photoresponsive biodegradable poly(carbonate)s with pendent o...

Photoresponsive Biodegradable Poly(Carbonate)s withPendent o-Nitrobenzyl Ester

Huihui Shen, Yingchun Xia, Zhouliang Qin, Juan Wu, Li Zhang,

Yanbing Lu, Xinnian Xia, Weijian Xu

Institute of Polymer Science, College of Chemistry & Chemical Engineering, Hunan University, Changsha 410082, China

Correspondence to: Y. Lu (E-mail: [email protected])

Received 27 March 2017; accepted 24 May 2017; published online 00 Month 2017

DOI: 10.1002/pola.28679

ABSTRACT: Photoresponsive amphiphilic diblock poly(carbon-

ate)s mPEG113-b-PMNCn with pendent o-nitrobenzyl ester group

were synthesized through ring-opening polymerization (ROP)

using 1,8-diazabi-cyclo[5.4.0]undec-7-ene (DBU) as catalyst and

monomethoxy poly(ethylene glycol) (mPEG) as macroinitiator.

In aqueous solution, the copolymers can self-assemble to spheri-

cal micelles with a PC core and a PEG shell. The critical micelle

concentration (CMC), size, and morphology of the micelles were

demonstrated by means of fluorescence spectroscopy, transmis-

sion electron microscopes (TEM), and dynamic light scattering

(DLS). Under UV light irradiation, the amphiphilic copolymer

micelles disassembled because of the photocleavage of o-NB

ester, and the light-controlled release behaviors of payload Nile

red were further proved. This study provides a convenient way

to construct smart poly(carbonate)s nanocarriers for controlled

drug release. VC 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A:

Polym. Chem. 2017, 00, 000–000

KEYWORDS: polycarbonates; stimuli-sensitive polymers; self-

assembly; photoresponsive; amphiphilic block copolymer

INTRODUCTION In the clinical therapy of diseases such ascancer, it is a great challenge to regulate drug delivery andrelease behaviors which aim to improve the therapeutic efficacyand reduce side effects of drugs. Therefore, the design and prep-aration of environmentally responsive carriers have attractedsignificant attention over the past decades because of theirpotential applications in the biomedical fields.1–3 Amphiphiliccopolymers composed of hydrophilic and hydrophobic blockscan self-assemble to different polymorphism of morphologies inwater such as micelles, star micelles, vesicles, and complexsupramolecular aggregates.4,5 In particular, self-assembled poly-meric micelles are designed to improve bioavailability and bio-distribution of small drugs in tumor tissues by means ofretaining prolonged blood circulation and then extravasatedinto tumor tissues through enhancing permeability and reten-tion (EPR) effect.6–8 Various stimuli-sensitive polymeric micellesresponding to environmental stimuli such as pH, temperature,light, redox potential, and enzymes have been actively stud-ied.9–21 Among these smart polymers, photoresponsive poly-mers have attracted great attentions due to the accuratelyadjusted remote, spatiotemporal control, and easy opera-tion.22–24 o-Nitrobenzyl (o-NB) esters are a good candidate forconstructing light-responsive materials as they are efficientlycleavable upon UV exposure.25 Historically, the photocleavagecompounds containing o-NB groups have gained tremendous

attention, which was used as photolabile protecting groups inthe area of synthetic organic chemistry.25,26 It is based on thephotocleavage of an o-NB ester into a corresponding o-nitroso-benzaldehyde and releasing a free carboxylic acid upon irradia-tion of UV light.27 Since then, the photocleavage compoundshave been employed to kinds of other applications, such as con-trolled release, photoreconfigurable hydrogels, labels for cellsignaling pathway investigations, and microfabrication.3 Earlyworks by Jing et al. explored poly(carbonate)s bearing o-NBesters as photolabile chromophore, which can be cleaved by UVlight to the corresponding carboxylic acid.28 Later, Zhao et al.turned their focus to o-NB group as a functional pendent in lightresponsive amphiphilic block copolymer and proved its light-cleavage nicely. The amphiphilic block copolymer composed ofpoly (ethylene oxide) and poly(2-nitrobenzyl methacrylate)(PNBM) via atom transfer radical polymerization (ATRP). UponUV irradiation, 2-nitrobenzyl ester moieties were cleaved fromthe side chain, and the amphiphilic block copolymer turned intoa double hydrophilic block copolymer, finally the micelles disas-sembled.29 Lee et al. synthesized an amphiphilic block copoly-mer PEO-b-[2-(1-azidobutyryloxy)ethyl methacrylate] (PEO-b-PAzHEMA) via ATRP, and then alkyne-functionalized pyrenewith a photocleavable 2-nitrobenzyl moiety was added to thehydrophobic block of PEO-b-PAzHEMA via click chemistry toobtained the desired block copolymer. Upon irradiation of UV

Additional Supporting Information may be found in the online version of this article.

VC 2017 Wiley Periodicals, Inc.

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light, 2-nitrobenzyl ester moieties were selectively cleaved, lead-ing to the block copolymer released a model drug, 1-pyrenebutyric acid. Then, they encapsulated coumarin 102,another model drug into the core of micelles physically, studyingthe two drug models released at the same time under UV irradi-ation.30 Wang et al. synthesized a dual-stimuli-sensitive amphi-philic block copolymer, poly(N-isopropylacrylamide)-block-poly-(2-nitrobenzyl methacrylate) (PNIPAM-b-PNBM) with aphotosensitive PNBM segment and a temperature-sensitive PNI-PAM segment.16 In spite of many different strategies for the con-struction of photoresponsive polymers with o-NB esters aredeveloped,31–40 there have been limited reports to deal withthe biocompatible and biodegradable polymers, especiallypoly(carbonate)s with pendent o-NB group applied to drugdelivery system.

As important biodegradable materials, aliphatic poly(ester)s, ali-phatic poly(carbonate)s (PC) have received considerable atten-tion for years. These materials are excellent candidates forpharmaceutical applications as a consequence of their low toxic-ity, biocompatibility, and biodegradability.41,42 To construct func-tional polycarbonates, the introduction of functional pendantgroup into the cyclic carbonate monomers before ring openingpolymerization (ROP) or further modifications following the poly-merization is the common choice. Stimuli-sensitive polycarbonatenanocarriers were designed and showed ideal applications indrug delivery system.43–51 Hu et al. synthesized light-responsivepoly(carbonate)s with a spiropyran chromophore in the sidechain via copper-catalyzed azide-alkyne cycloaddition reaction.Under alternative UV and visible light irradiation, the amphiphilecarried on a reversible micelle transition in water due to the pho-toisomerization between SP and MC form.8 Recently, severalstimuli-responsive poly(carbonate)s have been developed. Yanget al. synthesized diblock copolymers of hydrophilic poly(ethyl-ene glycol) (PEG) and hydrophobic biodegradable polycarbonatefunctionalized with GSH-sensitive disulfide bonds and pH-responsive carboxylic acid groups with PEG as macroinitiatorthrough organocatalytic ring-opening polymerization of func-tional cyclic carbonates.52 Xie et al. designed a novel triple-stimuli-sensitive graft copolymer which responds to the changes

in temperature, reducing agent, and light. The copolymer con-sisted of thermoresponsive tetraethylene glycolyl poly(trimethy-lene carbonate) (P(MTC-4EG)) as backbone and light-sensitivepoly(2-nitrobenzyl methacrylate) (PNBM) as side chain linked byan intervening disulfide bond.53 However, there are few reportsto construct light-responsive pendent of o-NB group in poly(car-bonate)s biomaterials used as drug delivery systems.

In this work, we developed a versatile method to prepare photo-responsive amphiphilic diblock poly(carbonate)s mPEG113-b-PMNCn with pendent o-NB ester group. The o-NB ester groupwas introduced to the cyclic carbonate monomer 5-Methyl-5-(2-nitro-benzoxy-carbonyl)-1,3-dioxan-2-one (MNC) first and thenphotoresponsive amphiphilic copolymers were obtained throughring-opening polymerization with DBU as catalyst and mPEG113as macroinitiator (Scheme 2). The amphiphilic copolymer couldself-assemble into spherical micelles in aqueous solution becauseof the photo-cleavage of o-NB ester, the obtained micelles disas-sembled, leading to the change of micelles size and morphologyand the efficiently controlled release of payloads NR. This studyprovides a convenient way to construct smart poly(carbonate)snanocarriers for controlled drug delivery.

EXPERIMENTAL

MaterialsEthyl chloroformate was purchased from Xiya Reagent andused as received. 2,2-Bis(hydroxymethyl)propionic acid, 2-nitrobenzyl bromide, 3-[4,5-dimethylthialzol-2-yl]-2,5-diphe-nyltetrazolium bromid (MTT), and 1,8-diazabi-cyclo[5.4.0]un-dec-7-ene (DBU) were purchased from Energy Chemical andused as received. mPEG(MWs5 5 kDa, mPEG113) and Nilered (NR) were purchased from Sigma-Aldrich and used asreceived. Dimethylformamide (DMF) was dried by commer-cially available 4 Å molecular sieves. Tetrahydrofuran (THF)was distilled utilizing sodium and benzophenone beforeused. Dichloromethane (DCM) and triethylamine (TEA) weredried over calcium hydride for 24 h at room temperatureand distilled under reduced pressure.

Measurements1H NMR spectra were recorded on an INOVA-400 NMRinstrument (Varian Inc., Palo Alto, CA, USA) using tetrame-thylsilane (TMS) as an internal standard in DMSO-d6.

SCHEME 1 Illustration of mPEG113-b-PMNCn micelles loading

with drugs and photocontrolled release. [Color figure can be

viewed at wileyonlinelibrary.com]

SCHEME 2 Synthesis of o-NB-containing cyclic carbonate and

amphiphilic block copolymer with o-NB pendant group.

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Fourier-transform infrared (FT-IR) spectra were gained by aWQF-410 spectrophotometer (Beijing Rayleigh AnalyticalInstrument Co., Beijing, China). Ultraviolet–visible (UV–vis)absorption spectra were obtained on a UV Bluestar Plus UV-Vis spectrophotometer (Beijing LabTech Instruments Co.,Ltd., Beijing, China). Fluorescent spectra were performed ona HITACH F-7000 fluorescence spectrophotometer (HitachiHigh-Technologies Co., Minato-ku, Tokyo, Japan) at room tem-perature, the excitation and emission slit widths were 5.0and 5.0 nm, respectively. The number-average molecularweight (Mn) and molecular distribution (Ð) of the obtaineddiblock copolymers were determined by a Waters 1515 Gelpermeation chromatography (GPC) instrument (Waters Co.,Milford, MA, USA) in DMF, calibrated with standard polysty-rene with a flow rate of 1 mL min21. Transmission electronmicroscopy (TEM) images were obtained on an H-7000 NARtransmission electron microscope (Hitachi High-TechnologiesCo., Minato-ku, Tokyo, Japan) at 100 kV. The mean size ofthe micelles was determined by dynamic light scattering(DLS) measurements using a Nano-ZS90 zeta-potential andparticle analyzer (Malvern Instruments Ltd, Malvern, UK).For the UV-light irradiation, the sample (3 mL of solution)was placed vertically under a high-pressure mercury lamp(wavelength: 365 nm, power: 125 W) with the distance of10 cm.

Synthesis of 2-Nitrobenzyl 2,2-Bis(Hydroxymethyl)PropionateKOH (3.96 g, 71.0 mmol) and 2,2-bis(hydroxymethyl)propionicacid (8.25 g, 61.5 mmol) were dissolved in DMF (75 mL). Thesolution of 2-nitrobenzyl bromide (12.00 g, 55.5 mmol) inDMF (20 mL) was then added dropwise, and the mixture wasstirred at 100 8C for 20 h. After fully reacted, deionized water(200 mL) was added into the solution, and the mixture wasextracted with DCM (2 3 100 mL). The resulting mixture waswashed with deionized water (4 3 100 mL). The organic phasewas then dried over MgSO4 and concentrated in vacuum toyield crude product, which was purified by column chromatog-raphy (ethyl acetate/petroleum ether5 1/1, v/v). Yield: 8.36 g(56.1%). 1H NMR (400 MHz, d6-DMSO, TMS): d 8.11 (d, J5 8.2Hz, 1H, aromatic), 8.04–7.69 (m, 2H, aromatic), 7.61 (t, J5 7.3Hz, 1H, aromatic), 5.43 (s, 2H, CH2O), 4.77 (t, J5 5.3 Hz, 2H,2CH2OH), 3.57 (dd, J5 10.3, 5.5 Hz, 2H, CH2OH), 3.47 (dd,J5 10.3, 5.5 Hz, 2H, CH2OH), 1.11 (s, 3H, CCH3).

Synthesis of 5-Methyl-5-(2-Nitro-Benzoxy-Carbonyl)-1,3-Dioxan-2-One (MNC)In a sealed vessel, 2-nitrobenzyl 2,2-bis(hydroxymethyl)-pro-pionate (5.00 g, 18.9 mmol) was dissolved in THF (150mL)at 0 8C and ethyl chloroformate (6.15 g, 56.7 mmol) wasadded. After stirring for 1 h, triethylamine (5.74 g, 56.7mmol) was added dropwise, and the whole process was car-ried out under nitrogen atmosphere. The mixture wasallowed to stir for 1 h, after that it was reacted at 30 8C for6 h. The reaction mixture was then filtered and evaporatedin vacuum. The crude product was recrystallized from DCMand diethyl ether, finally obtaining white crystals.Yield:5.05 g (90.5%). 1H NMR (400 MHz, d6-DMSO, TMS): d

8.14 (d, J5 8.1 Hz, 1H, aromatic), 7.80 (t, J5 7.5 Hz, 1H, aro-matic), 7.76–7.59 (m, 2H, aromatic), 5.54 (s, 2H,CH2O), 4.60(d, J5 10.3 Hz, 2H, CCH2O), 4.40 (d, J5 10.3 Hz, 2H, CCH2O),1.22 (s, 3H, CCH3).

Typical Procedure for the Synthesis of mPEG113-b-PMNCnThe ring-opening polymerization (ROP) method was used to syn-thesize the mPEG113-b-PMNCn block copolymer, the commerciallyavailable monomethoxy poly(ethylene glycol) (mPEG113) acted asthe macroinitiator. Polymerizations were conducted under aninert atmosphere of nitrogen using standard Schlenk-line techni-ques. Taking the preparation of mPEG113-b-PMNC50 for example,MNC (1.0000 g, 3.39 mmol), mPEG113(0.3396 g, 0.07 mmol),DBU (0.0088 g, 0.06 mmol), and dried DCM (10 mL) were placedin a dried Schlenk tube. The mixture solution was furtherdegassed by three vacuum-nitrogen purging cycles to removetrace moisture and oxygen. After stirring at room temperaturefor 7 h, the resulting mixture was precipitated in cold diethylether. After centrifugation, the resulting product was dried invacuum to yield a white solid. Yield: 1.2284 g (91.7%). 1H NMR(400 MHz, d6-DMSO, TMS): d 8.04 (d, J5 7.8 Hz, aromatic),7.69 (s, aromatic), 7.69–7.33 (m, aromatic), 5.40 (s, CH2O),4.23 (dd, J5 19.5, 11.2 Hz, OCH2CCH2O), 3.51 (s, OCH2CH2O),1.14 (d, J5 24.0 Hz, CCH3).

Preparation of the Polymeric Nanoparticles andDetermination of Critical Micelle ConcentrationMicelles of mPEG113-b-PMNC25 and mPEG113-b-PMNC50 wereprepared by the solvent exchange method. Ten milligrams ofamphiphilic copolymer was first dissolved in 2 mL DMF atroom temperature, and then 20 mL deionized water wasslowly added dropwise into the solution under vigorous stir-ring. After vigorous stirring for another 2 h at room temper-ature, the micelles were obtained and further dialyzedagainst deionized water in a dialysis membrane (Molecularweight cutoff of 3500 Da) for 48 h to remove DMF. In a50 mL volumetric flask, the final copolymer concentrationwas adjusted to 0.2 mg mL21 by adding deionized water.

The critical micelle concentration (CMC) values of themicelles were obtained by using NR as probe molecule. NRin THF (0.1 mg mL21, 30 lL) was added to 10 glass vialsthrough the use of a microsyringe. After THF was completelyevaporated, 4 mL micellar solution with different concentra-tion was added into the glass vials. The concentration of themicellar solution was confected varying from 0.2 to 2 31024 mg mL21. Then, the solution was stirred overnight.Finally, fluorescence measurements were recorded at an exci-tation wavelength of 550 nm and the fluorescence emissionspectrum was monitored from 580 to 750 nm.

Photoresponsiveness ExperimentsTo demonstrate the light-induced cleavage of the o-NBgroups, the mPEG113-b-PMNC25 and mPEG113-b-PMNC50micelles (0.2 mg mL21, 3mL) were irradiated with UV lightat 365 nm. As a control experiment, photochemical cleavageof the o-NB groups of the monomer MNC was carried out soas to evaluate the photolytic cleavage behavior. A solution ofmonomer MNC (0.2 mg mL21 in DMF, 3mL) was exposed to



UV light (365 nm). UV–Vis spectroscopy was used to recordthe whole process.

Encapsulation and Photo-Controlled ReleaseExperimentsThe amphiphilic copolymers were engineered as nanocarriersfor hydrophobic NR payload. NR in THF (0.1 mg mL21, 30 lL)was added to a glass vial through the use of a microsyringe.After THF was completely evaporated, 4 mL of 0.2 mg mL21

micellar solution was added into the glass vial. Then, the solu-tion was stirred overnight. Finally, micelles containing NR (3mL) were exposed to 365 nm UV light. Fluorescence spectros-copy was used to monitor the whole process at an excitationwavelength of 550 nm and the emission monitored from 580to 750 nm.

In Vitro Cytotoxicity AssayThe biocompatibility of blank micelles was estimated by MTTviability assays against the HeLa and HepG2 cells. In the MTTassay, The HeLa or HepG2 cells in 100 lL were incubated for48 h at 37 8C in 5% CO2 in a 96-well plate at an initial seedingdensity of 4 3 103 cells per well. Then, the original mediumwas removed and replaced with copolymer micelles in differ-ent concentration from 0.1 to 0.5 mg mL21 and incubated foranother 48 h. Then, the cells were washed with PBS solutionfor three times and 60 lL of MTT (0.5 mg mL21) was addedinto every well. After the plates were incubated for 3 h, the cul-ture medium was removed, and then 150 lL of DMSO wasadded to each well to dissolve intracellular MTT formazancrystals. Finally, the absorbance at 570 nm was measuredusing a microplate reader (Varioskan Flash, Thermo Scientific).Data were done as average6 SD (n5 5).

RESULTS AND DISCUSSION

Synthesis and Characterization of Block CopolymersThe cyclic carbonate monomer MNC were prepared by a two-step reaction according to the synthetic route showed inScheme 2. Amphiphilic copolymer mPEG113-b-PMNCn was pre-pared through the ROP of MNC at room temperature with DBUas catalyst and mPEG113 as macroinitiator [Scheme 2(b)]. Asshown in Table 1, the degrees of polymerization (DP) of theobtained mPEG113-b-PMNCn with three different monomer-to-initiator ratios were close to the theoretical values, and GPCanalysis displayed that the copolymer had a narrow distribu-tion, which demonstrated that very few side reactionsoccurred during the ROP, probably due to the high efficiency ofthe catalyst DBU during the ROP of cyclic carbonates.54 Thedegree of polymerization (DP) was calculated by comparing

the peak area of mPEG113. The1H NMR and FT-IR spectra pro-

vided evidence of the successful synthesis of the monomer (B)and corresponding copolymers (C) showed in Figures 1 and 2.As shown in Figure 1, compared with 2,2-bis(hydroxymethyl)-propionic acid (A), the spectra of monomer (B) appeared thenew peaks at 7.5–8.0 and 5.4 ppm representing the character-istic benzyl group peaks, whereas the peaks of hydroxyl at 4.6ppm and carboxyl at 12.0 ppm disappeared completely whichdemonstrated the successful introduction of o-NB ester groupto the monomer and the formation of cyclic carbonate. ThemPEG113-b-PMNCn (C) having just one more peak attributed tothe mPEG113 at 3.5 ppm compared with the monomer (B) indi-cated that the functionalized copolymers synthesized success-ful. Similarly, FT-IR spectra shown in Figure 2 provided furtherevidence. As compared with 2,2-bis(hydroxymethyl)propionicacid in Figure 2(A), the new absorption peaks at �3100 cm21assigned to the stretching vibration of the CAH in benzenering, 1600 cm21, 1570 cm21, and 1530 cm21 assigned to thestretching vibration of the C@C in benzene ring, 798 cm21 and739 cm21 assigned to the out-of-plane bending vibration ofthe CAH in monosubstituted benzenes appeared in the dashedframe of the spectra monomer MNC (B), and the characteristicalcoholic hydroxyl group and carboxylic hydroxyl groupabsorption peaks in Figure 2(A), respectively, at 3360 cm21

and 2850 cm21 completely disappeared, what is more, com-pared with monomer MNC (B), a blue shift of the carbanylgroup absorption peak at 1640 cm21 in Figure 2(A) wasobserved due to the existence of hydrogen bonding, all of

TABLE 1 Synthesis of mPEG113-b-PMNCn Copolymers

Entry DPa Mn,NMRa (kDa) Ðb Mn,GPC

b (kDa) CMCc (mg/mL) Dhd (nm)

mPEG113-b-PMNC25 24 12.4 1.31 17.4 0.0163 140.4

mPEG113-b-PMNC50 45 19.8 1.27 17.7 0.0133 159.5

a Determined by 1H NMR.b Determined by GPC calibrated with polystyrene standard.

c Obtained by fluorescence spectroscopy using Nile Red as probe.d Achieved by DLS.

FIGURE 1 1H NMR of 2,2-bis(hydroxymethyl)-propionic acid

(A), MNC (B), and mPEG113-b-PMNCn (C). [Color figure can be

viewed at wileyonlinelibrary.com]


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which illustrated successful synthesis of the monomer (B). Thespectra of copolymers (C) was similar to the monomer MNC(B) except for the stronger absorption peak at �2900 cm21resulting from the introduction of mPEG113 which confirmedthat the targeted block copolymers were indeed achieved. GPCresults in Figure 3 showed that mPEG113-b-PMNC50 had anarrow polydispersity of 1.27 and a number average molecularweight (Mn) of 17.7 kDa.

Self-Assembly Behaviors and Determinationof Critical Micelle ConcentrationAs a result of the molecular design of mPEG113-b-PMNCnrenders it amphiphilic, the photoresponsive copolymer canself-assemble into micelles in aqueous solution with hydro-phobic cored and hydrophilic PEG coronae showed inScheme 1. Self-assemblies of the copolymer were preparedthrough solvent exchange method and then characterized bymeans of fluorescence spectroscopy, TEM, and DLS. Themicelle formation and calculation of the CMC of mPEG113-b-PMNCn were confirmed by fluorescence technique, hydropho-bic dye NR was chosen as a fluorescence probe to be loadedinto the micelles based on the fact that its fluorescence wasignorable in water but increased crucially and blue shifted ina hydrophobic environment such as the core of micelle.29 Asshown in Figure 4, the curve was almost flat at very lowconcentration, indicating that the NR was in water and fewmicelles were formed. With increasing concentration ofamphiphile solution, the curve increased dramatically andblue shifted obviously, suggesting more NR were solubilizedin hydrophobic micelle core, which proving the self-assemblyof micelles. The CMC value of mPEG113-b-PMNCn was thenidentified through plotting the maximum fluorescence emis-sion intensity versus the log of micelle concentration ofmPEG113-b-PMNCn and the concentration of the inflectionpoint in the plot was CMC, showed in Figure 4. The CMC ofmPEG113-b-PMNC25 and mPEG113-b-PMNC50 were 0.0163 mgmL21 and 0.0133 mg mL21, respectively. mPEG113-b-PMNC50showed a smaller CMC value than that of mPEG113-b-

PMNC25 because that its longer hydrophobic chain increasedits hydrophobicity.55

The size and morphology of the self-assembled mPEG113-b-PMNC25 and mPEG113-b-PMNC50 micelles (0.2 mg mL

21) werefurther studied by DLS and TEM. The TEM image [Fig. 5(A,C)]manifested that mPEG113-b-PMNCn spontaneously self-assembled into spherical micelles of a core–shell structurewith a diameter of �130 nm. The DLS results [Fig. 6(A)]showed that the hydrodynamic diameter (Dh) of mPEG113-b-PMNC25 micelles in aqueous solution was 140.4 nm and thepolydispersity was 0.127, indicating that the micelles had anarrow size distribution. The average size of the sphericalmicelles determined by TEM was smaller than the results ofthe DLS analysis, which possibly because of the shrinkage ofthe PEG shell upon drying.56 Meanwhile, micelles of mPEG113-b-PMNC50 had a Dh of 159.5 nm and a polydispersity of 0.164[Fig. 6(B)]. The diameter of the micellar nanoparticles ofmPEG113-b-PMNC50 is larger than that of the mPEG113-b-PMNC25 as mPEG113-b-PMNC50 had a longer hydrophobicchain. The results demonstrated that the amphiphilesmPEG113-b-PMNCn can be self-assembled into sphericalmicelles in aqueous solution.

Photocleavage Experiments of o-NB GroupsAs shown in Scheme 1, the pendent o-NB ester group ofmPEG113-b-PMNCn carried out the photoisomerization into acorresponding o-nitrosobenzaldehyde upon UV irradiation,simultaneously releasing a free carboxylic acid. The mechanismof photocleavable properties of the copolymer under 365 nmUV irradiation were investigated in details.27,57 As a controlexperiment, the monomer MNC in DMF (0.2 mg mL21) was irra-diated under 365 nm UV light and the copolymers mPEG113-b-PMNC25 and mPEG113-b-PMNC50 in aqueous solution (0.2 mgmL21). The photoresponsive behavior was monitored by UV–Vis

FIGURE 2 FT-IR spectra of 2,2-bis(hydroxymethyl)propionic

acid (A), MNC (B), and mPEG113-b-PMNCn (C). [Color figure can

be viewed at wileyonlinelibrary.com]

FIGURE 3 GPC curves of mPEG113-b-PMNCn before and after UV

irradiation. [Color figure can be viewed at wileyonlinelibrary.

com]



http://wileyonlinelibrary.comhttp://wileyonlinelibrary.comhttp://wileyonlinelibrary.com

spectroscopy shown in Figure 7. Initially, the absorption peak at270 nm in mPEG113-b-PMNC25 and 265 nm in mPEG113-b-PMNC50 decreased gradually and then leveled off after beingirradiated for 100 min. Meanwhile, a following absorption peakat 318 nm in mPEG113-b-PMNC25 and 320 nm in mPEG113-b-PMNC50 appeared and increased relatively slowly, and both ofthem showed an isobestic point at 301 and 305 nm, respec-tively, indicating that the o-NB ester groups were cleaved fromthe copolymers. From Figure S3, we can know that the mono-mer MNC had the same behavior on UV irradiation.

These results tallyed with the report studied by Kim et al.,who found that the photo cleavage of the photosensitive unithad the feature that in UV–Vis spectroscopy existeddecreased and increased absorption peaks at 265 and310 nm, respectively, with an isobestic point at about 290nm at the same time.58

In addition, the color of the aqueous solution changedgradually from colourless to slightly brown with UV irradi-ation, which was caused by o-nitrosobenzaldehyde in thesolution.

1H NMR spectra and GPC were further used to verify thephoto cleavage of the o-NB ester groups. Figure 8 shows the1H NMR spectra of mPEG113-b-PMNC50 in DMSO-d6 underirradiation with 365 nm UV light for various times. To provethe photolysis, we used very high concentration solution (15mg mPEG113-b-PMNC50 dissolved in 0.6 mL DMSO-d6). Thepeak area of the methylenes in main chain of PMNC at 4.23ppm was set as 4.00; from Figure 8, we can see that thearea of peak at 5.4 ppm assigned to the protons of the meth-ylenes of o-NB groups decreased from 2.01 to 0.53 with theUV irradiation for 20 h, which was a strong proof of thephoto cleavage of the o-NB ester groups. Furthermore, thearea of the signals from the mPEG113 basically maintained10, suggesting the chain of PMNC was stable under UV irra-diation.59 GPC result of mPEG113-b-PMNC50 after UV irradia-tion showed that the number average molecular weightdecreased to 8.9 kDa with Ð about 1.46 (Fig. 3).

The DLS data of mPEG113-b-PMNC50 micelles after UV irradi-ation, and adjusting different pH between 11 and 3 also sug-gested the photo cleavage of the o-NB groups. As shown inFigure 9, after UV irradiation, pH of the solution decreased

FIGURE 4 Characterizations of the micelles: emission spectra of Nile Red (kexc 5 550 nm) in mPEG113-b-PMNC25 micelle solutions(A), plot emission intensity at 624.2 nm versus the log of micelle concentration of mPEG113-b-PMNC25 (B), emission spectra of Nile

Red (kexc 5 550 nm) in mPEG113-b-PMNC50 micelle solutions (C), and plot emission intensity at 621.0 nm versus the log of micelleconcentration of mPEG113-b-PMNC50 (D). [Color figure can be viewed at wileyonlinelibrary.com]


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from 5.5 to 3.5, when adjusted its pH to 11, predictably,there was no signal displayed in DLS test. It was becausethat the free carboxylic acid in the side chain of the

copolymer transformed to carboxylate, making the polymercompletely soluble in water. when adjusting the pH of thesolution back to 3, the micelles reformed.

FIGURE 5 TEM images of the micelles: mPEG113-b-PMNC25 (A) and mPEG113-b-PMNC25 after 365 nm UV irradiation for 60 min (B),

mPEG113-b-PMNC50 (C), and mPEG113-b-PMNC50 after 365 nm UV irradiation for 60 min (D). [Color figure can be viewed at

wileyonlinelibrary.com]

FIGURE 6 Mean size distributions of micelles before and after UV irradiation determined by DLS: mPEG113-b-PMNC25 (A)

and mPEG113-b-PMNC50 (B). [Color figure can be viewed at wileyonlinelibrary.com]



http://wileyonlinelibrary.comhttp://wileyonlinelibrary.com

The photocleavage of o-NB groups of mPEG113-b-PMNCn wasalso investigated by TEM and DLS. Surprisingly, as displayedin Figure 5(B,D), upon UV light irradiation, spherical micellarnanoparticles disappeared, and some irregularly shapednanoaggregates were formed. It may result from the fact thathydrophobic 2-nitrobenzyl ester was cleaved from the sidechain of the copolymer, transforming to hydrophilic carbox-ylic acid, leading to the primary hydrophilic–hydrophobicbalance of the micelles disrupted. The DLS data of the sizechanges upon stimulation with UV irradiation were shown inFigure 6. As shown in Figure 6(A), the average diameter ofthe mPEG113-b-PMNC25 micelles decreased from 140.4 to118.2 nm, and the polydispersity changed from 0.127 to0.152 after exposed to 365 nm UV light for 100 min. Mean-while, the mPEG113-b-PMNC50 micelles obtained the similartendency of average diameter decreasing from 159.5 to130.2 nm and polydispersity changing from 0.164 to 0.204

[Fig. 6(B)]. These results also illustrated that the photolysisof the o-NB esters leaded the photo-triggered disassembly ofthe nanoparticles.

On the basis of the results of TEM and DLS measurements, itcan be concluded that external stimuli such as UV light irradia-tion could adjust the morphology of the polymeric micellarnanoparticles, which might endow the nanoparticles with greatpotential to be a new nanocarriers for controlled cargo release.

Photo-Controlled Release of MicellesStimuli-responsive micelles have been widely used for con-trolled release because of the ideal nanocarriers for the con-trolled release of hydrophobic cargoes loaded in thehydrophobic core.16 The controlled release of cargoes NR,from the stimuli-responsive polymeric nanomicelle was inves-tigated by monitoring fluorescence spectroscopy. Supporting

FIGURE 7 Photoisomerization process of mPEG113-b-PMNC25 (A) and mPEG113-b-PMNC50 (B) micelles in aqueous solution irradi-

ated by 365 nm UV light, tracked by UV–vis spectroscopy. [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 8 1H NMR spectra of mPEG113-b-PMNC50 in d6-DMSO

under 365 nm UV irradiation for various times. [Color figure

can be viewed at wileyonlinelibrary.com]

FIGURE 9 Mean size distributions of mPEG113-b-PMNC50micelles determined by DLS: after UV irradiation, and then

adjusting its pH to 11 and adjusting back to 3. [Color figure can

be viewed at wileyonlinelibrary.com]


POLYMER SCIENCE



Information, Figure S4 showed the release profile of NRencapsulated in the mPEG113-b-PMNC25 and mPEG113-b-PMNC50, from which it can be seen that with the increase ofUV irradiating time, fluorescence emission intensity decreasegradually, demonstrating that drug model NR released suc-cessfully from the hydrophobic core of micelles, the reasonwas supposed to be that the primary hydrophilic–hydropho-bic balance of the micelles was destroyed because of theconversion from hydrophobic o-NB esters to hydrophilic car-boxylic acid due to UV light-induced cleavage and the hydro-phobicity of hydrophobic chain segment was weakened. Asshown in Figure 10, NR could be hardly released from themPEG113-b-PMNC25 nanoparticles without UV stimulation,indicating that the micellar assemblies were stable. The NRsurplus remained in mPEG113-b-PMNC25 and mPEG113-b-PMNC50 micelles only was 17.0% and 18.4%, respectively,when the irradiation time increased to 75 min, manifesting

that the drug model NR was fully released. The remaining NRamount in mPEG113-b-PMNC50 micellar was larger than thatof mPEG113-b-PMNC25 micellar, which was probably becauseof longer hydrophobic chain of mPEG113-b-PMNC50 causing itmore hydrophobic. Based on the above results, it can be con-cluded that the photo-controlled release of guest moleculesfrom the polymeric micellar nanocontainers may vary withthe irradiation time of the UV light and hydrophilic or hydro-phobic chain length.

Cell Cytotoxicity of mPEG113-b-PMNCn MicellesFor drug delivery system materials, the biocompatibility orcytotoxicity of the drug carrier is a pivotal issue. The bio-compatibility of mPEG113-b-PMNCn micelles were evaluatedby MTT assay against HeLa cells and HepG2 cells. As shownin Figure 11(A,B), the viabilities of HeLa cells and HepG2cells after incubated with different concentration of micellarsolution for 48 h were above 80%, even at a high concen-tration of 0.5 mg mL21. These results indicated that theprepared mPEG113-b-PMNC25 and mPEG113-b-PMNC50had low cytotoxicity, may be because of the excellentbiocompatibility of the PEG and poly(carbonate)s.Therefore, the light-responsive amphiphilic copolymersmPEG113-b-PMNCn have potential to be an ideal drug deliv-ery system materials.

CONCLUSIONS

In summary, we reported a simple synthetic route for the prep-aration of photoresponsive amphiphilic diblock copolymersmPEG113-b-PMNCn, which were composed of a hydrophilicmPEG block and a hydrophobic PMNC block with pendent o-NB ester groups. The functional group o-NB ester was firstintroduced to the six-membered cyclic carbonate monomer 5-methyl-5-(2-nitro-benzoxycarbonyl)-1,3-dioxan-2-one (MNC),and then proceeded ring-opening polymerization at room tem-perature with DBU as catalyst and mPEG113 as macroinitiator.The obtained amphiphilic copolymer mPEG113-b-PMNC25 andmPEG113-b-PMNC50 can self-assemble to spherical micelles of

FIGURE 10 Photo-controlled release profiles of NR encapsulated

in the micelles. [Color figure can be viewed at wileyonlinelibrary.

com]

FIGURE 11 Cytotoxicity of mPEG113-b-PMNCn micelles against HeLa cells (A) and HepG2 cells (B) for 48 h incubation. [Color figure

can be viewed at wileyonlinelibrary.com]




�150 nm with a PC core and a PEG shell in aqueous solution.The photocleavable properties were revealed by UV–Vis spec-troscopy, 1H NMR, GPC, TEM, and DLS. The o-NB ester groupcarried out the photoisomerization into free carboxylic acidand o-nitrosobenzaldehyde under 365 nm UV irradiation, lead-ing to the disassembly of micelles. The controlled release ofpayload NR from the stimuli-responsive polymeric nanomi-celles was investigated by fluorescence spectroscopy and thedrug model NR was fully released when irradiation timeincreased to 75 min. MTT assays showed that mPEG113-b-PMNCn micelles had excellent biocompatibility and were prom-ising materials for drug delivery as a whole.

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