binding capacities in comparison with their corresponding solid ones. In particular, the presence of PNIPAAm-b-PHEMA brushes on these hollow MIP particles allowed them to keep high surface hydrophilicity even above the lower critical solution temperature (LCST) of the PNIPAAm block because of the existence of external hydrophilic PHEMA layers. These positive characteristics (i.e., enhanced template binding capacities and high surface hydrophilicity even after the stimuli-responsivity of polymer brushes) make such hydrophilic hollow stimuli-responsive MIP microparticles superior to our previously developed ones and promising in such applications as controlled drug delivery and smart bioanalysis. The versatility of surface-initiated ATRP in the controlled grafting of tailor-made crosslinked and uncrosslinked polymer layers on various substrates, its suitability to a plethora of functional monomers and its mild polymerization conditions make this strategy highly applicable. To our knowledge, this is the first report on the hydrophilic hollow stimuli-responsive MIP particles with easily tunable template binding and release behaviors.

2. EXPERIMENTAL SECTION 2.1. Materials. Ethylene glycol dimethacrylate (EGDMA, Alfa Aesar, 98%), acetonitrile (Tianjin Jiangtian Chemicals, China, analytical grade (AR)), methanol (Tianjin Jiangtian Chemicals, AR), NIPAAm (Acros, 99%), and copper(I) chloride (CuCl, Tianjin Jiangtian Chemicals, AR) were purified prior to use according to our previously reported methods.22 2-Hydroxyethyl methacrylate (HEMA, Tianjin Institute of Chemical Reagents, China, 6


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chemical pure) was purified following a literature method.25 3-(N-Propyl)triethoxysilane 2-bromo-2-methylpropanamide (APTES-Br, Scheme S1) was prepared according to a reported method.26 Tris(2-(dimethylamino)ethyl)amine (Me6TREN) was synthesized following a literature approach.27 4-((4-Methacryloyloxy)phenylazo)pyridine (MAzoPy, Scheme S1) was obtained following our previous method.28 2,4-Dichlorophenoxyacetic acid (2,4-D, 98%, Scheme S1), tetraethylorthosilicate (TEOS, 98%), anhydrous copper(II) chloride (CuCl2, 98%), copper(II) bromide (CuBr2, 99%), and ethyl 2-chloropropionate (97%) were purchased from Alfa Aesar and used as received. Phenoxyacetic acid (POAc, Acros, 98+%, Scheme S1) and other chemicals were obtained commercially and used without further purification. 2.2. Synthesis of Uniform “Living” Silica Submicrospheres Bearing ATRP-Initiating Groups (or Briefly SiO2-Br). TEOS (10 mL) was added dropwise into a mixture of isopropanol (75 mL), methanol (25 mL), and an aqueous solution of ammonia (25%) (21 mL). The mixed solution was magnetically stirred (130 rpm) at 25 °C for 2 h, followed by the dropwise addition of APTES-Br (0.5043 g) under stirring. The reaction mixture was then stirred at 25 °C for 8 h. The resulting SiO2-Br particles (entry 1 in Table 1) were washed thoroughly with distilled water until the washing solution became neutral and then dried at 40 o

C under vacuum. 2.3. Synthesis of 2,4-D-Imprinted Core-Shell Submicrospheres with a Silica Core and

an Azo-Containing MIP Shell (i.e., SiO2@Azo-MIP) and Their Corresponding Control Core-Shell Submicrospheres (i.e., SiO2@Azo-CP). A mixture of SiO2-Br (0.3000 g), MAzoPy (0.9156 g), 2,4-D (0.7579 g), dried acetonitrile (150 mL), and methanol (50 mL) was magnetically stirred at 10 °C overnight in the dark to promote the complexation between 7



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Table 1. Characterization Data for SiO2-Br, the Ungrafted and Grafted Core-Shell MIP/CP, and Grafted Hollow MIP/CP Particles Dn a

Entry Sample

Ua

Mn,GPC b

Ðb

Contact angle (o) c

(μm)

a

1

SiO2-Br

0.565

1.02

-

-

77.9 ± 1.8

2

SiO2@Azo-MIP

0.632

1.04

-

-

125.4 ± 2.0

3

SiO2@Azo-CP

0.651

1.03

-

-

126.0 ± 1.7

4

SiO2@Azo-MIP@PNIPAAm

0.638

1.04

4350

1.35

74.2 ± 2.2

5

SiO2@Azo-CP@PNIPAAm

0.657

1.02

4760

1.48

74.5 ± 1.8

6

SiO2@Azo-MIP@(PNIPAAm-b-PHEMA)

0.649

1.05

10700

1.38

73.5 ± 3.5

7

SiO2@Azo-CP@(PNIPAAm-b-PHEMA)

0.670

1.04

11500

1.43

73.2 ± 2.4

8

H-Azo-MIP@(PNIPAAm-b-PHEMA)

-

-

-

-

74.2 ± 0.9

9

H-Azo-CP@(PNIPAAm-b-PHEMA)

-

-

-

-

72.8 ± 2.1

Number-average diameters (Dn) and size distributions (U) of the studied samples. bNumber-average

molecular weights (Mn,GPC) and molar-mass dispersities (Đ) of the free PNIPAAm or PHEMA generated during the surface-initiated ATRP processes as determined by GPC. c Static water contact angles of the polymer films.

MAzoPy and 2,4-D. To the above mixture were added EGDMA (1.9 mL) and Me6TREN (0.0823 g). After 10 min of argon bubbling for the reaction mixture in the ice bath, CuCl (11.9 mg) was added. The mixed solution was bubbled with argon for another 10 min and then stirred at 70 °C for 48 h in the dark. The obtained particles were washed successively with methanol, ethanol/acetic acid (9:1 v/v), and methanol to remove both the copper catalyst and template and then dried at room temperature under vacuum, leading to the desired SiO2@Azo-MIP particles (entry 2 in Table 1). The corresponding SiO2@Azo-CP particles (entry 3 in Table 1) were also synthesized following the same procedure, but without the addition of the template. 2.4. Synthesis of Core-Shell MIP and CP Submicrospheres Bearing PNIPAAm Brushes (i.e., SiO2@Azo-MIP@PNIPAAm and SiO2@Azo-CP@PNIPAAm). To an 8


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argon-purged (for 20 min in the ice bath) mixture of SiO2@Azo-MIP or SiO2@Azo-CP particles (0.2000 g), NIPAAm (2.4500 g), CuCl2 (5.2 mg), and isopropanol (10 mL) was added CuCl (38.0 mg). After 10 min of argon bubbling for the reaction mixture, Me6TREN (0.0960 g) was added. Ethyl 2-chloropropionate (1.35 μL) was then added into the reaction mixture after it was purged with argon for 10 min. The polymerization was then carried out at 25 °C for 24 h. The resulting particles were thoroughly washed with methanol and then dried at room temperature under vacuum to provide SiO2@Azo-MIP@PNIPAAm (entry 4 in Table 1) and SiO2@Azo-CP@PNIPAAm (entry 5 in Table 1). Some free PNIPAAm were generated in the above reaction solutions due to the presence of sacrificial initiator ethyl 2-chloropropionate there. They were obtained by the precipitation of the supernatant solutions (collected from the polymerization solutions through centrifugation and the copper catalysts inside them were removed by neutral aluminum oxide column purification) into ether, and then dried at room temperature under vacuum. 2.5.

Synthesis

of

Core-Shell

MIP

and

CP

Submicrospheres

Bearing

PNIPAAm-b-PHEMA Brushes (i.e., SiO2@Azo-MIP@(PNIPAAm-b-PHEMA) and SiO2@Azo-CP@(PNIPAAm-b-PHEMA)). To an argon-purged (for 20 min in the ice bath) mixture of SiO2@Azo-MIP@PNIPAAm or SiO2@Azo-CP@PNIPAAm particles (0.2000 g), HEMA (2.71 mL), CuBr2 (15.0 mg), methanol (3.25 mL), and distilled water (3.25 mL) was added CuCl (22.2 mg). After 10 min of argon bubbling for the mixed solution, 2,2’-bipyridine (0.0977 g) was added. Ethyl 2-chloropropionate (1.35 μL) was then added into the reaction solution after it was purged with argon for 10 min. The polymerization was then carried out at 25 °C for 12 h. The resulting particles were thoroughly washed with methanol,

and

then

dried

at

room

temperature

under

9


vacuum

to

provide


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(entry

6

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in

Table

1)

and

SiO2@Azo-CP@(PNIPAAm-b-PHEMA) (entry 7 in Table 1). Some free PHEMA were generated in the above reaction solutions due to the presence of sacrificial initiator ethyl 2-chloropropionate there. They were obtained by the precipitation of the supernatant solutions (collected from the polymerization solutions through centrifugation and the copper catalysts inside them were removed by neutral aluminum oxide column purification) into ether, and then dried at room temperature under vacuum. 2.6. Preparation of Hollow Azo-Containing MIP and CP Microparticles Bearing PNIPAAM-b-PHEMA

Brushes

(i.e.,


H-Azo-CP@(PNIPAAm-b-PHEMA)). SiO2@Azo-MIP@(PNIPAAm-b-PHEMA)

A or

mixture

and of


particles (0.25 mg/mL) and a 10% hydrogen fluoride (HF) solution in ethanol was incubated in a Teflon tube at 25 °C for 2 h. The resulting polymer particles were then washed thoroughly with methanol to remove the unreacted HF, and then dried at room temperature under vacuum to provide H-Azo-MIP@(PNIPAAm-b-PHEMA) (entry 8 in Table 1) and H-Azo-CP@(PNIPAAm-b-PHEMA) (entry 9 in Table 1).

2.7. Characterization. SiO2-Br particles, the ungrafted and grafted core-shell MIP/CP particles (i.e., core-shell MIP/CP particles without and with surface-grafted polymer (PNIPAAm or PNIPAAm-b-PHEMA) brushes), and grafted hollow MIP/CP particles (i.e., hollow MIP/CP particles with surface-grafted PNIPAAm-b-PHEMA brushes) were first characterized with a Nicolet Magna-560 FT-IR spectrometer and a scanning electron microscope (SEM, FEI Nova Nano 230). The diameters (Dn) and size distributions (U) of the

10


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studied particles were determined from their SEM images following the literature method.29 The molecular weights and molar-mass dispersities (Đ) of the free polymers obtained during the surface-initiated ATRP processes were characterized with the same gel permeation chromatograph (GPC) as described in our recent publication.22 Polystyrene (PS) was used as standards. Tetrahydrofuran (THF) and a solution of LiBr in N,N-dimethylformamide (DMF) (0.05 M) were utilized as the mobile phases for PNIPAAm and PHEMA, respectively, with their flow rates being 1 mL/min. The preparation of the films of SiO2-Br particles, the ungrafted and grafted core-shell MIP/CP particles, and grafted hollow MIP/CP particles and the determination of their static water contact angles as well as the water dispersion stability measurements of the ungrafted and grafted core-shell MIP/CP particles and grafted hollow MIP/CP particles in pure water (at 25 oC or 37 oC) were carried out according to the literature method.22 The equilibrium or selective binding properties of the MIPs/CPs were studied by incubating some MIP or CP particles with a solution of 2,4-D (0.02 mM) or a mixed solution of 2,4-D and its analogue POAc (C2,4-D or POAc = 0.02 mM) in different media (including acetonitrile and pure water) at 25 oC for 6 h in the dark. The mixed solutions were then centrifugated. The amounts of the analyte(s) bound to the MIPs/CPs were determined by first measuring those remaining in the supernatants with high performance liquid chromatography (HPLC, Scientific System Inc., USA) and then subtracting them from the initial analyte concentration(s). Note that the percentages of the amounts of the template bound to the

MIPs/CPs relative to those of the template in its initial solutions (i.e., Bound (%)) were used to express the bound amounts of the template in the equilibrium binding studies. The wavelength was 284 and 272 nm for measuring 2,4-D and the mixture of 2,4-D and POAc, 11



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respectively, and the corresponding mobile phases used were methanol/0.5% aqueous solution of acetic acid (4:1 v/v) and methanol/0.5% aqueous solution of acetic acid (3:2 v/v) (flow rate: 1 mL/min), respectively. Both “imprinting-induced promotion of binding” (IPB) 30 and imprinting factor (IF)31 were utilized to quantitatively evaluate the selectivity of the MIPs, and their definition can be found in the footnotes of Table S1 in Supporting Information. Photoresponsive template binding properties of the grafted hollow MIP/CP particles were studied by alternately switching on and turning off the UV light irradiation on the mixtures of MIP/CP particles (which were obtained by etching 3 mg of the corresponding grafted solid core-shell MIP/CP particles) and a pure aqueous solution of 2,4-D (0.02 mM, 0.5 mL) as described in the literature.22 The amounts of 2,4-D bound to the MIP/CP particles under different conditions were determined by HPLC, from which the photoregulated release and uptake behaviors of the MIP/CP could be evaluated. Thermoresponsive template binding properties of the grafted hollow MIP/CP particles were also evaluated by the first incubation of a pure aqueous solution of 2,4-D (0.02 mM, 0.5 mL) with some MIP/CP particles (which were obtained by etching 3 mg of the corresponding grafted solid ones) for 6 h in the dark at different temperatures, and the subsequent determination of the template bindings to the MIP/CP under different conditions. The stimuli-responsive template releases of the grafted hollow MIP/CP particles in pure water were also studied in detail by the first loading of 2,4-D onto the MIP/CP particles by their incubation with a 2,4-D solution in acetonitrile in the dark at 25 oC for 24 h and the subsequent template release study of the template-loaded MIP/CP particles (the template loading percentages (i.e., the weight percentages of the loaded template relative to MIP or CP) 12


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were found to be 0.59% and 0.46% for MIP and CP particles, respectively) in pure water under different conditions (i.e., at 25 oC or 37 oC in the dark or under the UV light irradiation) following the procedure as described in the literature,32 and the detailed template loading and release procedures can be found in Supporting Information. The stimuli-responsive template releases of the grafted hollow MIP particles in a real sample (i.e., the diluted bovine serum) were also studied (see Supporting Information). 3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of Hydrophilic Hollow Azo-Containing MIP/CP Microparticles Bearing PNIPAAm-b-PHEMA Brushes. It has been well established that hollow polymer particles can be readily fabricated by the first grafting of polymer layers onto some sacrificial core particles (e.g., silica or poly(styrene)) and the subsequent removal of the core.33 In particular, some hollow MIP particles have been prepared for different purposes by using the same strategy, which proved to have rather high binding capacities.34-36 In this work, we aim to develop a versatile method to prepare advanced water-compatible and stimuli-responsive MIP particles with enhanced template binding capacities, apparent photo- and thermoresponsive template binding and release behaviors, and high surface hydrophilicity (even above the LCST of PNIPAAm) in aqueous media. Hydrophilic hollow azo-containing MIP microparticles bearing thermoresponsive block copolymer PNIPAAm-b-PHEMA brushes were designed for this purpose, which were obtained by the first synthesis of uniform “living” silica microparticles bearing alkyl halide groups (i.e., ATRP-initiating groups) via a one-pot sol-gel method, their subsequent grafting of an azo-containing MIP layer (with a typical herbicide 2,4-D as the model template) and

13



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Page 14 of 37

PNIPAAm-b-PHEMA brushes via successive surface-initiated ATRP, and final removal of the silica core. Uniform “living” silica microparticles bearing alkyl bromide groups (briefly SiO2-Br particles) were prepared via a modified Stöber method in a one-pot way by the first sol-gel reaction of TEOS in isopropanol/methanol (3:1 v/v) with aqueous ammonia as the catalyst under stirring at 25 oC for 2 h, followed by the subsequent dropwise addition of APTES-Br and further reaction at 25 oC for another 8 h under stirring. In comparison with the previously reported multiple step synthetic protocol for silica particles bearing alkyl halide groups,37 this one-pot approach is more facile and efficient in preparing such uniform “living” silica microparticles under mild reaction conditions. Core-shell-structured 2,4-D-imprinted particles with a silica core and an azo-containing MIP shell (i.e., SiO2@Azo-MIP particles) were then prepared via surface-initiated ATRP with “living” SiO2-Br particles, CuCl/Me6TREN, 2,4-D, MAzoPy, EGDMA, and methanol/acetonitrile (1:3 v/v) as the immobilized ATRP initiator, catalyst, template, azo functional monomer, crosslinker, and porogenic solvent, respectively. The polymerization was performed at 70 °C under argon in the dark to keep azo moieties in their trans-rich state (Scheme 1). Me6TREN was used as the ligand because it could strongly interact with the copper catalyst, thus leading to better control over the polymerization. The corresponding control polymer (i.e., SiO2@Azo-CP) particles were also synthesized following the same procedure, but without the addition of the template. It is noteworthy that the living nature of the surface-initiated ATRP should result in “living” SiO2@Azo-MIP and SiO2@Azo-CP particles with surface-bound alkyl halide groups, in particular in a so diluted system.38

14


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Surface-initiated ATRP has proven very efficient in the controlled surface modification of a wide range of substrates,39 and it can be readily carried out at room temperature.40,41 In the present study, SiO2@Azo-MIP and SiO2@Azo-CP particles with PNIPAAm brushes (i.e., SiO2@Azo-MIP@PNIPAAm and SiO2@Azo-CP@PNIPAAm) were prepared via the surface-initiated ATRP of NIPAAm at 25

o

C with “living” SiO2@Azo-MIP and

SiO2@Azo-CP particles as the immobilized ATRP initiator and CuCl/CuCl2/Me6TREN as the catalyst. 10 mol% of CuCl2 relative to CuCl was used in the reaction system to better control the polymerization.42 Some sacrificial initiator ethyl 2-chloropropionate was also utilized in the above polymerization for characterizing the grafted polymer brushes.43 It is noteworthy that the resulting SiO2@Azo-MIP@PNIPAAm and SiO2@Azo-CP@PNIPAAm should also have “living” alkyl halide groups at the end of PNIPAAm brushes, thus making their further chain-extension possible. Surface-initiated ATRP of HEMA was then performed at 25 °C with the above-obtained SiO2@Azo-MIP@PNIPAAm and SiO2@Azo-CP@PNIPAAm particles as the immobilized ATRP initiator and CuCl/CuBr2/2,2’-bipyridine as the catalyst to prepare SiO2@Azo-MIP and

SiO2@Azo-CP

particles

with

PNIPAAm-b-PHEMA

brushes

(i.e.,

SiO2@Azo-MIP@(PNIPAAm-b-PHEMA) and SiO2@Azo-CP@(PNIPAAm-b-PHEMA)). Some sacrificial initiator ethyl 2-chloropropionate was also used in the polymerization systems to help characterize the grafted PHEMA brushes. Finally, hollow azo-containing MIP and CP particles with PNIPAAm-b-PHEMA brushes (i.e., H-Azo-MIP@(PNIPAAm-b-PHEMA) and H-Azo-CP@ (PNIPAAm-b-PHEMA)) were directly prepared by removing the silica core from SiO2@Azo-MIP@(PNIPAAm-b-PHEMA)

15



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Page 16 of 37

and SiO2@Azo-CP@(PNIPAAm-b-PHEMA) particles with a 10% HF solution in ethanol at ambient temperature. SEM study of the above-obtained SiO2-Br, the ungrafted core-shell MIP/CP (i.e., SiO2@Azo-MIP and SiO2@Azo-CP), grafted core-shell MIP/CP with different polymer brushes

(i.e.,

SiO2@Azo-MIP@PNIPAAm,

SiO2@Azo-CP@PNIPAAm,

SiO2@Azo-MIP@(PNIPAAm-b-PHEMA), and SiO2@Azo-CP@(PNIPAAm-b-PHEMA)), and

grafted

hollow

MIP/CP

(i.e.,


and

H-Azo-CP@(PNIPAAm-b-PHEMA)) particles showed that SiO2-Br and the ungrafted and grafted core-shell MIP/CP particles were all narrowly dispersed spherical microparticles (Figure 1a-g). In addition, surface imprinting and grafting of the polymer brushes led to an increase in their diameters, suggesting the successful surface modification (Table 1). Note that


and


particles were typically collapsed particles in their dry state (Figure 1h,i), which revealed the successful removal of the silica core.

Figure 1. SEM images of SiO2-Br (a), SiO2@Azo-MIP (b), SiO2@Azo-CP (c), SiO2@Azo-MIP@PNIPAAm

(d),

SiO2@Azo-CP@PNIPAAm

(e),

SiO2@Azo-MIP@(PNIPAAm-b-PHEMA) (f), SiO2@Azo-CP@(PNIPAAm-b-PHEMA) (g), H-Azo-MIP@(PNIPAAm-b-PHEMA) (h), and H-Azo-CP@(PNIPAAm-b-PHEMA) (i).

16


Page 17 of 37

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FT-IR characterization of SiO2-Br and the resulting MIP/CP particles (Figure 2) revealed that in addition to the characteristic peaks corresponding to Si-O-Si stretching band around 1110 cm-1 and Si-O vibration bands around 800 cm-1 and 470 cm-1, SiO2-Br also showed two rather weak peaks corresponding to amide I (1653 cm-1) and amide II (1542 cm-1) bands (Figure 2a), indicating its successful incorporation of APTES-Br and presence of ATRP-initiating groups. The presence of one significant peak around 1732 cm-1 (C=O stretching) and those around 1587 cm−1 (C=N stretching) and 1462 cm−1 (C=C stretching) from pyridine rings in the spectra of SiO2@Azo-MIP and SiO2@Azo-CP suggested their incorporation of poly(EGDMA) and poly(MAzoPy) (Figure 2b,c). In addition, some increase in

the

peaks

of

amide

I

and

II

bands

was

observed

in

the

spectra

of

SiO2@Azo-MIP@PNIPAAm and SiO2@Azo-CP@PNIPAAm in comparison with those of

Figure 2. FT-IR spectra of SiO2-Br (a), SiO2@Azo-MIP (b), SiO2@Azo-CP (c), SiO2@Azo-MIP@PNIPAAm

(d),

SiO2@Azo-CP@PNIPAAm

(e),

SiO2@Azo-MIP@(PNIPAAm-b-PHEMA) (f), SiO2@Azo-CP@(PNIPAAm-b-PHEMA) (g), H-Azo-MIP@(PNIPAAm-b-PHEMA) (h), and H-Azo-CP@(PNIPAAm-b-PHEMA) (i). 17



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Page 18 of 37

SiO2@Azo-MIP and SiO2@Azo-CP (Figure 2d,e), which confirmed the presence of PNIPAAm brushes on these core-shell MIP/CP particles. Furthermore, the O-H stretching band

around

3500

cm-1

could


be and

found

in

the

spectra

of


(Figure 2f,g), suggesting that PHEMA brushes were successfully grafted onto SiO2@Azo-MIP@PNIPAAm and SiO2@Azo-CP@PNIPAAm particles. In particular, the characteristic peaks of the silica core proved to be almost completely disappeared after HF etching

of


and

SiO2@Azo-CP@(PNIPAAm-b-PHEMA) particles, whereas the other peaks of the MIP/CP layer

and

polymer

brushes

still

remained

in

the

spectra

of

the

resulting

H-Azo-MIP@(PNIPAAm-b-PHEMA) and H-Azo-CP@(PNIPAAm-b-PHEMA) (Figure 2h,i), revealing that the silica core was effectively removed without affecting the MIP/CP layer and PNIPAAm-b-PHEMA brushes in the hollow polymer particles. Figure 3a shows that the static water contact angles of SiO2@Azo-MIP and SiO2@Azo-CP films were significantly higher than that of SiO2-Br film (see also Table 1), suggesting the successful grafting of hydrophobic azo-containing MIP/CP layer onto the silica core particles. Moreover, both the grafted core-shell MIP/CP particles bearing either PNIPAAm or PNIPAAm-b-PHEMA brushes and grafted hollow MIP/CP particles exhibited considerably higher surface hydrophilicity than the ungrafted core-shell MIP/CP particles, as revealed by the much lower water contact angles of the grafted polymer films (Table 1), again verified the attachment of hydrophilic brushes on these MIP/CP particle surfaces. Figure 3b and c shows the dispersion stability of the ungrafted and grafted core-shell MIP/CP particles and grafted hollow MIP/CP particles in pure water at 25 oC and 37 oC, 18


Page 19 of 37

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Figure 3. (a) Static water contact angles (at 25 oC) of the films of SiO2-Br (a0), SiO2@Azo-MIP

(a1),

SiO2@Azo-CP@PNIPAAm

SiO2@Azo-CP (a4),

(a2),

SiO2@Azo-MIP@PNIPAAm

(a3),


(a5),

SiO2@Azo-CP@(PNIPAAm-b-PHEMA) (a6), H-Azo-MIP@(PNIPAAm-b-PHEMA) (a7), and


(a8).

(b,c)

Dispersion

photographs

of

SiO2@Azo-MIP (b1,c1), SiO2@Azo-CP (b2,c2), SiO2@Azo-MIP@PNIPAAm (b3,c3), SiO2@Azo-CP@PNIPAAm (b4,c4), SiO2@Azo-MIP@(PNIPAAm-b-PHEMA) (b5,c5), SiO2@Azo-CP@(PNIPAAm-b-PHEMA)

(b6,c6),


(b7,c7), and H-Azo-CP@(PNIPAAm-b-PHEMA) (b8,c8) in pure water (1 mg/mL) after their ultrasonically dispersed solutions being settled down for 4 h at 25 oC (b) and 37 oC (c). respectively (see also Figure S1). The ungrafted core-shell MIP/CP particles showed much faster sedimentation than their corresponding grafted ones at 25

o

C, which further

demonstrated that hydrophilic polymer brushes were successfully grafted onto MIP/CP particles after the surface-initiated ATRP of hydrophilic monomers. Moreover, the sedimentation processes were almost the same for core-shell MIP/CP particles bearing PNIPAAm brushes and those bearing PNIPAAm-b-PHEMA brushes at 25 oC, which could 19



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Page 20 of 37

be ascribed to the rather similar hydrophilicty of PNIPAAm and PNIPAAm-b-PHEMA brushes (Table 1). Furthermore, the results also showed that the sedimentation processes of the core-shell MIP/CP particles with PNIPAAm brushes became much faster at 37 oC than at 25 oC, which could be explained by the increased surface hydrophobicity of MIP/CP particles due to the conformation change of PNIPAAm brushes from a hydrated and soluble state to a dehydrated and insoluble state above their LCST temperatures (~32 oC).44 In sharp contrast, the sedimentation processes of the core-shell MIP/CP particles with PNIPAAm-b-PHEMA brushes were hardly affected in aqueous media with increasing the temperature from 25 oC to 37 oC, which strongly demonstrated the success of our strategy to keep the surface hydrophilicity of MIP/CP particles by their grafting of PNIPAAm-b-PHEMA brushes. Similarly, the grafted hollow MIP/CP particles also showed good dispersion ability in water at both 25 oC and 37 oC, which is highly promising for their applications in controlled drug delivery and smart bioanalysis. It has been well demonstrated that the polymer brushes (grafted onto spherical particles) and free polymers generated during the surface-initiated ATRP processes (in the presence of sacrificial initiator) have rather similar molecular weights and molar-mass polydispersities (Ð).43,45 Therefore,

the

molecular

weights

(Mn,GPC)

of

PNIPAAm

brushes

on

SiO2@Azo-MIP@PNIPAAm and SiO2@Azo-CP@PNIPAAm particles were derived through GPC characterization of free polymers, which were 4350 and 4760, respectively, and their Ð values were 1.35 and 1.48, respectively (Table 1). Similarly, the Mn,GPC of the second block (i.e., PHEMA) of the block copolymer brushes were determined to be 10700 and 11500

for


20


and

Page 21 of 37

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SiO2@Azo-CP@(PNIPAAm-b-PHEMA) particles, respectively, and their Ð values were 1.38 and 1.43, respectively (Table 1). 3.2. Equilibrium Binding Properties of MIP/CP Particles. The equilibrium binding properties of the ungrafted core-shell MIP/CP, grafted core-shell MIP/CP (specifically refer to those with PNIPAAm-b-PHEMA brushes here and in the following study), and grafted hollow MIP/CP particles were studied. To accurately compare the template binding properties of the grafted hollow MIP/CP particles with those of the grafted solid ones, the concentrations of the grafted hollow MIP/CP particles were calculated by using the weights of their corresponding grafted solid ones before their etching. All the studied MIPs showed higher template binding capacities than their corresponding CPs in acetonitrile (Figure 4a). For instance, while 8 mg of ungrafted core-shell MIP, grafted core-shell MIP, and grafted hollow MIP bound 54%, 48%, and 59% of the template, respectively, their controls could only bind 37%, 35%, and 43%, respectively, which suggested the existence of imprinted

Figure 4. Uptake of 2,4-D by the ungrafted core-shell (square), grafted core-shell (circle), and grafted hollow (triangle) MIP (filled symbols)/CP (open symbols) particles in acetonitrile (a) and in pure water (b) at 25 oC (the concentration of 2,4-D solution was 0.02 mM, and those of the grafted hollow MIP/CP particles were calculated by using the weights of their corresponding grafted solid ones before etching). 21



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Page 22 of 37

cavities in such MIPs. As things turned out, the ungrafted core-shell MIP (SiO2@Azo-MIP) lost its specific template bindings (i.e., the binding differences between the MIP and its CP) in pure water and both SiO2@Azo-MIP and SiO2@Azo-CP had quite high template bindings (Figure 4b), which could be attributed to their rather hydrophobic surfaces.46 On the contrary, apparent specific template bindings were observed for the grafted core-shell and hollow MIPs in pure water due to their possessing quite hydrophilic surfaces, which indicated that water-compatible MIPs could be efficiently prepared by their grafting of hydrophilic polymer brushes. In particular, it is worth noting that the grafted hollow MIP particles showed obviously higher template binding capacities than their corresponding grafted solid ones (Figure 4a,b). For instance, in the aqueous solution of 2,4-D, while 8 mg of grafted hollow MIP bound 92% of the template, the corresponding grafted solid MIP bound only 45% (Figure 4b). These results might be attributed to the exposure of both the external and internal sides of the MIP layer on the grafted hollow MIP particles to the template molecules (whereas only the external side of the MIP layer on the grafted solid core-shell MIP particles was exposed to the template molecules). The binding selectivity of MIP/CP particles was evaluated by measuring their competitive bindings toward 2,4-D and its analogue POAc. As a valuable parameter for quantifying MIPs’ selectivity by normalizing the difference in the nonspecific bindings of MIPs (which are assumed to be equal to the bindings of CPs) toward different analytes, “imprinting-induced promotion of binding” (IPB) was first utilized here.30 On the basis of the competitive binding results of the studied MIPs/CPs toward 2,4-D and POAc in different solvents (Figure 5a and b), the IPB values of the MIPs were obtained (Figure 5, Table S1). Much larger IPB values were observed for the studied MIPs toward 2,4-D (IPB = 40%~48%) 22


Page 23 of 37

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Figure 5. Uptake of 2,4-D and POAc by the ungrafted and grafted core-shell MIPs/CPs and grafted hollow MIP/CP in their mixed solution (C2,4-D or POAc = 0.02 mM) in acetonitrile (a) and in pure water (b) (polymer concentration: 6 mg/mL; the concentrations of the grafted hollow MIP/CP particles were calculated by using the weights of their corresponding grafted solid ones before their etching). than toward POAc (IPB = 12%~14%) in acetonitrile (Figure 5a), suggesting their good template selectivity in the organic solvent. In contrast, only the grafted core-shell and hollow MIPs showed obviously higher IPB values toward 2,4-D (IPB = 41% and 35%, respectively) than toward POAc (IPB = 12% and 8%, respectively) in pure water, whereas the ungrafted core-shell MIP had a same IPB value (i.e., 6%) toward 2,4-D and POAc in pure water (Figure 5b), which demonstrated the presence of obvious template selectivity only for the grafted core-shell and hollow MIPs instead of the ungrafted core-shell MIP in the aqueous solution. Similarly, the imprinting factor (IF)31 data of the MIPs also revealed the same results (Figure 5a,b, Table S1). 3.3. Photo- and Thermoresponsive Template Binding Properties of the Grafted Hollow MIP Particles in Aqueous Media. The photoresponsive template binding properties of the grafted hollow MIP/CP particles in pure aqueous solutions were first studied. Figure 6a presents the dependence of the template bindings of the grafted hollow MIP/CP particles in

23



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Page 24 of 37

Figure 6. (a) Dependence of the 2,4-D bindings of H-Azo-MIP@(PNIPAAm-b-PHEMA) (filled symbol)/H-Azo-CP@(PNIPAAm-b-PHEMA) (open symbol) in the pure aqueous solution on photoswitching conditions (UV light on for 6 h and off for 18 h alternately at 25 o

C). (b) Photoresponsive specific template bindings of H-Azo-MIP@(PNIPAAm-b-PHEMA)

in pure water. The concentrations of the grafted hollow MIP/CP particles were calculated by using the weights of their corresponding grafted solid ones before their etching (6 mg/mL) and the initial template concentration in pure water was 0.02 mM. pure

water

on

photoswitching


conditions.

exhibited

As

apparently

things higher

turned template

out, binding

capacities than H-Azo-CP@(PNIPAAm-b-PHEMA) in pure water in the dark. In addition, photoregulated release and uptake of 2,4-D in pure water was clearly observed for the grafted hollow MIP. Its template bindings decreased from 90% to 81% after 6 h of UV light irradiation, and they increased from 80% to 91% upon turning off the UV light for 18 h. Such photoresponsive template binding processes proved to be fully reversible, suggesting that the binding site configuration and template affinity could be reversibly regulated during the photoswitching of azo groups. It is notable that H-Azo-CP@(PNIPAAm-b-PHEMA) also showed photoresponsive template binding behaviors, but they were in the opposite direction in comparison with H-Azo-MIP@(PNIPAAm-b-PHEMA). This phenomenon was also observed in our previous reports,22-24,28 which might be ascribed to the increase in the polarity 24


Page 25 of 37

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of the azo groups on the grafted hollow CP particle surfaces because of the trans- to cis-azo isomerization upon UV light irradiation (i.e., the polar cis-azo isomers on the CP particle surfaces might interact more strongly with 2,4-D (containing the polar carboxyl groups) than the non-polar trans-isomers, resulting in enhanced template bindings). Figure 6b shows the photoresponsive specific template bindings of H-Azo-MIP@(PNIPAAm-b-PHEMA), and more clear photoregulated template release and uptake could be observed in this case. Thermoresponsive template binding properties of the grafted hollow MIP/CP particles were also investigated. They are expected to show both thermoresponsivity and thermoresponsive template bindings in water because of the presence of PNIPAAm block in the grafted PNIPAAm-b-PHEMA brushes.21 The template binding capacities of the grafted hollow MIP particles were actually significantly influenced by the temperature, as revealed by their obvious decrease from 90% to 73% with increasing the temperature of the aqueous solutions from 20°C to 35°C (Figure 7a). This phenomenon might be explained by the

Figure 7. (a) Template uptake by the grafted hollow MIP (filled symbol)/CP (open symbol) particles in pure water at different temperatures. (b) Dependence of the specific template bindings of the grafted hollow MIP particles in pure water on the temperature. The concentrations of the grafted hollow MIP/CP particles were calculated by using the weights of their corresponding grafted solid ones before their etching (6 mg/mL) and the initial template concentration in pure water was 0.02 mM. 25



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Page 26 of 37

collapsing of PNIPAAm block brushes with elevated temperatures, which could lead to the blocking of binding sites (Scheme 1), as described by Hoffman and coworkers in a protein system.47 This hypothesis is further confirmed by the almost complete loss of the specific template bindings of the grafted hollow MIP particles at the higher temperatures (Figure 7b). Note that although our previously reported MIP particles with PNIPAAm or its random copolymer brushes also showed similar thermoresponsive specific template binding properties in water,21-24 their surface hydrophobicity considerably increased upon the increase in the temperature from 25 oC to a temperature above the LCST of PNIPAAm brushes (as revealed by the obviously increased template bindings of their corresponding CP particles upon the collapse of PNIPAAm brushes), which makes such thermosresponsive MIP particles inappropriate for drug delivery application in the aqueous media because their hydrophobic surfaces will lead to high nonspecific bindings of the organic compounds and proteins present in the biological media.

In sharp contrast, the presence of

PNIPAAm-b-PHEMA brushes on our presently developed MIP particles not only imparted them with thermoresponsive template binding properties, but also kept their high surface hydrophilicity even above the LCST of the PNIPAAm block because of the existence of external hydrophilic PHEMA layers (Figure 3c) (as suggested by the almost unchanged nonspecific template binding capacities of their CP particles upon the collapse of PNIPAAm brushes (Figure 7a)), thus allowing such MIP particles to function properly in the aqueous media even above the LCST of PNIPAAm. 3.4. Light- and Temperature-Controlled Template Release from the Grafted Hollow MIP Particles in Aqueous Media. With the grafted hollow MIP particles (i.e.,

26


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H-Azo-MIP@(PNIPAAm-b-PHEMA)) that show enhanced template binding capacities and apparent dual stimuli-responsive template binding properties in aqueous solutions in hand, we further studied their light- and temperature-controlled molecular release behaviors in pure water in detail. Figure 8a shows that the grafted hollow MIP particles exhibited much slower template release kinetics than the grafted hollow CP particles in pure water in the dark at ambient temperature, which could be attributed to the presence of strong interaction between the template and MIP binding sites, thus resulting in the more sustained template release from the MIP particles. For example, while the grafted hollow CP particles released 90% of the loaded template in 24 h, it took 96 h for the grafted hollow MIP particles to achieve the same release effect. In addition, the UV light irradiation caused a significant increase in the template release rate for the grafted hollow MIP particles, and the time to release 90% of the template from such MIP particles under this condition was reduced to 24 h, which is much faster than the time required in the dark (96 h), mainly because of the photoswitching of azo chromophores in the binding sites under the UV light irradiation and the resulting decrease in

Figure 8. (a) Release profiles of 2,4-D from the grafted hollow MIP (filled symbols)/CP (open symbols) particles in pure water under the UV light irradiation (diamond) and in the dark (cycle) at 25 oC, respectively. (b) Release profiles of 2,4-D from the grafted hollow MIP (filled symbols)/CP (open symbols) particles in pure water under the UV light irradiation (inverted triangle) and in the dark (triangle) at 37 oC, respectively. 27



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Page 28 of 37

their binding affinity toward the template. It is interesting to note that the template release rate of the grafted hollow CP particles became slower under the UV light irradiation than in the dark, which again might be stemmed from the increase in the polarity of the azo groups on the CP particle surfaces because of the transformation of non-polar trans-azo to polar cis-azo isomers under the UV light irradiation, thus resulting in their stronger nonspecific binding affinity toward the template (as demonstrated in Figure 6a). It is well known that the trans-cis isomer equilibrium of azo chromophores should move to the trans-direction upon elevating the temperature.48,49 Therefore, increasing the temperature of the grafted hollow MIP/CP particles with bound template (in the dark condition) might make the nonspecifically bound template release faster due to the presence of more trans-azo isomer (with weaker interaction with the template) on the MIP/CP particle surfaces. In addition, the elevated temperature may also increase the template diffusion coefficients, which might also lead to the more rapid template release. However, rather small amount of template was found to be released from the grafted hollow MIP/CP particles in the aqueous solution at 37 oC both under the UV light irradiation and in the dark (Figure 8b), which is in sharp contrast with the results observed at 25 oC. This phenomenon could be attributed to the entrapment of the template molecules by the collapsed PNIPAAm chains at 37 oC, which makes the effects of the transformation of cis- to trans-azo isomer and the increase in template diffusion coefficients (due to the increased temperature) on the template release negligible. The light- and temperature-controlled molecular release properties of the grafted hollow MIP particles were also studied in a real sample (i.e., a mixture of standard fetal bovine serum and pure water (1:4 v/v)) by fixing the release time at 16 h. Similar stimuli-responsive 28


Page 29 of 37

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template release behaviors were observed for the hydrophilic hollow MIP particles in this biological medium as in pure water (Figure S3). The above results demonstrated that the temperature-induced responsivity of polymer brushes is the dominating factor during the controlled template release processes of the hydrophilic hollow MIP particles in aqueous solutions, whereas the light-induced responsivity of azo moieties plays a subsidiary role in such processes (which can adjust the template release rates in the appropriate temperature). 4. CONCLUSIONS We have demonstrated for the first time the facile and efficient preparation of hydrophilic hollow azo-containing MIP microparticles bearing thermosensitive PNIPAAm-b-PHEMA brushes, which showed enhanced template binding capacities, obvious photo- and thermoresponsive template binding behaviors, and high surface hydrophilicity even above the LCST of PNIPAAm in aqueous media. Such advanced functional MIP microparticles have proven to be excellent environmentally tunable sustained chemical or drug delivery/release systems in aqueous solutions, with almost no template release at 37 oC, but relatively fast template release at ambient temperature (which can be further enhanced by UV light irradiation). Considering the easy preparation of uniform “living” silica particles with controllable micro- or nanometer sizes by the one-pot sol-gel method and high versatility of surface-initiated controlled/“living” radical polymerization techniques for the grafting of various MIP layers and hydrophilic block copolymer brushes under mild conditions, we believe that the established approach pave the way to the development of advanced water-compatible and stimuli-responsive hollow MIP microparticles for applications in controlled drug delivery and smart bioanalysis. Further development of advanced

29



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Page 30 of 37

stimuli-responsive hydrophilic hollow MIP drug delivery system that can have long circulation period at the body temperature (i.e., 37 oC), but can release the drugs at slightly higher temperatures (e.g., between 40~44 oC) is currently ongoing in our lab.

ASSOCIATED CONTENT Supporting Information Chemical structures of some reagents used in the study (Scheme S1), the detailed photographs for the dispersion of MIP/CP particles in pure water (Figure S1), HPLC elution profile of a mixed solution of 2,4-D and POAc (Figure S2), IPB and IF values of MIP particles (Table S1), the detailed template loading and light- and temperature-controlled template release procedures for the grafted hollow MIP particles in pure water, and the detailed procedure and result (Figure S3) for the studies on light- and temperature-controlled template release from the grafted hollow MIP particles in a real sample. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *H. Q. Zhang. E-mail: [email protected] Notes The authors declare no competing finical interest. ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support from National Natural Science Foundation of China (Grant Nos. 20744003, 20774044, 21174067, and 21574070), Research Fund for the Doctoral Program of Higher Education of China (Grant No. 20130031110018), and PCSIRT (IRT1257). 30


Page 31 of 37

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Hydrophilic Hollow Molecularly Imprinted Polymer Microparticles with

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