Synthesis, Self-Assembly Behavior, and BiologicalApplication of a New Photochromic Azo AmphiphilicDiblock Copolymer

Han Ding, Zheng Wang, Lijiao Sheng, Gongwu SongMinistry-of-Education Key Laboratory for the Synthesis and Application of Organic Functional Molecules,College of Chemistry and Chemical Engineering, Hubei University, Wuhan 430062, People’s Republic of China

Polymer (N,N-dimethyl-ethylamine methacrylate)-block-poly{6-[4-(4-methoxy phenyl-azo) phenoxy] hexylacrylate}p(DMAEMAm-b-AZOMn) was synthesized by successivereversible addition-fragmentation chain transfer poly-merization in the hydrothermal reactor. The productswere characterized by hydrogen nuclear magnetic reso-nance, differential scanning calorimetry, gel permeationchromatography, and ultraviolet and visible absorptionspectroscopy (UV-vis). In H2O/THF mixture, we foundamphiphilic p(DMAEMAm-b-AZOMn) self-assemblesoccurred. p(DMAEMA79-b-AZOM7) self-assembled intorods, p(DMAEMA79-b-AZOM5) self-assembled into giantmicrospheres with rods wind around, p(DMAEMA79-b-AZOM2) self-assembled into microspheres. Photochro-mic behaviors of the polymers in different environmentswere investigated. We found the colors of diblockcopolymers in films changed from yellow to orange afterirradiation by ultraviolet and visible (UV) light. The ratesof trans-cis photoisomerization in films were almost thesame for the three p(DMAEMAm-b-AZOMn) copolymers.The rates in aqueous micellar solutions were onlymarginally faster than those in films for all the threediblock copolymers. The observation of a sizable ratedifference in different environments for p(DMAEMAm-b-AZOMn) suggested that a rotational mechanism mightbe operative for these water-soluble amphiphilic diblockcopolymers. The self-assembly behaviors of threecopolymers and the application of p(DMAEMA79-b-AZOM2) microspheres in biochemistry were investigatedin present work. POLYM. ENG. SCI., 51:1662–1668, 2011.ª 2011 Society of Plastics Engineers

INTRODUCTION

Recently, much attention has been directed to the poly-

mer-based colloids because they are found to have some

potential usages in new areas such as microreactors,

targeted drug delivery, contrast enhanced imaging, and

mimic for biological membranes [1, 2]. Like small-mole-

cule surfactants, amphiphilic block copolymers can self-

assemble into micellar aggregates of various morpholo-

gies including star micelles, spheres, rods and vesicles in

selective solvents [3, 4]. Different factors, such as the ini-

tial copolymer concentration, the components of the

blocks and their ratio in the copolymers, the nature and

composition of the solvent, the temperature, the presence

of additives, and the polydispersity of the hydrophilic

block, provide control over the types and sizes of block

copolymer aggregates produced in solution [5, 6]. Many

efforts have been made to elucidate the formation of

block copolymer aggregates, with special emphasis on the

factors that control their types, the nature of the interface,

and their stability [7, 8].

Azobenzene-containing polymers (azo polymers for

short) have received considerable attention in recent years

because of their potential applications in the fields of opti-

cal data storage, liquid crystal displays, and holographic

surface relief gratings [9–11]. Upon light irradiation, azo

polymers show various photoinduced variations including

phase transition, chromophore orientation, surface-relief-

grating formation, and photomechanical bending, which

are based on the trans-to-cis and the cis-to-trans photoiso-merization of azo chromophores [12, 13]. The photores-

ponsive properties of azo polymers are connected with

the chemical structure of backbone and types of azo chro-

mophores. As a result, molecular design and synthesis of

azo polymers with desired photoresponsive properties

have aroused considerable research interest [14].

Functional polymer microspheres with carbonyl, hydroxyl,

and amino groups on their surfaces have been used to

covalently bind antibodies and other proteins onto micro-

spheres.

In this article, we report the synthesis of several azo

diblock copolymers of 2-(dimethylamino)ethyl methacry-

late (DMAEMA) monomer with azobenzene methacrylates

via reversible addition-fragmentation chain transfer (RAFT)

polymerization. To realize the anhydrous, high-pressure,

Correspondence to: Zheng Wang; e-mail: zwang805@yahoo.com.cn

Contract grant sponsor: Foundation of Hubei Department of Education;

contract grant number: D20091006; contract grant sponsor: Opened

Research Foundation of Ministry-of-Education Key Laboratory for the

Synthesis and Application of Organic Functional Molecules of Hubei

University; contract grant number: 2007-KL-002.

DOI 10.1002/pen.21956

Published online in Wiley Online Library (wileyonlinelibrary.com).

VVC 2011 Society of Plastics Engineers

POLYMER ENGINEERING AND SCIENCE—-2011

and nitrogen environment, we use hydrothermal reactor to

process the polymerization. The self-assembly behaviors of

amphiphilic azo polymer p(DMAEMAm-b-AZOMn) with

different degrees of functionalization are investigated. In

addition, the photochromism of azo polymers in the film

and in the H2O/THF suspension are also investigated in

this article. We then discussed the biological application

of p(DMAEMAm-b-AZOMn) microspheres by fluorescence

technique.

EXPERIMENTAL

Materials

N,N-dimethylformamide (DMF), tetrahydrofuran

(THF), hexachlorohexanol, and 2,20-azobisisobutyronitrile(AIBN) were purchased from Sinopharm Chemical Rea-

gent Co. Ltd. Acryloyl chloride was purchased from Alfa

Aesar. 2-(dimethylamino)ethyl methacrylate (DMAEMA)

was purchased from Aladdin Reagent Co. Ltd. All the

chemicals were of analytical grade. All the solvents were

freshly distilled and were dehydrated before use.

The monomer {2-[4-(4-methoxy phenyl azo) phenoxy]

hexyl acrylate (AZOM)} was synthesized according to the

procedure similar to the published method [15]. The addi-

tion-fragmentation chain transfer agent dithiobenzoate [S ¼C(Ph)S-Ph] was synthesized according to the literature [16].

The stock solution of bovine serum albumin (BSA)

was 1.0 3 1025 mol L21 and was kept at 0–48C. The ini-

tial solution of p(DMAEMA79-b-AZOM2) microspheres

was 1.5 mg mL21.

Preparation of the Block Copolymer and Its Film

The synthetic route of p(DMAEMAm-b-AZOMn) diblock

copolymer was outlined in Scheme1. The macro-RAFT

agent, poly(2-(dimethylamino)ethyl methacrylate) capped

with dithiobenzoate (PDMAEMA-SC(S)Ph), was synthe-

sized by RAFT polymerization using [S ¼ C(Ph)S-Ph] as

chain transfer agent in THF at 758C for 48 h. PDMAEMA-

SC(S)Ph was then reacted with hydrophobic azobenzene

monomer {2-[4-(4-methoxy phenyl azo) phenoxy] hexyl

acrylate (AZOM)} to obtain poly (N,N-dimethyl-ethylamine

methacrylate)-block-poly{6-[4-(4-methoxy phenyl-azo) phe-

noxy] hexylacrylate} p(DMAEMAm-b-AZOMn). The proc-

dure was described as follows: PDMAEMA-SC(S)Ph

(0.25g), AZOM (0.5 g, 0.26 g, and 0.128 g, respectively)

and AIBN were dissolved in anhydrous THF (5 mL). The

mixture was added to the Teflon-lined reactor with N2

protection, and was heated at 758C for 24 h. The polymer

was obtained by precipitation in excess of hexane. The

reprecipitation process was carried out for three times. The

polymer was dried in a vacuum oven at 308C. The % yields

of p(DMAEMA79-b-AZOM7), p(DMAEMA79-b-AZOM5),

and p(DMAEMA79-b-AZOM2) copolymers are 41.2%,

SCHEME 1. Synthetic route of the p(DMAEMAm-b-AZOMn) diblock copolymer.

DOI 10.1002/pen POLYMER ENGINEERING AND SCIENCE—-2011 1663

39%, and 31.7%, respectively. The film of the polymer was

obtained by dip-coating from THF solution (10 wt %) onto

a quartz surface.

The Self-Assembly of p(DMAEMAm-b-AZOMn) in theSolution

The self-assembly process was performed by adding

Milli-Q water at a rate of 10 lL/s, with stirring into a

THF solution of the diblock copolymer p(DMAEMAm-b-AZOMn) with the initial concentration of 2 mg/mL. The

volume ratio of H2O/THF was about 1:1. And the mixture

was left to equilibrate for 24 h.

Measurements

The hydrogen nuclear magnetic resonance (1H NMR)

spectra of the block copolymer was measured in CDCl3using a UNITY INOVA 600 NMR spectrometer. Thermal

phase transitions were examined using a Perkin-Elmer

DSC-7 differential scanning calorimeter with a heating rate

of 108C/min. An Agilent 1100 series gel permeation chro-

matography (GPC) system equipped with a LC pump, a

PLgel 5 lm Mixed-C column calibrated using linear poly-

styrene standards and a refractive index (RI) detector was

used to determine polymer molecular weights and molecu-

lar weight distributions. Transmission electron microscopy

(TEM) images of the polymer vesicles and cylindrical

micelles were obtained by using a Tecai/G2-20-S-TWIN

(Fillip) microscope. The fluorescence emission spectra and

resonance light scattering (RLS) spectra were obtained on a

RF-540 spectrofluorophotometer (Shimadzu, Kyoto, Ja-

pan). Ultraviolet and visible absorption spectroscopy (UV-

vis) were recorded on a UV-2300 PC spectrophotometer

(Techcomp Bio-Equipment LTD, Shanghai, China).

Spectroscopic and Transmission Electron MicroscopyMeasurements

Bovine serum albumin (BSA) concentration was fixed at

3.333 1026 mol L21 and the solution was titrated by succes-

sive additions of working solution of p(DMAEMA79-b-AZOM2)microspheres (1.533 1025mol L21). Titrations were

done manually by using a microinjector. The BSA

fluorescence emission spectra were then obtained at the

excitation of 280 nm in the wavelength range of 300–500 nm.

Resonance light scattering (RLS) spectra of p(DMAEMA79-b-AZOM2) microspheres, BSA, and microspheres-BSA systems

were obtained by simultaneously scanning the excitation and

emissionmonochromators (Dk¼ 0 nm) from 300 to 600 nm.

The morphologies of the aggregates self-assembled

from p(DMAEMA79-b-AZOM7), p(DMAEMA79-b-AZOM5) and p(DMAEMA79-b-AZOM2) copolymers were

characterized by TEM technique. Samples were first

stained with uranium acetate, and a drop of the samples

was then placed on a Formvar-coated copper grid which

was dried in air. The TEM images were obtained at 258Cat an electron acceleration voltage of 120 kV.

RESULTS AND DISCUSSION

Characterization of Polymers

The composition of the block copolymer p(DMAEMA79-

b-AZOM5) is determined by 1H NMR spectrum, which

is illustrated in detail in Fig. 1. In Fig. 1, each band in1H NMR spectra is listed in accordance with the ‘‘H’’ atoms

in the structure of copolymer (named from a to q).

Molecular weights (Mn) and polydispersity index (PDI)

of the macroinitiator p(DMAEMA-SC(S)Ph) and the

diblock copolymer p(DMAEMAm-b-AZOMn) are deter-

mined by GPC. Well-controlled copolymerizations of

hydrophobic azobenzene monomers are observed. The

results are summarized in Table 1.

The differential scanning calorimetry (DSC) heating

curves of diblock copolymer p(DMAEMA79-b-AZOM7)

(Fig. 2a) and p(DMAEMA79-b-AZOM5) (Fig. 2b) are

shown in Fig. 2. PAzoMA is known to have a smectic (S)

and nematic (N) phase [17, 18]. For p(DMAEMA79-b-AZOM7), the glass transition was observed to occur at Tg¼ 518C, the liquid crystalline transition at 888C, and the

isotropic transition at 1158C. The diblock copolymer

p(DMAEMA79-b-AZOM5) and p(DMAEMA79-b-AZOM2)

show similar DSC thermograms, but with lower corre-

sponding transitions temperatures, respectively, due to the

decrease of AZOM block. The phase transition tempera-

tures for 3 copolymers are included in Table 1.

Self-Assembly of p(DMAEMAm-b-AZOMn) in theH2O/THF Solution

In solution, the formation of block copolymer aggre-

gates of various morphologies is controlled by a forceFIG. 1. Hydrogen nuclear magnetic resonance (1H NMR) spectrum of

p(DMAEMA79-b-AZOM5) in CDCl3.

1664 POLYMER ENGINEERING AND SCIENCE—-2011 DOI 10.1002/pen

balance between three different factors: the degree of

stretching of the core-forming blocks, the interfacial ten-

sion between the micelle core and the solvent outside the

core, and the repulsive interactions among corona forming

chains [19]. The morphologies can be controlled through

variations in the copolymer composition, the initial copol-

ymer concentration, the nature of the common solvent,

the amount of water present in the solvent mixture, the

temperature and so on [20]. In this article, the self-assem-

bly behaviors of p(DMAEMAm-b-AZOMn) with different

degrees of functionalization are investigated in H2O/THF

dispersion media. Amphiphilic polymers can self-assem-

ble into an ordered architecture to minimize the interfacial

energy in suitable solvents. THF is used to dissolve

both the hydrophobic and hydrophilic blocks of

p(DMAEMAm-b-AZOMn) to form a diblock copolymer

solution. Water is then added as a precipitant for the

hydrophobic block to induce self-assemble. Figure 3a–3c

show the TEM images of the self-assembled aggregates

of p(DMAEMA79-b-AZOM7), p(DMAEMA79-b-AZOM5)

and p(DMAEMA79-b-AZOM2) in the H2O/THF disper-

sion media, respectively. Rods are observed in Fig. 3a.

And microspheres with different diameters are observed

in Fig. 3b and c. As the hydrophobic block length

decreases, the morphology of the diblock copolymer

p(DMAEMAm-b-AZOMn) changes from rods to micro-

spheres. Besides, it is interesting that the surface of

p(DMAEMA79-b-AZOM5) microspheres is smoother than

that of p(DMAEMA79-b-AZOM2) microspheres. From the

facts above, we can conclude that the block length ratio

of the hydrophilic block p(DMAEMA) to hydrophobic

block p(AZOM) provides control over the types, sizes

and microstructures of block copolymer aggregates pro-

duced in H2O/THF dispersion media. The higher the

block length ratio was of p(DMAEMA) to pAZOM, the

greater the tendency was to form microspheres. It may be

that as the length of the pAZOM block increases, the

morphology changes for equal p(DMAEMA) block

lengths, this results in a decrease in repulsion.

Photochromic and Photoisomerization of the DiblockCopolymer p(DMAEMAm-b-AZOMn)

The color of diblock copolymers in films changes from

yellow to orange after irradiation by ultraviolet and visi-

ble (UV) light because of the isomerization. We can see

from Fig. 4 that the absorbance at 460 increases with the

irradiation time.

The trans-to-cis photoisomerization of the

p(DMAEMAm-b-AZOMn) diblock copolymer in the film

and in the H2O/THF suspension are studied respectively.

The film and the stable suspension of the diblock copoly-

mer are irradiated with 365 nm ultraviolet and visible

FIG. 2. (a) Differential scanning calorimetry (DSC) heating curves of

diblock copolymer p(DMAEMA79-b-AZOM7). (b) Differential scanning

calorimetry (DSC) heating curves of diblock copolymer p(DMAEMA79-

b-AZOM5).

TABLE 1. Synthesis conditions and characteristics of p(DMAEMA79) homopolymer and p(DMAEMAm-b-AZOMn) diblock copolymers.

Sample Time (h) [AIBN] mol L21 1023 [Monomer] mol L21 Mn (GPC) PDI

Phase transition

temperature (8C)

p(DMAEMA79) 48 2 12,635 1.09

p(DMAEMA79-b-AZOM7) 24 1 0.524 15,228 1.25 g51 S88 N115

p(DMAEMA79-b-AZOM5) 24 1 0.282 14,742 1.20 g48 S79 N111

p(DMAEMA79-b-AZOM2) 24 1 0.134 13,412 1.16 g41 S78 N102

g, glassy phase, S, smectic phase, N, nematic phase.

DOI 10.1002/pen POLYMER ENGINEERING AND SCIENCE—-2011 1665

(UV) light until they reach the photostationary state. The

samples were then kept in the dark where the reverse

cis-to-trans isomerization occurred due to thermal energy

as the trans form was thermodynamically more stable.

The samples irradiated with 440 nm visible light can also

result in reverse cis-to-trans isomerization. In aqueous

solutions, the copolymers formed giant microspheres and

rods. The spectra changes with different irradiation time

and the reverse cis-trans thermal isomerization at differ-

ent time intervals are shown in Fig. 5 for p(DMAEMA79-

b-AZOM2) in films: (a) before and after ultraviolet and

visible (UV) irradiation at 366 nm reaching the photosta-

tionary state; (b) after in the dark; (c) irradiation at 460

nm reaching the photostationary state. The absorbance at

360 nm corresponds to the trans azobenzene (p-p* transi-

tion) and the maximum at 450 nm belong to the cis azo-

benzene. Similar behavior was observed in aqueous solu-

tions. The UV-vis spectra of p(DMAEMA79-b-AZOM7)

and p(DMAEMA79-b-AZOM5) show similar features. The

rate of isomerization was analyzed from the absorbance at

360 nm, corresponding to the p-p* transition. From the

equation ln(A!2At)/(A!2A0) ¼ 2kt [20], the first-order

rate constant of isomerization is determined, where At, A0,

and A! are the absorbance a 360 nm at time t, time zero,

and infinite time, respectively. Similar linear plots were

obtained for the three copolymers in aqueous solutions.

The corresponding first-order rate constants kt-c (for transto cis) obtained is shown in Table 2.

The photoisomerization rates were found to be about the

same for p(DMAEMA79-b-AZOM7), p(DMAEMA79-b-AZOM5) and p(DMAEMA79-b-AZOM2) films. The kt-c(s21) of above 3 copolymers in solutions are 10.857 31023, 11.5873 1023, and 12.0013 1023, respectively. The

kt-c (s21) of above 3 copolymers in films are 9.594 3 1023,

9.228 3 1023, and 10.362 3 1023, respectively. Interest-

ingly, the rates of photoisomerization were fractionally

slower for all the corresponding copolymers in solutions.

In aqueous solution, the diblock copolymers self-assembled

into microspheres and rods. Apparently, the structural

differences of the polymeric micelles and the films did not

FIG. 3. Transmission electron microscopy (TEM) images of

p(DMAEMAm-b-AZOMn): (a) p(DMAEMA79-b-AZOM7) (rods), (b)

p(DMAEMA79-b-AZOM5) (giant microspheres with tubes wind around),

and (c) p(DMAEMA79-b-AZOM2) (microspheres).

FIG. 4. Photochromic of the p(DMAEMAm-b-AZOMn) diblock copoly-

mer: (a) p(DMAEMA79-b-AZOM7); (b) p(DMAEMA79-b-AZOM5); (c)

p(DMAEMA79-b-AZOM2).

1666 POLYMER ENGINEERING AND SCIENCE—-2011 DOI 10.1002/pen

result in any significant differences in the rate of photoiso-

merization. which is similar to the previous results obtained

by Sin et al. [21]. In aqueous solution, the diblock

copolymer self-assemble into core-shell micelles with the

hydrophobic core formed by azo block. The formation of

micelles shield the core azobenzene from the solvent

water, and the core azobenzene is in a more hydrophobic

environment than in the film. It can be estimated that the

isomerization of p(DMAEMAm-b-AZOMn) proceeds via a

rotational mechanism.

The Application of p(DMAEMA79-b-AZOM2)Microspheres in Biochemistry

Drug and Biomacromolecules Delivery System. The

amphiphilic diblock copolymers can self-assemble into

microspheres which are composed of a hydrophobic core

and a hydrophilic shell and the microspheres can then

potentially act as drugs and biomacromolecules carriers.

They will interact with serum albumin, the most abundant

carrier protein in blood plasma. So it is important to

investigate the interaction of serum albumin with carriers.

In present work, fluorescence quenching technique [22] is

used to discuss the binding interaction between

p(DMAEMA79-b-AZOM2) microspheres and a model pro-

tein-BSA. The fluorescence quenching spectra of BSA in

the presence of p(DMAEMA-b-AZOM) microspheres are

shown in Fig. 6. The results revealed that with more

p(DMAEMA79-b-AZOM2) microspheres, the fluorescence

intensity of BSA at 350 nm (exited at 280 nm) decreased

FIG. 6. Effect of p(DMAEMA79-b-AZOM2) microspheres on fluores-

cence spectra of BSA (kex ¼ 280 nm). (a?g) c(BSA)¼ 3.33 3 1026

mol L21, working solution of spheres is 1.53 3 1025 mol L21, V(Azo

microspheres) (lL): 20, 40, 60, 80, 100, 120, and 140, respectively.

FIG. 5. UV-vis spectra of p(DMAEMA79-b-AZOM2) film (a) before

and after UV irradiation at 366 nm reaching the photostationary state,

(b) after in the dark, and (c) irradiation at 460 nm reaching the photosta-

tionary state.

TABLE 2. First-order rate constants, kt-c, for the trans-to-cisphotoisomerization of p(DMAEMA79-b-AZOM7), p(DMAEMA79-b-

AZOM5), and p(DMAEMA79-b-AZOM2) in film and aqueous solution.

Diblock copolymers 103kt-c (s21)

Dip-coated films

p(DMAEMA79-b-AZOM7) 9.594

p(DMAEMA79-b-AZOM5) 9.228

p(DMAEMA79-b-AZOM2) 10.362

Aqueous solutions

p(DMAEMA79-b-AZOM7) 10.857

p(DMAEMA79-b-AZOM5) 11.587

p(DMAEMA79-b-AZOM2) 12.001

DOI 10.1002/pen POLYMER ENGINEERING AND SCIENCE—-2011 1667

significantly with a slight blue shift and the emission band

of azobenzene at 470 nm emerged step by step

(Fig. 6a?g), indicating that microspheres could act as

quenchers and the binding interaction between BSA and

microspheres occurred with complex formation. The

above phenomenon revealed that p(DMAEMA79-b-AZOM2) microspheres can be potentially applied as pro-

tein carriers and the inherent binding information such as

binding mode, binding sites, interaction force, etc., can be

investigated in detail in future studies.

Detection of Proteins. We also used RLS spectra to

discuss the interaction and the RLS spectra of

p(DMAEMA79-b-AZOM2) microspheres-BSA system

were displayed in Fig. 7. The RLS intensities of both

BSA and azo microspheres in the wavelength range of

300–600 nm were rather weak. When mixed together,

however, an enhanced peak could be observed at 370 nm

and the RLS signals increased with increasing BSA con-

centration. According to the RLS theory [23], the

enhanced RLS signals in this work could further prove

that the micron sized complexation of azo microspheres

and BSA occurred. Apart from that, the results indicated

that the amphiphilic diblock copolymer p(DMAEMA79-b-AZOM2) and the microspheres can be potentially used as

fluorescence probes in the determination of BSA by RLS

method. The optimization of experimental conditions in

the detection can be further discussed in future papers.

CONCLUSION

A new amphiphilic azobenzene diblock copolymer

p(DMAEMAm-b-AZOMn) was synthesized via reversible

addition-fragmentation chain transfer polymerization in

the hydrothermal reactor. Colloidal spheres, rods and

microspheres are obtained through gradual hydrophobic

aggregation of azobenzene diblock in H2O/THF disper-

sion media. With the decrease of length of the pAZOM

block, p(DMAEMAm-b-AZOMn) self-assembly morphol-

ogy changes from rods to microspheres. The colour of

diblock copolymers in films changes from yellow to or-

ange after irradiation by ultraviolet and visible (UV) light.

The lower photoisomerization rate constant of

p(DMAEMAm-b-AZOMn) in the film than in the H2O/

THF suspension indicates the rotational mechanism of the

isomerization of p(DMAEMAm-b-AZOMn). Apart from

that, p(DMAEMAm-b-AZOMn) can be potentially used in

the drug delivery system and the determination of BSA.

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FIG. 7. Resonance light scattering (RLS) spectra of p(DMAEMA79-b-

AZOM2) microspheres-BSA system. (1) 1.4 3 1026 mol L21 BSA;

(2?6) c(microspheres) ¼ 2.04 3 1027 mol L21; c(BSA)(31026 mol

L21): 0, 0.2, 0.6, 1, 1.4.

1668 POLYMER ENGINEERING AND SCIENCE—-2011 DOI 10.1002/pen