Spiropyran-Based Hyperbranched Star Copolymer: Synthesis

Jul 3, 2012 - Jaber Keyvan RadAli Reza MahdavianSamideh KhoeiSakine Shirvalilou. ACS Applied Materials & Interfaces 2018 10 (23), 19483-19493...
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Spiropyran-Based Hyperbranched Star Copolymer: Synthesis, Phototropy, FRET, and Bioapplication Ying Wang, Chun-Yan Hong,* and Cai-Yuan Pan* CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, 230026, Anhui, P. R. China S Supporting Information *

ABSTRACT: Photo- and pH-responsive amphiphilic hyperbranched star copolymers, poly(6-O-methacryloyl-1,2;3,4-diO-isopropylidene-D-galactopyranose)[poly(2-(N,N-dimethylaminoethyl) methacrylate)-co-poly(1′-(2-methacryloxyethyl)3′,3′-dimethyl-6-nitro-spiro(2H-1-benzo-pyran-2,2′-indoline))]ns [HPMAlpGP(PDMAEMA-co-PSPMA)n], were synthesized by reversible addition−fragmentation chain transfer (RAFT) polymerization of the DMAEMA and the SPMA using hyperbranched PMAlpGP as a macro RAFT agent. In aqueous solution, the copolymers self-assembled to form core−shell micelles with HPMAlpGP core and PDMAEMA-co-PSPMA shell. The hydrophobic fluorescent dye nitrobenzoxadiazolyl derivative (NBD) was loaded into the spiropyran-containing micelles. The obtained micelles not only have the photochromic properties, but also modulate the fluorescence of NBD through fluorescence resonance energy transfer (FRET), which was also observed in living cells. Slight fluorescence intensity decrease of the spiropyran in merocyanine (ME) form was observed after five UV−visible light irradiation cycles. The cytotoxicity of the HPMAlpGP(PDMAEMA-co-PSPMA)n micelles was lower than that of 25k PEI. All the results revealed that these photoresponsive nanoparticles are a good candidate for cell imaging and may find broad applications in biological areas such as biological diagnosis, imaging, and detection.

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INTRODUCTION

assemblies of redox-sensitive amphiphilic hyperbranched star copolymers are biodegradable and have potential applications. Recently, fluorescent nanoparticles have attracted great attention for their advantages over conventional dyes. They are not only very bright and exhibit improved photostability, but also feature versatility in their design and synthesis. In the past few years, optical imaging of living cells with fluorescent polymeric nanoparticles (NPs) has aroused a great deal of interest. 27,28 Spiropyran, as one of the photochromic compounds, adopts the colorless spiropyran (SP) form in the dark or under visible light (vis), whereas it can convert to the colored and fluorescent merocyanine (ME) form upon UV irradiation.29−31 By employing this special property of spiropyran-containing molecules, nanoparticles have found interesting applications including data recording, optical and electrical switching, and light-actuated nanovalves.32−39 In a notable example, Li et al.39 prepared the polymer nanoparticles with SP−ME dyes incorporated into hydrophobic cavities. The resultant NPs are capable of undergoing reversible fluorescence photoswitching. Compared to single-color fluorescent nanoparticles, photoswitchable dual ones are more desirable because they can be applied in the fluorescence resonance energy transfer (FRET) process. The same research group introduced

In the last two decades, various polymeric micelles have been extensively studied for potential application as delivery vehicles of drugs,1−4 and they are generally formed by self-assembling of amphiphilic copolymers in aqueous solution, and then loading hydrophobic drug in the hydrophobic core is performed.5−10 Very recently, the micelles formed from the amphiphilic hyperbranched star copolymers have been investigated because of their unique characteristics in comparison with those obtained by linear block copolymers, such as morphology varieties, self-assembly mechanism, unusual mechanical properties, excellent template ability, facile functionalization, and smart responsibility.10 In addition, the hyperbranched star copolymers can provide many nanocavities for drug encapsulation. These advantages pave the way for biomedical applications of amphiphilic hyperbranched star copolymers assemblies. The micelles of stimuli−sensitive polymers responding to environmental stimuli such as ultrasound,11 pH,12−15 redox,16−20 or temperature21−23 are highly attractive for their potential applications in the controlled release of the loaded drug or gene. Redox-sensitive polymers, which usually contain disulfide linkages, are widely used in drug delivery systems.16−20 A tripeptide, glutathione (GSH), within the cells is found at millimolar concentrations, and it can be used as a water-soluble reducing agent to degrade disulfide-containing polymers to their corresponding thiols.24−26 Therefore, the © 2012 American Chemical Society

Received: May 28, 2012 Revised: July 1, 2012 Published: July 3, 2012 2585

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Scheme 1. Synthesis of Hyperbranched PMAlpGP(PDMAEMA-co-PSPMA)n

They can be applied in the FRET process, and, via alternative UV/vis irradiation, the photo−switching between green and red fluorescence can be facilely achieved. These photochromic properties and photoswitchable FRET process were also observed in living cells. Thus the fluorescent nanoparticles studied in this article have potential applications in biological diagnosis and drug delivery synchronously.

not only spiropyran (FRET acceptors) but also perylene derivatives (FRET donor species) into the latex particles, and via alternative UV/vis irradiation, the photoswitching between green and red fluorescence can be facilely achieved, which were also used in cell imaging.38 Later on, Wu et al.32,33 synthesized amphiphilic core−shell nanoparticles covalently attached with spiropyran moieties. After introduction of hydrophobic nitrobenzoxadiazloyl derivatives into these spiropyran-containing nanoparticles, reversible photoswiching between green and red fluorescence emission was also realized. Moreover, in recent years, owing to the sustained interest on the application of fluorescent nanoparticles in biology, the development of nontoxic, biocompatible, and usable fluorescent nanoparticles in aqueous medium is a topic of great interest. To the best of our knowledge, no paper about the biodegradable photochromic hyperbranched star polymers has been reported. Herein, we prepared the novel spiropyran-based amphiphilic hyperbranched star copolymers containing biodegradable disulfide linkages, HPMAlpGP(PDMAEMA-co-PSPMA) n (Scheme 1). They can be self-assembled to form core−shell micelles in aqueous media and then encapsulate the hydrophobic fluorescent dye NBD. In the micelles, the core hyperbranched PMAlpGP endues its biodegradation and biocompatibility, while PDMAEMA-co-PSPMA in the shell endues the pH- and photoresponsive ability. These spiropyrancontaining micelles are capable of reversible fluorescence photo-switching by alternating UV (365 nm) and visible (590 nm) light. After NBD was loaded into the micelles, photoswitchable dual color fluorescent nanoparticles were formed.

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EXPERIMENTAL SECTION

Materials. 6-O-methacryloyl-1,2;3,4-di-O-isopropylidene-D-galactopyranose (MAlpGP) was prepared as reported,40 and its 1H NMR (300 MHz, CDCl3, δ, ppm): 1.21−1.60 (m, 12H, −CH3), 1.95 (s, 3H, −CH3), 4.08, 4.19−4.42, 4.63, and 5.54 (7H, sugar moiety), 5.57 and 6.14 (s, 2H, CH2). Disulfide-based dimethacrylate (DSDMA) was synthesized according to previously reported literature,41 and its 1H NMR (300 MHz, CDCl3, δ, ppm): 1.88 (s, 6H, −CH3), 2.88−2.93 (t, 4H, −S−CH2−), 4.31−4.36 (t, 4H, −O−CH2−), 5.52 and 6.07 (s, 4H, CH2). Nitrobenzoxadiazolyl derivative (NBD) was prepared as reported.33,42 2-(N,N-dimethylaminoethyl) methacrylate (DMAEMA, 97%, Alfa) was purified by being passed through basic alumina columns, then vacuum-distilled and stored at −20 °C prior to use. 10(2-Hydroxyethyl)-30,30-dimethyl-6- nitrospiro(2H-1-benzopyran2,20-indoline) (SP−OH) was purchased from Tianjin SaiTeRui Science and Technology Development Co. SPMA was synthesized according to the literature,34 and its 1H NMR (300 MHz, CDCl3 δ, ppm): 1.16 (s, 3H, −CH3), 1.28 (s, 3H, −CH3), 1.91 (s, 3H, −CH3), 3.37−3.62 (m, 2H, −N−CH2−), 4.3 (t, 2H, −O−CH2−), 5.56 and 6.07 (s, 2H, CH2), 5.87 (d, 1H, −CH), 6.67−6.80 (q, 2H, Ar−H and −CH), 6.86−6.95 (q, 2H, 2Ar−H), 7.06−7.13 (d, 1H, Ar−H), 7.17−7.24 (m, 1H, Ar−H), 7.97−8.05 (m, 2H, 2Ar−H). Toluene and tetrahydrofuran (THF) were refluxed over sodium for 24 h and distilled prior to use. Azobis(isobutyronitrile) (AIBN, Aldrich) was 2586

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Table 1. Preparation of Hyperbranched Star Copolymers, HPMAlpGP(PDMAEMA-co-PSPMA)ns samplea

macro-RAFT agent

molar ratiob in feed

Mn,GPCc(g/mol)

Mw/Mnc

MAlpGP:DMAEMA:SPMAd in polymer (molar ratio)

H1 H2

HPMAlpGP[SC(S)Ph]n HPMAlpGP[SC(S)Ph]n

300:6:1 300:3:1

30800 31200

1.17 1.21

20:123:1 30:190:1

a H1 and H2 represent HPMAlpGP(PDMAEMA-co-PSPMA)n1 and HPMAlpGP(PDMAEMA-co-PSPMA)n2, respectively. Polymerization temperature: 70 °C; time: 20 h. bMolar ratio of DMAEMA/SPMA/macro−RAFT agent in the feed. cDetermined by GPC calibrated with polystyrene standards. dDetermined by 1H NMR.

recrystallized from ethanol. Cyanoisopropyl dithiobenzoate (CPDB) was prepared according to the literature,43,44 and 1H NMR (300 MHz, CDCl3, δ, ppm): 1.94 (6H, m, −CH3), 7.31−7.97 (5H, m, aryl H). All other reagents with analytical grade were purchased from Shanghai Chemical Reagent Co. and used without further purification. Preparation of Hyperbranched PMAlpGP (HPMAlpGP). Into a 5 mL polymerization tube, MAlpGP (0.32 g, 1.0 mmol), DSDMA (21.2 mg, 0.075 mmol), AIBN (2.0 mg, 0.012 mmol), CPDB (10.8 mg, 0.05 mmol), and toluene (1.3 mL) were added. The mixture was degassed through three freeze−pump−thaw cycles. The polymerization tube was then flame−sealed under vacuum, and the sealed tube was immersed into an oil bath thermostatted at 75 °C. After 26 h, the polymerization tube was cooled to room temperature rapidly, and the polymer was obtained by precipitation from n-hexane. The obtained product was dried overnight in a vacuum oven at room temperature (conversion = 91%, Mn,GPC = 9000 g/mol, Mw/Mn = 1.48). Preparation of Hyperbranched Star Copolymer HPMAlpGP(PDMAEMA-co-PSPMA)n. Into a 5 mL polymerization tube, HPMAlpGP (Mn,GPC = 9000 g/mol, 65 mg, 0.0089 mmol), DMAEMA (0.3 g, 1.9 mol), SPMA (16 mg, 0.037 mmol), AIBN (0.2 mg, 0.0012 mmol), and THF (0.8 mL) were added. After three freeze−vacuum− thaw cycles, the polymerization tube was sealed under vacuum. The polymerization was carried out at 70 °C for a prescribed time. The polymerization tube was cooled to room temperature rapidly, then the reaction mixture was precipitated into an excess of n-hexane. The obtained product was dried overnight in a vacuum oven at room temperature. Micellization of Hyperbranched Star Copolymers. HPMAlpGP(PDMAEMA-co-PSPMA)n (Mn = 30 800 g/mol, 10 mg) was dissolved in THF (1 mL). Under vigorously stirring, deionized water was added slowly. Then the dispersion was stirred for another 5 h and THF was removed by dialysis [molecular weight (Mw) cutoff: 3500 Da] against deionized water for 24 h. In Vitro Cytotoxicity Measurement. Cell viability was examined by the MTT assay. Hela cells were seeded in a 96-well plate at an initial density of 5000 cells/well in 100 μL of DMEM complete medium. After incubating for 24 h, DMEM was replaced with fresh medium, and the cells were treated with HPMAlpGP(PDMAEMA-coPSPMA)n micellar solution at a given concentration. The treated cells were incubated in a humidified environment with 5% CO2 at 37 °C for 24 h, MTT reagent (in 20 μL PBS buffer, 5 mg/mL) was added to each well, and the cells were further incubated for 4 h at 37 °C. The culture medium in each well was removed and replaced by 100 μL dimethyl sulfoxide (DMSO). The plate was gently agitated for 15 min, and the absorbance values were recorded at a wavelength of 490 nm upon using a Thermo Multiskan flash. The cell viability is calculated as A490,treated/A490,control × 100%, where A490,treated and A490,control are the absorbance values with or without the addition of micelles, respectively. Each experiment was done in quadruplicate. The data are expressed as average ± standard deviations (±SD). Cell Incubation and Imaging. The prepared cell lines, Hela cells (5 × 104) were seeded on coverslip in 24-well plate and then incubated in 300 μL Dulbecco’s Modified Eagle’s Medium (DMEM, containing 10% Hyclone fetal bovine serum, 50 units mL−1 penicillin and 50 units mL−1 streptomycin) medium containing free and NBDloaded HPMAlpGP(PDMAEMA-co-PSPMA)n micellar solution (final micelle concentration was 80 μg/mL), respectively for 3 h at 37 °C. After removing the medium, the Hela cells were washed three times with phosphate-buffered saline (PBS) and incubated in 300 μL DMEM, then the samples were observed by an LSM510 confocal laser

scanning microscope (Carl Zeiss, Germany) with a 20× objective before and after UV (365 nm) irradiation.

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RESULTS AND DISCUSSION Preparation of Hyperbranched Star Copolymer HPMAlpGP(PDMAEMA-co-PSPMA)n. Similar to the synthetic strategy of dendrimer-star copolymers in our previous reports,45,46 the hyperbranched star copolymers are synthesized according to Scheme 1. The first step is to prepare the macroRAFT agent, HPMAlpGP[SC(S)Ph]n, which was prepared by RAFT polymerization of MAlpGP with DSDMA as the divergent agent and CPDB as the RAFT agent. The feed molar ratio of [CPDB]/[DSDMA]/[MAlpGP]/[AIBN] was fixed at 1:1.5:20:0.25, and HPMAlpGP[SC(S)Ph]n with MnGPC = 9000 g/mol and Mw/Mn = 1.48 was obtained. Then the HPMAlpGP[SC(S)Ph]n was used as a macro-RAFT agent in the subsequent RAFT polymerization of SPMA and DMAEMA, forming the hyperbranched star copolymers, HPMAlpGP(PDMAEMA-co-PSPMA)ns. The detailed polymerization conditions and results are listed in Table 1. The results reveal that the polymerization activity of SPMA is lower than that of DMAEMA, probably due to the bulky side group of SPMA. The expected structures of the macro-RAFT agent and the hyperbranched star copolymers were verified by their 1H NMR spectra. As shown in 1H NMR spectrum of the HPMAlpGP(SC(S)Ph)n in Figure 1A, the signal at δ = 3.01 (c) is attributed

Figure 1. 1H NMR spectra of the hyperbranched PMAlpGP[SC(S)Ph]n and HPMAlpGP(PDMAEMA-co-PSPMA)n1.

to the methylene groups next to the disulfide linkage in the DSDMA units, and the signals of anomeric protons in the MAlpGP units appear at δ = 3.9−4.5 ppm (h,j,e,f), 4.62 ppm (i), and 5.50 ppm (g), respectively. The phenyl proton signals of dithiobenzoate units at δ = 7.37−7.93 ppm (m) can be clearly seen, and the ascription of other proton signals is marked in the figure. All these facts indicate that the 2587

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hyperbranched PMAlpGP[SC(S)Ph]n was successfully synthesized. The molar ratio of MAlpGP and DSDMA units in the resultant polymers is 19:1, which was calculated based on the integral values of the signals at δ = 5.50 ppm (g) and δ = 3.01 ppm (c). 1 H NMR spectra of the hyperbranched star copolymers was also used to confirm their structure, and Figure 1B is a typical 1 H NMR spectrum of the hyperbranched star copolymer HPMAlpGP(PDMAEMA-co-PSPMA)n1. The characteristic signals of both HPMAlpGP and PDMAEMA-co-PSPMA are visible and labeled in the figure. Besides the characteristic signals of HPMAlpGP, we can see the characteristic signals of PDMAEMA at 2.28 ppm (m′), 2.58 ppm (i′) and 4.08 ppm (j′), which are attributed to the methyl protons of dimethylamino group, the methylene protons adjacent to the dimethylamino group, and ester methylene protons, respectively. The signals of methylene protons adjacent to the nitrogen atom in PSPMA appear at δ = 3.40 ppm (d). The 1H NMR data support the formation of HPMAlpGP(PDMAEMAco-PSPMA)n. In addition, the compositions of hyperbranched star copolymers HPMAlpGP(PDMAEMA-co-PSPMA)ns were calculated based on the integration of proton signals at δ = 2.56 ppm (i′), δ = 5.50 ppm (g), and δ = 8.0 ppm (b″). The results are listed in Table 1. The molecular weight and molecular weight distribution of the resultant HPMAlpGP and HPMAlpGP(PDMAEMA-coPSPMA)ns were characterized by GPC, and the results are shown in Figure S1. The GPC trace of HPMAlpGP is bimodal, which is the general phenomenon for the controlled radical copolymerization of the mono- and divinyl monomers because the hyperbranched polymers are formed via linear polymer chains to branched polymer chains to hyperbranched chains.47,48 In addition, the GPC traces of the hyperbranched star copolymers, HPMAlpGP(PDMAEMA-co-PSPMA)ns are completely shifted to the higher molecular weight region. Thus, the hyperbranched star copolymers HPMAlpGP(PDMAEMAco-PSPMA)ns were successfully synthesized and used in the following studies. Micelle Formation of Hyperbranched Copolymers in Water and Incorporation of NBD Dye in Micelles. The HPMAlpGP(PDMAEMA-co-PSPMA)ns contain hydrophobic HPMAlpGP and hydrophilic PDMAEMA-co-PSPMA, thus they can self-assemble to form micelles with a HPMAlpGP core and a PDMAEMA-co-PSPMA corona in water. Formation of the micelles was performed by slowly adding water into the copolymer solution in THF, and then THF was removed by dialysis against deionized water for 24 h. With a similar procedure, NBD, which was prepared by the reaction of nitrobenzoxadiazolyl with n-octylamine, was encapsulated in the micelles for investigation of the FRET phenomenon. The transmission electron microscopy (TEM) images of the micelles without NBD and with NBD are shown in Figure 2. In comparison with the TEM image of the micelles without NBD in Figure 2A, the contrast of the micelles with NBD in Figure 2B is higher, which is reasonable because the NBD is a dye. From both images, their average diameters (D) were measured and are almost the same (about 120 nm). The NBD dye exhibits very low solubility in pure water, and, as a result, its absorption and fluorescence emission are very weak as shown in Figure S2 (Supporting Information). However, with the incorporation of the dye into the micelles, the dispersion exhibited a prominent absorption at ca. 460 nm and a strong fluorescent emission at 530 nm (Figure S2A and

Figure 2. TEM images of the micelles without NBD (A) and with NBD (B) respectively obtained from self-assembling of HPMAlpGP(PDMAEMA-co-PSPMA) n 1 and HPMAlpGP(PDMAEMA-coPSPMA)n1 containing NBD (2 mg/mL).

Figure S2B). Compared to absorption of NBD in water, a blueshift for the dye in the micelle system was observed, indicating that the NBD dye molecules were actually incorporated into the micelles, and the strong fluorescence intensity of the dye suggests that the dye molecules reside in a more hydrophobic environment, in this case probably at the interface between the hydrophilic PDMAEMA/PSPMA shell and the hydrophobic HPMAlpGP core. The Phototropy of the Spiropyran-Containing Micelles and Photoreversible Modulation (Switching) of Fluorescence of the NBD Dye. As we mentioned above, the spiropyran molecules have two stable states: the ring-closed state, known as the “spiro (SP) form” and the ring-opened state, known as the zwitterionic “merocyanine (ME) form”.29−31 Under UV irradiation, the spiropyran molecules adopt the colored and fluorescent ME form, while under visible light, they adopt the colorless SP form. Figure 3A shows the absorption and fluorescence spectra of the aqueous micellar solution prepared from the HPMAlpGP(PDMAEMA-coPSPMA)n upon visible and UV (365 nm) irradiation. Under visible light, spiropyran moieties in the micelles are assumed to be the SP form and exhibited no absorption from 450 to 700 nm, whereas under UV irradiation, a new absorption band at ca. 550 nm appeared due to the formation of the ME form (Figure 3A1). Under irradiation at λex = 560 nm, the spiropyran molecules in ME form emitted strong fluorescence as shown in Figure 3A2. When the NBD was loaded in the micelles, the micelles containing both the donor (NBD dye) and acceptor (spiropyran) may serve as the scaffolds for the FRET process. Figure 3B shows the absorption spectrum and fluorescence intensity of the NBD in the micelle systems upon visible and UV light irradiation, respectively. In Figure 3B1, under visible light irradiation, spiropyran moieties in the SP form exhibited no absorption from 500 to 700 nm, only an absorbance band of NBD at 460 nm was observed. After UV (365 nm) irradiation, a new absorbance band at 550 nm can be observed, which is ascribed to the ring-opened ME form in the SPMA units. After irradiation by visible light, only fluorescence emission of NBD with a maximum at 530 nm was observed in Figure 3B2, whereas, after irradiation by UV (365 nm) light, a new emission band at λ = 610 nm, which was emitted by the spiropyran moieties in ME form, appeared. On the other hand, compared to that before UV irradiation, the characteristic fluorescence emission of NBD at 530 nm decreased significantly. This is ascribed to the quenching of NBD fluorescence by the ME 2588

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Figure 3. (A) Absorption and fluorescence spectra (λex = 560 nm) of H1 micelles dispersion without NBD. (B) Absorption and fluorescence spectra (λex = 490 nm) of NBD-loaded micelle dispersion upon UV- and vis-light irradiation. The insets in A2 and B2 show color changes of the dispersions upon UV and vis irradiation.

with panel D in Figure 4, we can find that the FRET efficiency is affected by the pH of the micelle solution. It is well known that the FRET efficiency strongly depends on the relative distance between fluorescent donors and acceptors; the distance decrease can considerably improve the FRET efficiency. For these NBD-loaded spiropyran-containing micelles we studied, the shell is the PDMAEMA-co-PSPMA, and the PDMAEMA is a weak polybase with pKa of about 7.0. It is soluble in water of pH = 7.0, but its solubility in aqueous solution of pH= 9.0 decreases. Thus, at pH = 9.0, the PDMAEMA chains in the corona of micelles collapsed, and the relative distance between spiropyran and NBD decreased. So, the FRET efficiency improved under UV irradiation because the spiropyran molecules adopt the ME forms. To find the potential applications of the fluorescent nanoparticles prepared in this study in biological diagnosis, imaging, and detection, we studied the fluorescence response behavior of the hyperbranched star copolymer micelles. Figure 5A shows a typical fluorescence response behavior of the NBDloaded HPMAlpGP(PDMAEMA-co-PSPMA)n1 micellar solution upon irradiation by UV and visible light. Under irradiation of 365 nm light, the fluorescence intensity of NBD gradually decreased over 5 min and then leveled off, indicating it was quenched by the ring-opened ME form of SPMA. Under irradiation with 590 nm light, the NBD fluorescence recovered to the original value within 4 min, which is in agreement with previous reports by Wu et al.32,33 This switching behavior is due to the photochemical conversion between the two states of the spiropyran moieties. In addition, we also investigated the

form of SPMA moieties, i.e., the FRET process occurred. Moreover, we found that the fluorescence of the NBD−loaded micelle solution can be reversibly quenched and recovered by alternating irradiation with the visible and UV light, and, as a result, the colors of the dispersion also reversibly changed as shown in the inset of Figure 3B2. A similar color change was also observed in the inset of Figure 3A2 for the aqueous solution of the HPMAlpGP(PDMAEMA-co-PSPMA)n micelles. In addition, we also investigated the influencing factors on the FRET process. Figure 4 illustrates the modulation of fluorescence intensity of NBD dye incorporated in spiropyranbased micelles prepared from H1 and H2 by UV irradiated at different conditions. For all of these NBD-loaded micelle solutions, after irradiation by UV light (365 nm, 0.16 W/cm2), the characteristic fluorescence emission of the NBD at 530 nm was decreased, and a new emission band at around 610 nm appeared under excitation wavelength of 490 nm, which is the emission of the spiropyran moieties in ME form, as shown in Figure 4. For the spiropyran−containing micelles of the copolymer H1, the results of Figures 4A and 4B reveal that the amount of NBD loaded in the micelles affected the FRET degree, and the lower concentration (1.5 × 10−6 M) of NBD in the micelles displayed a higher quench degree of NBD. Figure 4B,C shows the emission spectra of the micelles prepared respectively from H1 and H2 with the same concentration of NBD, and the results demonstrate that the amount of spiropyran moieties in the shell of micelles affected the FRET degree, and the higher amount of spiropyran moieties in H1 shows a higher quench degree of NBD. Comparing panel A 2589

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Figure 4. The fluorescence intensities of NBD-loaded micelles prepared from H1 or H2 (see Table 1) and various concentrations of NBD at various pH values, after UV (365 nm) irradiation. (A) A micelle solution prepared from H1 and NBD with concentration of 6.0 × 10−6 M, pH = 7.0; (B) a micelle solution prepared from H1 and the NBD of 1.5 × 10−6 M, pH = 7.0; (C) a micelle solution prepared from H2 and the NBD of 1.5 × 10−6 M, pH = 7.0; (D) a micelle solution prepared from H1 and the NBD of 6.0 × 10−6 M, pH = 9.0. The contents of the spiropyran moiety are 7.0 × 10−3 M (H1) and 4.5 × 10−3 M (H2), respectively.

Figure 5. Fluorescence response (excited at 490 nm and emitted at 530 nm) of the NBD-loaded micelles of H1 to the irradiation with UV (365 nm, 0.16 W/cm2) and visible (590 nm, 0.23 W/cm2) light (A), and the fluorescence intensity change for the NBD-loaded micelles of H1 under alternative irradiation by UV and visible light (B). (Concentration: 2 mg/mL; Temperature: 25 °C; Volume: 1 mL.)

reversible nature of the fluorescence modulation of the NBD loaded micelles via cycled irradiation with UV (365 nm) and visible (590 nm) light, and the results are shown in Figure 5B. After five cycles, we observed an increase in the fluorescence intensity of the “off state” (upon UV irradiation) and a decrease in the fluorescence intensity of the “on state” (upon visible light irradiation), which indicates the presence of irreversible photodamage of some NBD molecules and spiropyran moieties under repeated UV irradiation.32,49−51 Cytotoxicity of HPMAlpGP(PDMAEMA-co-PSPMA)n Micellar Nanocarriers. The cytotoxicity of free and NBDloaded HPMAlpGP(PDMAEMA-co-PSPMA)n micelles was

evaluated by MTT assay using Hela cells. The cells were incubated with various amounts of micelles for 24 h. PEI with a molecular weight of 25 kDa was used as a control. The results in Figure 6 show that the free and NBD-loaded micelles were less toxic than 25 kDa PEI. It should be noted that the micelles reserved over 80% cell viability until a concentration of 25 μg/ mL, indicating relatively low cytotoxicity of this cationic micelles. The Phototropy of the Spiropyran-Based Micelles and Photoreversible Modulation (Switching) of Fluorescence of the NBD Dye in Living Cells. We also investigated the phototropy capability of the spiropyran−based micelles in 2590

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spiropyran brought by 405 nm laser. Under brief exposure to UV illumination, the spiropyran was converted to its colored and fluorescent ME form, so the red images of the cells was observed as shown in Figure 7B. When Hela cells were treated with the NBD-loaded HPMAlpGP(PDMAEMA-co-PSPMA)n micelles, before UV irradiation, the green cells were observed as shown in Figure 7C because the spiropyran moieties in the SP form had no FRET effect with NBD, so, the NBD emitted strong green fluorescence under irradiation of λex = 488 nm. After brief exposure of these Hela cells under UV light, the red Hela cells in Figure 7D were clearly seen because the SP form of the spiropyran was converted to its colored and fluorescent ME form under UV light, and the FRET effect with NBD occurred, resulting in significant decrease of NBD fluorescence and marked increase of the fluorescence of the spiropyran in ME form. These results proved that the phototropy and the photoswitchable FRET process can take place in living cells.

Figure 6. Relative cell viability values of Hela cells evaluated by MTT assay after incubation with various concentrations of NBD0-free and NBD-loaded micellar solution of HPMAlpGP(PDMAEMA-coPSPMA)n1, and the 25K PEI was used as control at 37 °C.

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the commonly used cell lines, living Hela (human cervical cancer cell) cell lines. After free and NBD-loaded hyperbranched star copolymer HPMAlpGP(PDMAEMA-coPSPMA)n micelles were delivered into Hela cells by incubation with the cells, the fluorescence images of Hela cells treated with the micelles were acquired using a 405 nm laser as shown in Figure 7. For the NBD-free HPMAlpGP(PDMAEMA-coPSPMA)n micelles, before UV illumination, the SP form of the spiropyran moieties in the micelles gave no fluorescence, but the images of the cells in Figure 7A still showed very weakly red fluorescence because of the part ring-opening of the

CONCLUSION

The hyperbranched star copolymers, HPMAlpGP(PDMAEMA-co-PSPMA)ns have been successfully prepared by two-step RAFT polymerization: first the hyperbranched polymer, HPMAlpGP, was prepared, and then the resultant HPMAlpGP was used as the macro−RAFT agent in the subsequent RAFT copolymerization of DMAEMA and SPMA. The self-assembling of HPMAlpGP(PDMAEMA-co-PSPMA)ns in water formed spherical spiropyran-containing micelles with HPMAlpGP core and PDMAEMA-co-PSPMA shell. The spiropyran-containing micelles have the phototropy, and there is a slight decrease of the fluorescence intensity after five UV−

Figure 7. Fluorescence microscopy images of Hela cells treated with NBD-free and NBD-loaded HPMAlpGP(PDMAEMA-co-PSPMA)n micellar solution at 37 °C for 3 h before and after UV illumination. (A) NBD-free micelles, before UV illumination; (B) NBD-free micelles, after UV illumination; (C) NBD-loaded micelles, before UV illumination; (D) NBD-loaded micelles, after UV illumination. 2591

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vis irradiation cycles. The FRET effect was observed in the NBD-loaded micelles of the hyperbranched star copolymers, so the fluorescence of NBD can be modulated through the FRET process. These photochromic properties and the photoswitchable FRET process were also observed in living cells, and the NBD-free and NBD-loaded micelles of the hyperbranched star copolymers revealed less cytotoxicity in comparison with 25K PEI. These biocompatible and photoresponsive micelles may have applications in biological areas such as drug delivery, imaging, and detection.

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

S Supporting Information *

Supplementary experimental details and figures, including characterization, GPC traces of hyperrbanched PMAlpGP, the hyperbranched star copolymers, HPMAlpGP(PDMAEMA-coPSPMA)n1 and HPMAlpGP(PDMAEMA-co-PSPMA)n2 (Figure S1), and absorption and fluorescence intensities of NBD in the aqueous micelle solution and in pure water (Figure S2). This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author

*E-mail: [email protected] (C.-Y.H.); [email protected] (C.Y.P.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support by National Natural Science Foundation of China (Nos. 20974103, 21074121, and 21090354), the Fundamental Research Funds for the Central Universities, and Program for New Century Excellent Talents in University (NCET-08-0520) is greatly acknowledged.

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