Synthesis of Hemoglobin Conjugated Polymeric ... - ACS Publications

Jul 24, 2015 - Eric Duverger , Fabien Picaud , Louise Stauffer , and Philippe Sonnet. ACS Applied ... Angewandte Chemie 2018 130 (36), 11694-11704 ...
1 downloads 0 Views 2MB Size
Download PDF
Article pubs.acs.org/Biomac

Synthesis of Hemoglobin Conjugated Polymeric Micelle: A ZnPc Carrier with Oxygen Self-Compensating Ability for Photodynamic Therapy Shasha Wang,† Fang Yuan,† Kui Chen,‡ Gaojian Chen,*,‡ Kehua Tu,† Hongjun Wang,† and Li-Qun Wang*,† †

MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, P. R. China ‡ Center for Soft Condensed Matter Physics and Interdisciplinary Research, Soochow University, Suzhou, 215006, P. R. China S Supporting Information *

ABSTRACT: Photodynamic therapy (PDT) is a promising singlet oxygen (1O2) mediated clinical treatment for many tumors. As the source of 1O2, oxygen plays an important role in the curative effect of PDT. However, the facts of photochemical depletion of oxygen and the intrinsic hypoxic microenvironment of tumors remain the major challenges. In this work, a novel photosensitizer carrier with oxygen self-compensating ability was designed for PDT. It was synthesized via chemical conjugation of hemoglobin (Hb) to polymeric micelles formed by triblock copolymers of poly(ethylene glycol)-block-poly(acrylic acid)-block-polystyrene (PEG-b-PAA-b-PS). The PEG-b-PAA-b-PS and resultant micelles in aqueous solution were comprehensively characterized by means of FTIR, 1H NMR, GPC, DLS, TEM, and fluorescence spectroscopy. The oxygen-binding capacity and antioxidative activity of the Hb conjugated micelles were evaluated via UV−vis spectroscopy. In addition, compared with the control micelles without Hb, the Hb conjugated photosensitizer carrier was able to generate more 1O2 and exert greater photocytotoxicity on Hela cells in vitro. particularly PDT.22 Hetzel et al. found that the hypoxia of a tumor could be effectively compensated by a hyperoxygenation technique, and the therapeutic effect of a tumor could be significantly improved.19 Therefore, it is more attractive and effective in PDT if an oxygen carrier with encapsulation of hydrophobic photosensitizer and ability to compensate the oxygen depletion could be developed. Hemoglobin (Hb), the most abundant protein in blood, is responsible for oxygen transport around the body.23 During the past few decades, diverse nanoscale carriers have been developed for physical encapsulation24−26 or chemical conjugation27,28 of Hb, which were known as hemoglobin-based oxygen carriers (HBOC).29,30 For example, poly(L-lysine)block-poly(L-phenylalanine) (PLL-b-PPA) diblock copolymers were self-assembled into polypeptide vesicles to encapsulate Hb, which were proven to be more stable than free Hb.24 Besides physically encapsulated Hb, the chemical conjugation method was also proved to improve the stability of Hb in HBOC. Huang et al. synthesized MPEG−PMPC−PLA copolymers to conjugate azide-functionalized Hb on PMPC segments using click chemistry to form Hb-bearing nanomicelles, which showed appropriate stability and oxygen carrying capacity.31 Besides, they also prepared PEG− PMCC−PLA copolymers to obtain Hb-conjugated micelles

1. INTRODUCTION Recently, photodynamic therapy (PDT) has been developed as an effective clinical treatment for superficial tumors1,2 and agerelated muscular degeneration.3,4 In PDT, a photosensitizer is first administrated and activated to its triplet state by light irradiation at a certain wavelength. Subsequently, the triplet state can interact with molecular oxygen to produce singlet oxygen (1O2) or other reactive oxygen species (ROS) that exert oxidative damage to tumor cells.4−6 However, most of photosensitizers utilized in PDT are hydrophobic, which makes intravenous injection problematic.7 As a result, various strategies have been proposed to overcome this limitation, including conjugation of photosensitizer to water-soluble polymers6,8,9 and encapsulation in nanoparticle carriers10−12 such as polymeric micelles.13−15 The micellar systems have been extensively designed to enable the hydrophobic photosensitizers to circulate in blood and target solid tumors by enhanced permeability and retention (EPR) effect,5 due to their good biocompatibility and thermodynamic stability.16 It is now generally believed that oxygen is highly involved in the process of PDT.5,17 The yields of singlet oxygen, the primary ROS inducing cellular toxicity in PDT, has been proven to increase with oxygen concentration.17,18 However, the photochemical depletion of oxygen through the generation of 1O2 can cause hypoxia and limit curative effect of PDT.19 Moreover, the microenvironment of most malignancies is hypoxic.20,21 The hypoxic tumors are more invasive, metastatic, and resistant to standard cytotoxic anticancer therapies, © XXXX American Chemical Society

Received: April 29, 2015 Revised: July 21, 2015

A

DOI: 10.1021/acs.biomac.5b00571 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

equipped with a 4.0 mW He−Ne laser (λ = 632.8 nm) at a scattering angle of 173 o. All samples were filtered with a 0.45 μm filter before analysis. The morphology of particles was observed by JEOL JEM1200EX transmission electron microscope (TEM) operating at an acceleration voltage of 80 kV. Samples were deposited onto carbon coated copper grids and left to dry in air. Steady-state fluorescence excitation and emission spectra were obtained on a PerkinElmer LS55 luminescence spectrometer. UV−vis spectra were collected using a Shimidazu UV-1800 spectrophotometer. 2.3. Synthesis of PEG-b-PAA-b-PS Triblock Copolymer. 2.3.1. Synthesis of PEG-Br Macroinitiator. First, mPEG (10 g, 2 mmol) was dissolved in 50 mL of toluene and refluxed at 110 °C for 3 h, followed by azeotropic distillation to remove traces of water. Then, 60 mL of anhydrous THF and 1.1 mL of triethylamine (8 mmol) were added, and the solution was cooled to 0 °C. BIBB (1 mL, 8 mmol) in 15 mL of anhydrous THF was added dropwise and the reaction mixture was stirred at 34 °C for 24 h. After the reaction, the mixture was filtered and most of the solvent was removed by rotary evaporation. The rough product was dissolved in dichloromethane and extracted with saturated NaHCO3 and NaCl each for three times. The organic phase was collected and dried over anhydrous Na2SO4, followed by filtration and precipitation into cold diethyl ether. Finally, the white powder was obtained with a yield of 80%. 1H NMR (400 MHz, D2O, ppm): 4.35 (m, −CH2OCO− of mPEG), 3.67 (m, −OCH2CH2− of mPEG), 3.35 (m, CH3O− of mPEG), 1.93 (m, −C(CH3)2Br). 2.3.2. Synthesis of PEG-b-PtBA. PEG-b-PtBA was synthesized using PEG-Br as a macroinitiator via ATRP of tBA in bulk. Typically, a glass ampule was charged with 1 g (0.2 mmol) of PEG-Br and 40.2 mg (0.28 mmol) of CuBr and degassed by three freeze−pump−thaw cycles. Afterward the deoxygenated PMDETA (58.46 μL, 0.28 mmol) and tBA (2.903 mL, 20 mmol) were added to the ampule. The polymerization of tBA was conducted at 50 °C for 7 h and then stopped by exposure of the solution to air. The reaction mixture was diluted in THF and passed through a column of activated neutral alumina to remove the copper catalyst. The diblock copolymer with a yield of 90% was isolated by precipitation in cold hexane twice and dried under vacuum to a constant mass. 1H NMR (400 MHz, CDCl3, ppm): 4.35 (m, −CH2OCO− of mPEG), 3.64 (m, −OCH2CH2− of mPEG), 3.38 (m, CH 3 O− of mPEG), 2.12−2.33 (br, − CH2CHCOO− of PtBA), 1.48−2.00 (br, −CH2CHCOO− of PtBA), 1.25−1.48 (br, −(CH3)3 of PtBA). 2.3.3. Synthesis of PEG-b-PtBA-b-PS. PEG-b-PtBA-b-PS was synthesized by ATRP of styrene using PEG-b-PtBA as a macroinitiator. PEG-b-PtBA (1 g, 0.056 mmol), styrene (1.29 mL, 11.23 mmol), and PMDETA (23.44 μL, 0.112 mmol) were dissolved in 8 mL chlorobenzene. After being degassed by three freeze−pump−thaw cycles, the above solution was added to a deoxygenated glass ampule which charged with 16.1 mg (0.112 mmol) of CuBr. The polymerization of St was conducted at 110 °C for 24 h and then stopped by exposure of the solution to air. The residual copper catalyst in the solution was removed by activated neutral alumina column chromatography using heated THF as eluent. The triblock copolymer with a yield of 30% was obtained by precipitation in cold methanol twice and dried under vacuum to a constant mass. 1H NMR (400 MHz, CDCl3, ppm): 6.85−7.23 (br, ortho- and para-H from the aromatic ring), 6.30−6.85 (br, meta-H from the aromatic ring), 4.32 (m, −CH2OCO− of mPEG), 3.64 (m, −OCH2CH2− of mPEG), 3.38 (m, CH3O− of mPEG), 2.12−2.33 (br, −CH2CHCOO− of PtBA), 1.48−1.57, 1.72−2.00 (br, −CH2CHCOO- of PtBA), 1.57−1.72 (br, the backbone of PS segments). 1.25−1.48 (br, −(CH3)3 of PtBA). 2.3.4. Synthesis of PEG-b-PAA-b-PS. The triblock copolymer of PEG-b-PtBA-b-PS was dissolved in chloroform and stirred with 5 equivalence of TFA (with respect to the tert-butyl group) at room temperature for 24 h. After removal of excess TFA and chloroform by rotary evaporation, the triblock copolymer of PEG-b-PAA-b-PS with a yield of 85% was obtained by precipitating in cold hexane and dried under vacuum to a constant mass. 1H NMR (400 MHz, DMSO-d6 ppm): 6.80−7.36 (br, ortho- and para-H from the aromatic ring), 6.24−6.80 (br, meta-H from the aromatic ring), 3.26−3.58 (m,

by condensation reaction of carbonyl of PMCC moiety with the amino group of Hb. It was found that the positon of conjugated Hb could be regulated through the reassembling of copolymers at the isoelectric point (pI) of Hb.32 Using the same condensation reaction, we have recently reported a thermoresponsive hemoglobin−polymer conjugate named Hb-Dex-gPNIPAAm, which was confirmed to have redox activity and oxygen-binding capacity.33 Compared to free Hb, the conjugated Hb in Hb-Dex-g-PNIPAAm exhibited a better stability at 37 °C, probably due to the protection from the aggregative PNIPAAm chains above its lower critical solution temperature. In this work, a novel strategy of adapting HBOC as a zinc phthalocyanine (ZnPc) carrier was proposed, which had oxygen self-compensating ability for photodynamic therapy. First, a well-defined triblock copolymer of poly(ethylene glycol)-blockpoly(acrylic acid)-block-polystyrene (PEG-b-PAA-b-PS) was synthesized by atom transfer radical polymerization (ATRP). It could self-assemble to form polymeric micelles for covalent conjugation of Hb via carbodiimide chemistry. Herein, the HBOC was testified to possess good oxygen-binding capacity and antioxidative activity. And then zinc phthalocyanine, a second-generation photosensitizer frequently utilized in PDT,34 was encapsulated into the hemoglobin conjugated PEG-b-PAAb-PS micelles to prepare a novel oxygen-carrying polymeric PDT agent. The resultant photosensitizer carrier was confirmed to generate more singlet oxygen and give rise to greater photocytotoxicity to Hela cells in vitro, compared to ZnPcloaded micelles without Hb.

2. EXPERIMENTAL SECTION 2.1. Materials. The monomers, both tert-butyl acrylate (tBA, Aldrich, 98%) and styrene (St, Sigma-Aldrich, ≥ 99%) were passed through an activated alkaline alumina column prior to use. Copper(I) bromide (CuBr, Adamas, 99%) was successively washed with 2% acetic acid aqueous solution (v/v) and acetone, and then dried in vacuum. Poly(ethylene glycol) methyl ether (mPEG, Aldrich, number-average molecular weight (Mn) 5000), 2-bromoisobutyryl bromide (BIBB, Adamas, > 98%), N,N,N′,N″,N″-pentamethyl diethylene triamine (PMDETA, TCI, > 98%), trifluoroacetic acid (TFA, Adamas, 99%), N(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC, Aladdin, 98.5%), zinc phthalocyanine (ZnPc, Aldrich, 97%), anthracene-9,10-bis-methyl-malonate (ADMA, Aldrich), cell counting kit-8 (CCK-8, 7sea biotech) and Dulbecco’s modified eagle medium (DMEM/high glucose, HyClone) were used without further purification. Bovine hemoglobin (FW: ∼ 68000) was purchased from Shanghai Yuanju Biological Technology Co. Ltd. The dialysis membrane made of regenerated cellulose was purchased from Spectrumlabs Co. Anhydrous tetrahydrofuran (THF, Sinopharm Chemical Reagent Co.) was obtained by refluxing over sodium under argon atmosphere and distilled immediately before use. All other solvents were purchased from Sinopharm Chemical Reagent Co. and purified according to standard methods. 2.2. Characterization. FTIR spectra were recorded on a Bruker Vector 22 FTIR spectrometer over the region of 4000−400 cm−1. 1H NMR was performed on a Bruker DMX-400 NMR spectrometer operating at 400 MHz with a 1.0 s delay between pulses using D2O, CDCl3, or deuterated dimethyl sulfoxide (DMSO-d6) as solvent. Molecular weight distribution was obtained by Waters PL-GPC-50 instrument at 60 °C. Samples were eluted through an Aglient PLgel 5 μm mixed C column using HPLC-grade dimethylformamide (DMF) with LiBr at a concentration of 0.05 M as the mobile phase at the rate of 1 mL/min. Additionally, the results were obtained by calibrating with poly(methyl methacrylate) (PMMA) standard. The particle size and polydispersity index (PDI) were measured by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS size analyzer B

DOI: 10.1021/acs.biomac.5b00571 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

Scheme 1. Synthesis of ZnPc-Loaded HbMs: (A) tBA, CuBr, PMDETA, 50 °C; (B) St, CuBr, PMDETA, chlorobenzene, 110 °C; (C) TFA, chloroform, 25 °C

−OCH2CH2− of mPEG), 3.24 (m, CH3O−- of mPEG), 1.18−2.30 (br, the CH and CH2 of polymer backbone), 12.24 (m, −COOH of PAA moieties). 2.4. Preparation of the Micellar Solution. PEG-b-PAA-b-PS copolymer (200 mg) was dissolved in 20 mL of DMF and the solution was dialyzed (MWCO = 3500) against phosphate buffer (10 mM, pH = 7.4) for several days to form micelles. Critical aggregation concentration (CAC) was obtained by the reference method35 using pyrene as a fluorescence probe. The polymeric solutions with varying concentration ranging from 10−4 to 0.5 mg/mL were diluted from a 1 mg/mL stock solution. And then 10 mL of the solutions were put in volumetric flasks containing a certain amount of pyrene making sure that the pyrene concentration in the final sample solutions was 6 × 10−7 mol/L. The samples were incubated for 12 h at 25 °C prior to measurement. The fluorescence excitation spectra (300−350 nm) of pyrene at room temperature were monitored using an emission wavelength of 372 nm and excitation bandwidth of 3 nm. Thus, the CAC values were determined as the onset concentration of the plot of intensity ratio (I339 /I333). 2.5. Preparation of Hemoglobin Conjugated Micelles (HbMs). The covalent conjugation of Hb to micelles was performed via a standard carbodiimide chemistry.33,36 First the pH value of micellar solution of PEG-b-PAA-b-PS (0.25 mM, 40 mL) containing 1 mmol carboxyl group was adjusted to near 7.0. Calculated amount of EDC (191.6 mg, 1 mmol) was added as the coupling agent, and the mixture was stirred at 0 °C for 15 min. Then Hb (13.6 mg, 200 nmol) dissolved in 4 mL of Na2HPO4 buffer (100 mM, pH = 7.0) was dropwise added. The mixture was then allowed to react at 25 °C for 24 h and exhaustively dialyzed (MWCO = 100 kDa) against phosphate buffer (10 mM, pH = 7.4) for several days to obtain the Hbconjugated micelles named as HbMs. 2.6. Oxygen-Carrying Capacity and Stability of the Oxygenated HbMs (Oxy-HbMs). The oxygenated HbMs were prepared as follows: first the solution of HbMs was reduced by calculated amount of sodium ascorbate (2-fold molar of Hb) under CO atmosphere. And then oxygen gas was allowed to flow over the solution of CO stabilized HbMs (CO-HbMs) under visible light irradiation in order to obtain the oxygenated HbMs. The whole conversion process was monitored via UV−vis spectroscopy. The Oxy-HbMs could be gradually oxidized to the methemoglobin form (met-HbMs) by exposing Oxy-HbMs to air atmosphere at room temperature. Thus, the stability of Oxy-HbMs in air was characterized by the plot of Oxy-HbMs percentage versus time (see Figure 5b). When the absorbance of 405 nm reached the maximum value, the Oxy-HbMs were considered to have been converted to its met-form.

The oxidation curve and t1/2 (the time at which 50% of the Hb molecules were oxidized) were used to characterize the stability of the Oxy-HbMs. 2.7. Preparation of ZnPc-Loaded HbMs. The lyophilized HbMs (19 mg) and ZnPc (1 mg) were dissolved in 10 mL of DMF. Afterward, the solution was added dropwise to 100 mL of phosphate buffer (10 mM, pH = 7.4) under vigorous ultrasonic agitation. The residual DMF was removed by exhaustive dialysis (MWCO = 3500) against phosphate buffer (10 mM, pH = 7.4). The micelle solution was filtered with a syringe filter (pore size: 0.45 μm) to eliminate the polymer and ZnPc aggregates. The final concentration of ZnPc in the filtrated solutions for further experiments as well as the loading content (LC) of ZnPc were quantified by the same method as described in the next paragraph. Additionally, the micelle solution for characterizing the generation of 1O2 was dialyzed against deuteroxide containing 10 mM phosphate buffer (pH = 7.4) to displace water. Similarly, ZnPc was also loaded in the PEG-b-PAA-b-PS micelles as control. Two milligrams of freeze-dried ZnPc-loaded micelles was dissolved in DMSO (0.05 mg/mL), and the solution was magnetically stirred for 1 h to break up the micelles and release ZnPc into DMSO. The ZnPc concentration was then analyzed via UV−vis spectrophotometry to determine the loading content (LC) and encapsulation efficiency (EE) of ZnPc. And the absorption and fluorescence emission spectra (λex = 610 nm) of the ZnPc-loaded micelles in DMSO were recorded at room temperature. The spectra of free ZnPc were measured in DMSO for comparison. 2.8. Generation of 1O2 by Light Irradiation. The HbMs and PEG-b-PAA-b-PS micelles in deuteroxide, containing 5 μM of ZnPc, were mixed with a chemical quencher of ADMA (50 μM) to characterize the generation of 1O2. After bubbled with argon for different time, these solutions were irradiated using a 150 W halogen lamp equipped with a 610 nm filter at the power density of 20 mW/ cm2. Then the absorbance decay of ADMA was followed at 379 nm via UV−vis spectroscopy. 2.9. Phototoxicity. Photo and dark toxicity of ZnPc-loaded micelles were evaluated by WST-8 (water-soluble tetrazolium salts) assay using CCK-8 reagent. The HeLa cells (1 × 104 cells per well) were seeded on a 96-well plate in 200 μL of culture medium and incubated for 1 day at 37 °C in a 5% CO2 atmosphere. Then 100 μL of micelle solutions prepared in culture medium at 5 μM ZnPc were added in each well. After 4 h incubation, the cells were exposed to a 150 W halogen lamp for 2 min at the power density of 20 mW/cm2, followed by 24 h incubation. Then 100 μL of fresh culture medium and 10 μL of CCK-8 reagent were added in each well and the cells C

DOI: 10.1021/acs.biomac.5b00571 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

Figure 1. (a) FTIR spectra of PEG-Br, PEG-b-PtBA, PEG-b-PtBA-b-PS, and PEG-b-PAA-b-PS over the region 4000−400 cm−1; (b) 1H NMR spectra of PEG-Br, PEG-b-PtBA, PEG-b-PtBA-b-PS, and PEG-b-PAA-b-PS in D2O, CDCl3, and DMSO-d6, respectively. were incubated for 1 h. Finally, the absorbance (450 nm for soluble dye and 650 nm for viable cells) were recorded on a microplate reader (Thermo Fisher Scientific, Inc.).

Table 1. Characterization of Triblock Copolymers Synthesized via ATRP copolymers

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of Triblock Copolymer. The triblock copolymer PEG-b-PAA-b-PS was synthesized as outlined in Scheme 1. Brominate-terminated mPEG (PEG-Br) was first synthesized by a reaction between mPEG and BIBB. Then the monomers of tBA and styrene were polymerized via two-step ATRP to yield PEG-b-PtBA-b-PS using PEG-Br as a macromolecular initiator. The final amphiphilic triblock copolymer of PEG-b-PAA-b-PS was obtained by hydrolysis of PtBA into PAA using TFA in chloroform. As shown in Figure 1a, the peak at 1730 cm−1 in Curve A belonged to the CO stretching vibration of PEG-Br, and this peak in Curve B was further enlarged owing to the superposition of CO stretching vibration of tBA moieties in PEG-b-PtBA and that of PEG-Br. Additionally, the peaks located at 1601 cm−1 and 2029−1789 cm−1 in Curve C were assigned to the C−C stretching and comb vibration of benzene ring respectively, suggesting the successful polymerization of styrene. And the broad peak located at 3500 cm−1 in Curve D was ascribed to the COO-H stretching vibration of PEG-bPAA-b-PS. The success of the whole synthetic process was further verified by 1H NMR (Figure 1b), in view of a clear observation of the peaks ascribed to the methyl protons of mPEG (a, 3.38 ppm), methyl protons of BIBB (c, 1.83 ppm), methyl protons of tBA (f, 1.44 ppm) and methine protons in benzene ring (g, 6.3−7.2 ppm). According to the ratio of integrals Sa:Sc:Sf:Sg = 1:2:300:125, the triblock copolymer of PEG112-b-tBA100-b-PS75 was thus determined successfully (Table 1). After hydrolysis of the PtBA, the disappearance of signal f indicated a complete removal of tert-butyl groups and the Mn of the triblock copolymer of PEG-b-PAA-b-PS was calculated to be ∼20000. In addition, the successful conjugation of hemoglobin and PEG-b-PAA-b-PS was characterized via a conventional Tris-glycine SDS-PAGE procedure (Figure S1, Supporting Information). The gel permeation chromatography (GPC) traces of PEGBr, PEG-b-PtBA, and PEG-b-PtBA-b-PS are shown in Figure 2. These sharp and symmetric peaks with narrow PDI (Table 1) indicate the homogeneous and controllable polymerization of PtBA and PS.

PEG-Br PEG-bPtBA PEG-bPtBA-bPS

conversiona (%)

n(NMR)b

Mn(NMR)c (g/mol)

Mn(GPC)d (g/mol)

PDI(GPC)d

100

112 100

5000 17800

8700 18800

1.12 1.10

37.5

75

25600

26500

1.14

a

Conversion of monomer calculated by 1H NMR. bDegree of polymerization calculated by 1H NMR. cNumber-average molecular weight calculated by 1H NMR. dNumber-average molecular weight and polydispersity index determined by GPC.

Figure 2. GPC spectra (in DMF) of PEG-Br, PEG-b-PtBA, and PEGb-PtBA-b-PS.

3.2. Formation and Characterization of Micelles. The triblock copolymer PEG-b-PAA-b-PS was assumed to form micelles with core−shell−corona structure in aqueous media due to its amphiphilic nature.37 The water-soluble mPEG chains served as hydrophilic corona, the PS chains formed the hydrophobic core, and the PAA segments were considered to be in the shell phase due to the partial ionization of carboxyl groups in relatively basic environment (pH = 7.4). The CAC of PEG-b-PAA-b-PS was determined using pyrene as a fluorescence probe.35 The intensity ratio I339/I333 in the pyrene excitation spectrum was very sensitive to the polarity of the microenvironment where pyrene was located.38 As shown in Figure S2, the I339/I333 values were relatively small at lower concentrations. However, the values increased significantly when the concentration exceeded a critical level, indicating that D

DOI: 10.1021/acs.biomac.5b00571 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules Table 2. Physicochemical Characterization of the Micelles DLSa PEG-b-PAA-b-PS HbMs ZnPc loaded HbMs a b

Dh (nm)

PDI

ζ-potential (mV)

TEMb diameter (nm)

66.01 ± 0.95 54.01 ± 0.77 72.35 ± 1.58

0.093 ± 0.01 0.148 ± 0.02 0.188 ± 0.01

−24.97 ± 0.51 −19.63 ± 0.36 −13.67 ± 0.29

37.67 ± 3.47 34.10 ± 3.29 45.56 ± 3.64

Number mean diameter (Dh), polydispersity index (PDI), and ζ-potential were determined in phosphate buffer (10 mM, pH = 7.4) by DLS. Statistical diameter was determined by TEM.

the Dh of ZnPc-loaded HbMs was found to be larger than that of free HbMs, probably because of the thickened hydrophobic PS/ZnPc complex core. ζ-Potential changed little after the modifications. The stability of ZnPc-loaded HbMs in fetal bovine serum was further monitored by DLS (Figure S4 in the Supporting Information). Results show that the Dh of samples changed little indicating that they are stable in serum at 4 °C for at least 1 week. In addition, isolated and uniform spherical nanoparticles were clearly viewed in the TEM images (in Figure 4). The average diameters of the three micelles observed by TEM were 37.67 nm, 34.10 nm, 45.56 nm respectively, which were smaller than that obtained by DLS (Figure 3), on account of rapid dehydration in drying process for preparation of TEM specimens.39 3.3. Oxygen Binding Capacity and Stability of the Oxy-HbMs. Hemoglobin is a major protein in mammalian blood, and is responsible for storage and transport of O2 and other gaseous ligands (carbon monoxide, nitric oxide, etc.) in red blood cells.33 Thereinto, CO has about 200 times the affinity of O2 for Hb, and CO only can be displaced by O2 under high oxygen concentration.24,40 Therefore, met-Hb conjugated micelles were first reduced and protected by CO in the present work, allowing for easy storage and further processing. The state conversion could be monitored via UV− vis spectrophotometry. As shown in Figure 5a, met-Hb conjugated micelles were transformed into the ferrous COHbMs in the presence of ascorbic acid under CO atmosphere, which was confirmed by a red shift of the Soret band from 405 to 417 nm. After being fed pure oxygen, the CO-HbMs were converted into the oxygenated state, accompanying with a blue shift of the Soret band from 417 to 412 nm, which is the characteristic absorption peak of Oxy-Hb. In addition, the existence of secondary peaks between 500 and 600 nm in the spectra of CO-HbMs and Oxy-HbMs further verified that metHb in the micelles was reduced and reacted with CO and O2 successively. The Oxy-Hb could be gradually oxidized to its ferric state (met-Hb) in air, which is fatally unable to deliver oxygen to tissues. Therefore, the stability of Oxy-HbMs in air was

the amphiphilic copolymer formed micelles and the pyrene moieties were loaded into the hydrophobic core. Therefore, the CAC value of PEG-b-PAA-b-PS determined from the graphical intersecting point was extremely low (3.46 μg/mL). Herein, the HbMs were obtained by covalent conjugation of Hb onto the PAA moieties of PEG-b-PAA-b-PS micelles in aqueous meida via carbodiimide chemistry, and then ZnPcloaded HbMs could be readily prepared. The number mean diameter (Dh), polydispersity index (PDI) and ζ-potential of the three micelles in this work were characterized by DLS (Table 2 and Figure 3). Samples were filtered before analysis,

Figure 3. Number mean diameter and ζ-potential of PEG-b-PAA-b-PS micelles, HbMs and ZnPc-loaded HbMs measured by DLS in phosphate buffer (10 mM, pH = 7.4).

the count rate (scattered intensity) after filtration decreased slightly (Table S1 in the Supporting Information), indicating that small amount of particles or dust have been removed and there is no obvious bigger aggregates to be removed by filtration. The Dh of PEG-b-PAA-b-PS micelles was 66.01 ± 0.95 nm with a narrow PDI of 0.093 ± 0.01. After conjugation of Hb to the PEG-b-PAA-b-PS micelles, the Dh of HbMs decreased to 54.01 ± 0.77 nm, probably due to the partial consumption of COOH and the reduced electrostatic repulsion effect between COO− groups. After the encapsulation of ZnPc,

Figure 4. TEM images (scale bar = 100 nm) and statistical analysis (in the upper right inset) of (a) PEG-b-PAA-b-PS micelles, (b) HbMs, and (c) ZnPc-loaded HbMs. E

DOI: 10.1021/acs.biomac.5b00571 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

Figure 5. (a) UV−vis spectra of Hb conjugated PEG-b-PAA-b-PS micelles under various atmospheres. (b) Stability of Oxy-Hb in Hb conjugated PEG-b-PAA-b-PS micelles at 25 °C.

characterized by plotting the percentage of residual Oxy-Hb versus time31,32 (see Figure 5b). The percentage of Oxy-Hb was defined as 100% when the maximum absorption peak located at 412 nm. Then the percentage declined over time since the ferrous protoporphyrin in Oxy-HbMs was gradually oxidized to its inactive ferric state in the atmosphere. When the maximum absorption peak shifted to 405 nm, the residual OxyHb was denoted as 0. The oxidation curve was obtained with a half time of about 7.7 h. As Huang et al. reported previously, the oxidation curve of the Oxy-Hb conjugated micelles containing hydrophilic PEG chains showed two stages.32 Additionally, the PEG segments on the surface of the micelles were assumed to provide a good protection to Oxy-Hb and improved its stability in air. 3.4. Encapsulation of ZnPc. ZnPc was encapsulated in the HbMs in this work due to its excellent hydrophobic character. The encapsulation efficiency (EE) of ZnPc in the HbMs for 5% theoretical loading densities was 81.6% (weight%) with a small standard deviation of ±1.7%, which was slightly higher than that of ZnPc loaded poly(lactic-co-glycolic acid) (EE = 70%) reported previously.11 The stability of the obtained ZnPcloaded HbMs was also evaluated for 4 weeks (data not shown), and there were no significant changes in the size distribution and spectroscopic properties. These results confirmed that the HbMs could provide an effective strategy for solubilization and encapsulation of ZnPc. Also, the absorption and fluorescence emission spectra of these ZnPc-loaded HbMs (Figure S5) showed similar spectroscopic behaviors to the ZnPc standard (5 μM of free ZnPc in DMSO). 3.5. Generation of 1O2 by Light Irradiation. Singlet oxygen, generated by excitation of photosensitizers using light of appropriated wavelength and power, has been proven as the predominant species leading to significant cellular apoptosis in photodynamic therapy.5,19 In this work, generation of singlet oxygen for the ZnPc-loaded micelles was monitored by the photobleaching of ADMA,41 a useful chemical trap to 1O2. Additionally, the yield of singlet oxygen is proportional to the decay rate of chemical quencher.42 The natural lifetime of 1O2 state in aqueous solution is too short to observe significant bleaching of ADMA (data not shown) owing to the solvent deactivation.17 Therefore, the decay of ADMA for the two ZnPc-loaded micelles with or without Hb was shown in Figure 6 in deuteroxide after being bubbled with argon for different times with constant argon velocity. The slopes of the ADMA

Figure 6. Comparison of the decay rates of ADMA. (a) ZnPc-loaded HbMs (open triangle and dash line, red), Y = 99.647 − 0.908X, R2 = 0.996; (b) ZnPc-loaded micelles without Hb (solid triangle and straight line, red), Y = 100.056 − 0.882X, R2 = 0.996; (c) ZnPc-loaded HbMs treated with argon for 5 min (open square and dash line, black), Y = 99.564 − 0.34X, R2 = 0.993; (d) ZnPc-loaded micelles without Hb treated with argon for 5 min (solid square and straight line, black), Y = 100.328 − 0.288X, R2 = 0.995; (e) ZnPc-loaded HbMs treated with argon for 12 min (open circle and dash line, green), Y = 99.791 − 0.144X, R2 = 0.988; (f) ZnPc-loaded micelles without Hb treated with argon for 12 min (solid circle and straight line, green), Y = 99.93 − 0.07X, R2 = 0.994.

decay were determined by linear regression fitting. As shown in Table 3, the absolute value of slope for the decay of ADMA decreased with prolonged time of feeding argon. This could be explained by the fact that oxygen is involved in the photooxidation process, and the rate of 1O2 generation increases with increasing oxygen concentration.17,18 Therefore, the production of singlet oxygen was suppressed for both the Table 3. Slopes of the ADMA Decay after Bubbled with Argon for Different Time

0 min 5 min 12 min

ZnPc-loaded micelles without Hb

ZnPc-loaded HbMs

slope ratioa

−0.882 −0.288 −0.07

−0.908 −0.34 −0.144

1.03 1.18 2.06

a

The ratio of slope for ZnPc-loaded HbMs and ZnPc-loaded micelles without Hb.

F

DOI: 10.1021/acs.biomac.5b00571 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

Figure 7. (a) Comparison of photo and dark toxicity of Hela cells to HbMs, ZnPc-loaded PEG-b-PAA-b-PS micelles, and ZnPc-loaded HbMs. (b) The photo and dark toxicity of Hela cells to ZnPc-loaded HbMs that were exposed to air for various times. Each sample for phototoxicity assay was further equally illuminated for 2 min.

Supporting Information. The Soret band shifted gradually from 417 to 412 nm by increasing the exposure time in air. After 30 h, the Oxy-Hb was oxidized irreversibly to methemoglobin, which is totally unable to bind oxygen.

ZnPc-loaded micelle solutions due to the deaeration by argon.17 Interestingly, however, the ZnPc-loaded HbMs presented larger decay rate compared to the control micelles without Hb after being bubbled with argon for the same time. After being bubbled with argon for 12 min, the absolute value of slope for e (ZnPc-loaded HbMs) was twice of that for f (ZnPc-loaded micelles without Hb). This could be attributed to the supplement of oxygen molecule carried by Hb to generate more 1O2 than the control micelles without Hb. 3.6. Photocytotoxicity. The photo and dark cytotoxicity of Hela cells were evaluated by WST-8 assays using CCK-8 reagent. As shown in Figure 7a, the viabilities of Hela cells (>93%) were not affected after incubation in the two ZnPcloaded micelles without light irradiation, indicating that both of them were biocompatible and had no significant dark cytotoxicity in cultivation. After exposure to red light, both of the ZnPc-loaded micelles were subjected to a dramatic decline in cell viability since the excited ZnPc could interact with molecular oxygen to generate 1O2 and cause irreversible damage to target tissues. Interestingly, the cell viability of ZnPc-loaded HbMs was nearly 30% lower than that of ZnPcloaded micelles without Hb. The significant increase in cytotoxicity might be due to the interaction between the extra molecular oxygen carried by Hb and ZnPc under illumination to produce more 1O2 and kill more Hela cells. In addition, the photocytotoxicity of the ZnPc-free HbMs was also monitored, and the cellular viability was not affected (>94%). The Oxy-HbMs utilized in the cytotoxicity experiments were obtained by exposing CO-HbMs to the atmosphere for a period. And for phototoxicity assays, samples was further equally illuminated for 2 min. The cytotoxicity of ZnPc-loaded HbMs that were exposed to air for different periods of time was also measured. As shown in Figure 7b, there was no significant change in the dark cytotoxicity; however, the photocytotoxicity gradually increased with the prolonged periods of time in contact with air. After being exposed to air for 24 h, the cell viability of the ZnPc-loaded HbMs had dropped by about 40% relative to that of samples without previous exposure. This could probably be ascribed to the enhanced oxygenation level of hemoglobin due to the prolonged exposure time in air. After 30 h, the ZnPc-loaded HbMs suffered a slight increase in cell viability, probably due to the partial oxidation of Oxy-Hb. The explanation could also be verified by Figure S7 in the

4. CONCLUSIONS In summary, we have synthesized a novel photosensitizer carrier for PDT, which was equipped with oxygen selfcompensating ability. The nanoparticle carrier was obtained via chemical conjugation of Hb onto the micelle of triblock copolymer PEG-b-PAA-b-PS which was prepared by ATRP. The HBOC could form monodisperse stable nanoparticles with relatively low CAC value. Simultaneously, Hb incorporated into the polymeric micelles was confirmed to retain its oxygenbinding capacity. After ZnPc was encapsulated into the HBOC, the resulting photosensitizer carrier was confirmed to generate more singlet oxygen and exert greater photocytotoxicity on Hela cells than ZnPc-loaded micelles without Hb in vitro.

■

ASSOCIATED CONTENT

* Supporting Information S

SDS-PAGE of conjugates, Plot of intensity ratio (I339/I333) versus log C to determine CAC, absorption, and fluorescence emission spectra of samples, count rate in DLS test before and after filtration, stability of sample in serum, and UV−vis spectral change of Oxy-HbMs during the exposure to air. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b00571. (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]; Fax: +86 512 69155837; Tel: +86 512 65884406. *E-mail: [email protected]; Fax: +86 571 87952596; Tel: +86 571 87952596. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We acknowledge Prof. Hong Chen, Mr. Zhonglin Lv, Mr. Yuqi Yuan and Mr. Wei Lu in Soochow University for the assistance G

DOI: 10.1021/acs.biomac.5b00571 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules in cytotoxicity assays. This research is financially supported by the National Natural Science Foundation of China (21274124, 21374069).

■

(32) Li, T. H.; Jing, X. B.; Huang, Y. B. Polym. Adv. Technol. 2011, 22, 1266−1271. (33) Wang, S.; Yuan, F.; Chen, G.; Tu, K.; Wang, H.; Wang, L.-Q. RSC Adv. 2014, 4, 52940−52948. (34) Liu, J.-Y.; Lo, P.-C.; Jiang, X.-J.; Fong, W.-P.; Ng, D. K. Dalton Trans. 2009, 4129−4135. (35) Wilhelm, M.; Zhao, C. L.; Wang, Y.; Xu, R.; Winnik, M. A.; Mura, J. L.; Riess, G.; Croucher, M. D. Macromolecules 1991, 24, 1033−1040. (36) Thilakarathne, V.; Briand, V. A.; Zhou, Y.; Kasi, R. M.; Kumar, C. V. Langmuir 2011, 27, 7663−7671. (37) Wang, R.; Cherukuri, P.; Duque, J. G.; Leeuw, T. K.; Lackey, M. K.; Moran, C. H.; Moore, V. C.; Conyers, J. L.; Smalley, R. E.; Schmidt, H. K.; et al. Carbon 2007, 45, 2388−2393. (38) Zhang, J. X.; Qiu, L. Y.; Jin, Y.; Zhu, K. J. J. Biomed. Mater. Res., Part A 2006, 76, 773−780. (39) Lu, J.; Zhang, W.; Richards, S.-J.; Gibson, M. I.; Chen, G. Polym. Chem. 2014, 5, 2326−2332. (40) Li, B.; Chen, G.; Meng, F. B.; Li, T. H.; Yue, J.; Jing, X. B.; Huang, Y. B. Polym. Chem. 2012, 3, 2421−2429. (41) Wang, L.; Li, J.; Zhang, W.; Chen, G.; Zhang, W.; Zhu, X. Polym. Chem. 2014, 5, 2872−2879. (42) Hoebeke, M.; Damoiseau, X. Photoch. Photobio. Sci. 2002, 1, 283−287.

REFERENCES

(1) Derycke, A. S.; de Witte, P. A. Adv. Drug Delivery Rev. 2004, 56, 17−30. (2) Dolmans, D. E.; Fukumura, D.; Jain, R. K. Nat. Rev. Cancer 2003, 3, 380−387. (3) Verteporfin In Photodynamic Therapy Study Group1A . Am. J. Ophthalmol. 2001, 131, 541−560. (4) Detty, M. R.; Gibson, S. L.; Wagner, S. J. J. Med. Chem. 2004, 47, 3897−3915. (5) Nishiyama, N.; Morimoto, Y.; Jang, W.-D.; Kataoka, K. Adv. Drug Delivery Rev. 2009, 61, 327−338. (6) Lu, J.; Zhang, W.; Yuan, L.; Ma, W.; Li, X.; Lu, W.; Zhao, Y.; Chen, G. Macromol. Biosci. 2014, 14, 340−346. (7) van Nostrum, C. F. Adv. Drug Delivery Rev. 2004, 56, 9−16. (8) Wu, D.-Q.; Li, Z.-Y.; Li, C.; Fan, J.-J.; Lu, B.; Chang, C.; Cheng, S.-X.; Zhang, X.-Z.; Zhuo, R.-X. Pharm. Res. 2010, 27, 187−199. (9) Kim, W. L.; Cho, H.; Li, L.; Kang, H. C.; Huh, K. M. Biomacromolecules 2014, 15, 2224−2234. (10) Ideta, R.; Tasaka, F.; Jang, W.-D.; Nishiyama, N.; Zhang, G.-D.; Harada, A.; Yanagi, Y.; Tamaki, Y.; Aida, T.; Kataoka, K. Nano Lett. 2005, 5, 2426−2431. (11) Ricci-Júnior, E.; Marchetti, J. M. Int. J. Pharm. 2006, 310, 187− 195. (12) Kojima, C.; Toi, Y.; Harada, A.; Kono, K. Bioconjugate Chem. 2007, 18, 663−670. (13) Li, B.; Moriyama, E. H.; Li, F.; Jarvi, M. T.; Allen, C.; Wilson, B. C. Photochem. Photobiol. 2007, 83, 1505−1512. (14) Xu, J.; Zeng, F.; Wu, H.; Hu, C.; Wu, S. Biomacromolecules 2014, 15, 4249−4259. (15) Gibot, L.; Lemelle, A.; Till, U.; Moukarzel, B.; Mingotaud, A.-F.; Pimienta, V.; Saint-Aguet, P.; Rols, M.-P.; Gaucher, M.; Violleau, F.; et al. Biomacromolecules 2014, 15, 1443−1455. (16) Cohen, E. M.; Ding, H.; Kessinger, C. W.; Khemtong, C.; Gao, J.; Sumer, B. D. Otolaryngol.–Head Neck Surg. 2010, 143, 109−115. (17) Lindig, B. A.; Rodgers, M. A.; Schaap, A. P. J. Am. Chem. Soc. 1980, 102, 5590−5593. (18) Maree, M. D.; Kuznetsova, N.; Nyokong, T. J. Photochem. Photobiol., A 2001, 140, 117−125. (19) Chen, Q.; Huang, Z.; Chen, H.; Shapiro, H.; Beckers, J.; Hetzel, F. W. Photochem. Photobiol. 2002, 76, 197−203. (20) Fukumura, D.; Jain, R. K. J. Cell. Biochem. 2007, 101, 937−949. (21) Vaupel, P.; Kallinowski, F.; Okunieff, P. Cancer Res. 1989, 49, 6449−6465. (22) Yu, M.; Dai, M.; Liu, Q.; Xiu, R. Cancer Treat. Rev. 2007, 33, 757−761. (23) Lehninger’s Principles of Biochemistry, 4th ed.; Nelson, D. L., Lox, M. M., Eds.; W. H. Freeman and Company: New York, 2004; p 162. (24) Sun, J.; Huang, Y. B.; Shi, Q.; Chen, X. S.; Jing, X. B. Langmuir 2009, 25, 13726−13729. (25) Rameez, S.; Alosta, H.; Palmer, A. F. Bioconjugate Chem. 2008, 19, 1025−1032. (26) Arifin, D. R.; Palmer, A. F. Biomacromolecules 2005, 6, 2172− 2181. (27) Mudhivarthi, V. K.; Cole, K. S.; Novak, M. J.; Kipphut, W.; Deshapriya, I. K.; Zhou, Y.; Kasi, R. M.; Kumar, C. V. J. Mater. Chem. 2012, 22, 20423−20433. (28) Chen, K.; Merkel, T. J.; Pandya, A.; Napier, M. E.; Luft, J. C.; Daniel, W.; Sheiko, S.; DeSimone, J. M. Biomacromolecules 2012, 13, 2748−2759. (29) Greenburg, A. G.; Kim, H. W. Crit. Care 2004, 8, S61−S64. (30) Kim, H. W.; Greenburg, A. G. Artif. Organs 2004, 28, 813−828. (31) Li, B.; Li, T.; Chen, G.; Li, X.; Yan, L.; Xie, Z.; Jing, X.; Huang, Y. Macromol. Biosci. 2013, 13, 893−902. H

DOI: 10.1021/acs.biomac.5b00571 Biomacromolecules XXXX, XXX, XXX−XXX