Fluorescent Copolymer-Based Prodrug for pH-Triggered Intracellular

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Fluorescent copolymer-based prodrug for pH-triggered intracellular release of DOX Xu Jia, Xubo Zhao, Kun Tian, Tingting Zhou, Jiagen Li, Ruinian Zhang, and Peng Liu Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b01070 • Publication Date (Web): 13 Oct 2015 Downloaded from http://pubs.acs.org on October 15, 2015

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Fluorescent copolymer-based prodrug for pHtriggered intracellular release of DOX Xu Jia, Xubo Zhao, Kun Tian, Tingting Zhou, Jiagen Li, Ruinian Zhang, and Peng Liu* State Key Laboratory of Applied Organic Chemistry and Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China ABSTRACT: A novel water-soluble pH stimuli-responsive fluorescent copolymer of P(PEGMA-b-(MAH-co-Rh6GEAm))

was

synthesized

by

two-step

sequential

RAFT

polymerization. The prodrug with drug content of 0.1560 mg/mg was prepared by coupling doxorubicin (DOX) onto the copolymer via acid-cleavable hydrazone bond, formed between the carbonyl group of DOX and abundant hydrazide functional groups in the copolymer. The amphiphilic DOX-conjugated prodrug (P(PEGMA-b-(MAH-DOX-co-Rh6GEAm))) could easily form micelle in water with Dh of less than 100 nm. It could be transported into HepG2 cells and release DOX without burst release, while the leakage of DOX can be avoided in the simulated normal physiological media. Furthermore, its fluorescence intensity experienced the reversibly change with the transformation of the media pH. The better biocompatibility, pH stimuliresponsiveness and the strong fluorescence at low pH media make the nanoparticles potential platform for the controlled release of anticarcinogen and real-time fluorescent imaging of tumor tissues.

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Keywords: Prodrug; intracellular release; pH-triggered controlled release; fluorescent copolymer; RAFT

INTRODUCTION Doxorubicin (DOX) is widely used anticarcinogen for a variety of malignant tumors. However, its poor selectivity to tumor cells and inherent multi-drug resistance (MDR) effect are still significant problems to be solved.1,2 The ideal drug delivery system (DDS) should possess the function of targeted delivery with smart release, so that the loaded drug could be selectively transported and released in desired tissues or cells.3,4 To overcome the shortcoming of DOX in the process of cancer therapeutic, several polymeric DDSs have been explored intensively.5 The DOX-loading mode mainly includes electrostatic interaction,6 π-π stacking interaction,7 hydrophobic interaction,8,9 Schiff base or hydrazone bond.10-12 The difference of pH between normal and tumor tissues has been universally accepted as one of the significantly feature,13 which declared that the accurate detection of pH played a crucial role in the diagnosis and treatment of cancer. So it can realize specific release in tumor tissues or cells. Due to the weak acidic media in tumor tissues,14 the conjugates of DOX via acidic cleavable Schiff base or hydrazone linkage could decrease the leakage in the blood circulation, and subsequently reduce the side effect to the normal cells or tissues.15 Park et al has synthesized a bi-block copolymer composed of poly(L-lactic acid) and methoxy-poly(ethylene glycol) in which DOX was chemically conjugated to the terminal end groups via hydrazone bond.16 Ulbrich and co-workers described that DOX was bonded to the poly(ethylene oxide)-block-poly(allyl glycidyl ether) via acid-cleavable hydrazone linkage, and the prodrug can reduce the systemic toxicity and prolong the circulation time.17,18 Zhan et al has also reported a pH responsive DDS based on acid-cleavable hydrazone bond, the drug release was achieved in acid solution.19 Because of the


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instability of hydrazone bond, these prodrugs can reach the goal of controlled release of the loaded drug in the tumor sites without drug leakage in normal tissues.13,15 Furthermore, multifunctional drug carriers have great potential by integrating the therapeutic and diagnostic functions. The magnetic nanoparticles coated with the substance of drug loading capability showed better prospect in application.20 For instance, Chen et al reported a mesoporous silica nanoparticle coupled with small lanthanide magnetic nanoparticles as anticancer DDS which could be visualized by magnetic resonance imaging in vivo.21 The DDSs with fluorescence also attracted the attention of researchers. Zhao synthesized a pH-sensitive fluorescence and hepatocyte-targeting carrier via a layer-by-layer assembly of fluorescein isothiocyanate (FITC)-modified chitosan and sodium hyaluronate on polystyrene sulfonate templates with galactosylated chitosan (GC) as the outermost layer, and it was expected to be used for the diagnosis and therapy of hepatic cancer.22 Lin reported the methotraxate-PEGchitosan-iron oxide nanoparticles-Cy5.5 for fluorescence imaging.23 In this work, a novel hydrazide-containing block copolymer poly(polyethylene glycol methyl ether

methacrylate-b-(methylacryloylhydrazide-co-N’-Rhodamine

6G-ethyl-acrylamide))

(P(PEGMA)-b-(MAH-co-Rh6GEAm)) was synthesized by RAFT polymerization as drug delivery system. Then DOX, an antitumor antibiotic, was conjugated onto the side groups of the copolymer

through

acylhydrazone

bond

between

the

hydrazide

groups

of

the

methylacryloylhydrazide (MAH) units in the block copolymer and the carbonyl group of DOX to form the pH-sensitive fluorescent polymer-based prodrug P(PEGMA-b-(MAH-DOX-coRh6GEAm)) (Scheme 1). Herein, PEG was deemed to an ideal polymer for reducing the chance of both immunological and kidney clearance and elongating circulation time in vivo.24,25 Rhodamine 6G is a fluorochrome with remarkable pH sensitivity, the fluorescence intensity experienced the reversibly switch with the transformation of pH.26-28 The acylhydrazone linkage


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bond between the anti-cancer drug and DDS was acid-sensitive. It could be cleaved in acidic environment and would hydrolyze to release the drug loaded. However, the structure is more stable in neutral media, which could avoid the drug leakage from the prodrug during the blood circulation. Therefore, the designed prodrug with better biocompatibility, pH stimuliresponsiveness and strong fluorescence at acidic media are expected for the diagnosis and chemotherapy of cancers.

Scheme 1. Illustration of pH-sensitive fluorescent polymer-based prodrug for the intracellular release of DOX triggered by the acidic micro-environment inside the tumor cells.

EXPERIMENTAL SECTION Materials and reagents. Poly(ethylene glycol) methyl ether methacrylate (PEGMA) with number average molecular weight of 475 was got from Aladdin Chemistry Co. Ltd. It was purified by passing through a basic alumina column before use. Doxorubicin hydrochloride (DOX·HCl) was bought from Beijing Huafeng United Technology Co. Ltd. Methacryloyl chloride was provided from Tianjin Heowns Company. Acetone oxime was got from Sinopharm


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Chemical Reagent Co., Ltd. Rhodamine 6G was purchased from Dongsheng Chemical Reagent Company. Aliquot 336 (methyltrioctylammonium chloride) was provided by Maya Reagent Company. 1-Dodecanethiol was bought from Shanghai Shanpu Chemistry Co. Ltd. 2,2'Azoisobutyronitrile (AIBN) was bought from Tianjin Guangfu Technology and Development Co. Ltd. All other reagents were analytical reagent grade and used directly without further purification. Deionized water was used throughout.

Synthetic procedures. N’-ethylamino- Rhodamine 6G (Rh6GEA). Rhodamine 6G (9.60 g, 20 mmol) was dissolved in 120 mL ethanol, the solution was stirred at ambient temperature, and then 5 mL ethylenediamine was dropped into the solution within 30 min. The solution was refluxed till the fluorescence disappeared. After the reaction solution was cooled, the solvent was removed by rotatory evaporator. The residue was washed by cold ethanol (20 mL× 3) and recrystallized in acetonitrile and then dried in vacuum to yield Rh6GEA as a pink solid (6.39 g, 70% yield).29,30

N’-Rhodamine 6G-ethyl-acrylamide (Rh6GEAm). Rh6GEA (5.70 g, 12.5 mmol) was dissolved in 200 mL dichloromethane, and then triethylamine (2.1 mL, 15 mmol) was added into the solution with stirring in ice bath. The solution of acryloyl chloride (2.72 g, 30 mmol) in 150 mL dichloromethane was added dropwise to the aforementioned solution while stirring. After being stirred in ice bath for 8 h, the solution was washed with saturated sodium bicarbonate solution (100 mL × 3) and water (100 mL × 3), the organic layer was dried over anhydrous MgSO4 and filtered, the solvent was removed by rotatory evaporator. The solid was recrystallized in acetonitrile and then dried in vacuum to yield Rh6GEAm as a white solid (9.34 g, 61% yield).30


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Methacryloylacetone oxime (MAO). A solution of acetone oxime (2.33 g, 31.9 mmol) and triethylamine (3.55 g, 35.1 mmol) in dichloromethane (50 mL) was cooled in an ice bath. The solution of methacryloyl chloride (5.0 g, 47.8 mmol) in 50 mL dichloromethane was added dropwise into the aforementioned solution while stirring, and then the reaction solution was stirred for another 8 h. The solution was washed with saturated sodium bicarbonate solution (100 mL × 3) and water (100 mL × 3), and the organic layer was dried over anhydrous MgSO4 and filtered, the solvent was removed by rotatory evaporator. Finally, the yellow oily product, methacryloylacetone oxime (MAO), was obtained with column chromatography (ethyl acetate/petroleum ether, v/v, 3/7) (4.31 g, 96%).31

S-1-Dodecyl-S’-(α,α’-dimethyl-α’’-aceticacid) trithio-carbonate (DDMAT). 1-Dodecanethiol (40.38 g, 0.20 mol), acetone (96.2 g, 1.66 mol), and Aliquot 336 (tricaprylylmethylammonium chloride) ( 3.25 g, 8 mmol) were mixed in a reaction flask, the solution was cooled to 10 °C under a nitrogen atmosphere. Sodium hydroxide solution (50%) (16.77 g, 0.21 mol) was added dropwise into the aforementioned solution while stirring. Carbon disulfide (15.21 g, 0.20 mol) in 26 mL acetone was added into the reaction solution within 20 min after the reaction was stirred for another 15 min. Ten minutes later, chloroform (35.63 g, 0.30 mol) was added in one portion, followed by dropwise addition of 50% sodium hydroxide solution (80 g, 1 mol) within more than 30 min. The mixture was reacted under stirring overnight. Finally water (300 mL) was added, followed by 100 mL of concentrated hydrochloric acid to acidify the aqueous solution. The solid was collected with a Buchner funnel and then dissolved in 500 mL of isopropanol. The filtrate was collected and the undissolved solid was filtered off. The solvent isopropanol was removed by rotatory evaporator, and the resulting solid was recrystallized from hexanes to afford yellow crystalline solid.32


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P(PEGMA) homopolymer. PEGMA (2.57 g, 5.42 mmol ), DDMAT (0.1974 g, 0.54 mmol ) and AIBN (0.0148 g, 0.0903 mmol ) were dissolved in 7 mL of DMF, the mixture was added into the Schlenk flask. The mixture was degassed by three freeze–vacuum–thaw cycles. The polymerization was processed in oil bath (70 °C) for 24 h, and stopped by cooling the mixture in ice water and exposed to air. The produced polymer was precipitated into ethyl ether, centrifuged, and dried overnight in vacuum.33

P(PEGMA-b-(MAO-co-Rh6GEAm)) block copolymer. P(PEGMA) (2.60 g, 0.62 mmol), MAO (0.80 g, 5.67 mmol), Rh6GEAm (0.30 g, 0.58 mmol) and AIBN (0.015 g, 0.093 mmol) were dissolved in DMF (12.5 mL), and the mixture was degassed by three freeze–vacuum–thaw cycles. The polymerization was processed in oil bath (70 °C) for 24 h, and stopped by cooling the mixture in ice water and exposed to air. The copolymer was precipitated into petroleum ether, centrifuged, and dried under vacuum.33

P(PEGMA-b-(MAH-co-Rh6GEAm)) block copolymer. P(PEGMA-b-(MAO-co-Rh6GEAm)) (1.1 g) was dissolved in 15 mL of DMF, then hydrazine hydrate (5 mL) was added into the solution under a nitrogen atmosphere. The mixture was proceeded for 48 h at r.t. The block copolymer P(PEGMA-b-(MAH-co-Rh6GEAm)) was purified by extensive dialysis against DI water (MWCO 1000) and collected by lyophilization.33

P(PEGMA-b-(MAH-DOX-co-Rh6GEAm))

prodrug.

P(PEGMA-b-(MAH-co-Rh6GEAm))

(0.6509 g, 0.117 mmol) was dispersed in 10 mL of methanol. After 2 drops of acetic acid glacial were added into the solution, DOX·HCl (0.1356 g, 0.234 mmol) was added into the mixture under a nitrogen atmosphere. The mixture was proceeded at 50 °C for 48 h, and then dialyzed


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against deionized water for 4 days (MWCO 1000). The product was collected by lyophilization and gave a red residuum. To measure the DOX content, 1 mg of the prodrug was dealt with 50 mL of 1 mol/L HCl at ambient temperature for 36 h. The content of free DOX was determined by UV-vis at 480 nm.19

Cytotoxicity. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) assay was selected to test the cytotoxicity of the prodrug. For the MTT assay, the cells were implanted into the 96-well plate with the density of 1 ×105 cells per well. Then, a series of concentrations of the prodrug or corresponding polymer precursor were added into the cells, and incubated for 48 h at 37 °C. Then, 20 µL of MTT solution (5 mg/mL) was added into each well, and incubated for another 4 h. After the solution was removed carefully, 150 µL of DMSO was added. The plates were shaken for 20 min. The absorbance of each well was measured by the Enzyme-linked Immunosorbent Assay Appliance at the wavelength of 490 nm.

Cellular uptake. The cellular uptake was exhibited by fluorescence microscope (Olympus BX43) using HepG2 cells after 12 h incubation in the presence of the P(PEGMA-b-(MAH-DOXco-Rh6GEAm)) prodrug. The location of intracellular fluorescence was validated with excitation wavelengths of 480 nm for DOX and 405 nm for Hoechst. The cell nucleus was stained with Hoechst 33258.

In vitro release. The release of DOX from the prodrug was conducted at 37 °C in three different buffer solutions individually: pH 5.0 acetate buffer; pH 6.0 acetate buffer and pH 7.4 phosphate buffer. Each sample was then transferred into a dialysis bag with molecular weight cut off of 3500, then immersed into 130 mL corresponding buffer and mildly shaken at 37 °C, respectively.


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At desired stage, 5 mL of the solution was taken out termly from the solution, and the mass of the released DOX was detected by UV-vis at 480 nm. To keep the solution volume constant, an equal volume of corresponding fresh media was replenished after each sampling. All the data of drug release were averaged over three experiments.

Instruments and characterization. For the Fourier transform infrared (FT-IR) spectra within 4000-400 cm-1 wavenumber, a Nicolet 360 FTIR spectrometer with the KBr pellet technique was employed. 1

H NMR spectra were characterized by JEOL ECS 400M instrument at 400 MHz.

The number average molecular weight (Mn) and polydispersity (PDI) of the P(PEGMA-b(MAO-co-Rh6GEAm)) were determined by gel permeation chromatography (GPC) by employing tetrahydrofuran as the eluent at the flow rate of 1.0 mL/ min at 35 °C after calibration with polystyrene standard. The critical micelle concentration (CMC) of the P(PEGMA-b-(MAH-DOX-co-Rh6GEAm)) was measured by testing the fluorescence intensities of the solution of pyrene containing the copolymer at specific concentrations with a fluorescence spectrophotometer (Hitachi F-7000). The morphology and diameter of the prodrug were measured by a JEM-1200EX transmission electron microscopy (TEM). The hydrodynamic diameter of the prodrug in different pH conditions was measured by the dynamic scattered light (DLS，BI-200SM) in water. The absorbance of DOX was tested on a TU-1901 UV–vis absorption spectrophotometer. The steady-state emission spectra of the prodrug at different pH media were measured by a Hitachi F-4500 spectrofluorometer.


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RESULTS AND DISCUSSION Synthesis and characterization of P(PEGMA-b-(MAH-co-Rh6GEAm)). On the basis of an active ester monomer methacryloylacetone oxime (MAO), biocompatible monomer PEGMA and pH-sensitive fluorescent monomer Rh6GEAm, the block copolymer of poly(polyethylene glycol methyl ether methacrylate-b- (methylacryloylhydrazide-co-N’-Rhodamine 6G-ethyl-acrylamide)) (P(PEGMA)-b-(MAH-co-Rh6GEAm))

was

synthesized

by

two-step

sequential

RAFT

polymerization. As shown in Scheme 1, the macro-RAFT agent P(PEGMA) (Mn = 4165, Xn = 8 from NMR analysis) was synthesized by DDMAT-mediated RAFT polymerization with AIBN as the initiator in DMF at 70 °C.

Scheme 2. Outline for the synthesis of the P(PEGMA-b-(MAH-co-Rh6GEAm)) block copolymer by RAFT polymerization and conjugation of DOX via an acylhydrazone bond. Then P(PEGMA) was extended with MAO and Rh6GEAm to acquire the block copolymer P(PEGMA-b-(MAO-co-Rh6GEAm) (Mn

NMR

= 5804). The number-average polymerization

degree (Xn) of MAO was worked out to be 8 by comparison of the integration of peak at 3.5-3.8 ppm to that of peaks (e, f, and h) at 1.75–2.25 ppm in the 1H NMR spectrum (Figure 1).31,33 The


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number average molecular weight (Mn) of the P(PEGMA-b-(MAO-co-Rh6GEAm)) was determined to be 8124 with the polydispersity (PDI) of 1.48 (Figure S2). Its relatively low PDI value after two-step sequential RAFT polymerization indicated the characteristic of the “living”/controlled radical polymerization.

Figure 1. 1H NMR spectra of P(PEGMA) homopolymer (a), P(PEGMA-b-(MAO-coRh6GEAm)) block copolymer (b), P(PEGMA-b-(MAH-co-Rh6GEAm)) block copolymer (c) and P(PEGMA-b-(MAH-DOX-co-Rh6GEAm)) prodrug (d). Then the activated acetoxime esters in the P(PEGMA-b-(MAO-co-Rh6GEAm) block copolymer were converted into corresponding hydrazides by the reaction with an excess amount of hydrazine hydrate, a hydrophilic P(PEGMA-b-(MAH-co-Rh6GEAm)) block copolymer was


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prepared which contained hydrazide bonds. The transformation of acetoxime ester to hydrazide was verified by the sharply decreased intensity of the signal at 2.0 ppm corresponding to methyl protons of oxime units in the 1H NMR spectrum (Figure 1).27,29 In the infrared spectrum (Figure S1), the appearance of an amide band at 1673 cm-1 indicated the transformation of acetoxime ester to hydrazide.

Relativity fluorescence intensity/a.u.

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pH=3 pH=4 pH=5 pH=6 pH=7 pH=8 pH=9 pH=10 pH=11

3000

2000

1000

0 550

600

650

Wavelength (nm)

Figure 2. Fluorescence emission spectra (excitation at λex = 510 nm) of P(PEGMA-b-(MAH-coRh6GEAm)) block copolymer as a function of the media pH values. The inset is the image of the copolymer in acid (pH 5.0) (a) and neutral (pH 7.4) (b) solutions under 365 nm-UV light. Due to the existence of the Rh6GEAm units, the part of the nonfluorescent spiro structure transform reversibly into fluorescent ring-opened amide form in acidic environment (Figure 2).2628

The P(PEGMA-b-(MAH-co-Rh6GEAm)) block copolymer could also be served as pH sensors.

When the pH of the media was above 6, the solution of the copolymer was colorless. Because the construction of the Rh6GEAm was spiro, the copolymer was essentially nonfluorescent. While the color of the solution changed from colorless to pink in the daylight, and turned from colorless to yellow under ultraviolet lamp at the wavelength of 365 nm under pH < 6, due to that the spiro form was induced to change into the ring-opened amide form by the hydrogen ion. Thus, this


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characteristic is expected to be useful for the diagnosis of cancers because of the different pH values between the tumor and normal cells.

Synthesis of P(PEGMA-b-(MAH-DOX-co-Rh6GEAm)) prodrug. The reversible hydrazone linkage between hydrazide and carbonyl groups has been implemented in the generation of biologically active substances, self-healing gels, and pH responsive micelles as drug carriers.34-36 As a carbonyl-containing antitumor antibiotics to effectively inhibit the synthesis of RNA and DNA, DOX has been involved in the formation of hydrazone and Schiff base.37 Here, the mixture solution of the P(PEGMA-b-(MAH-co-Rh6GEAm)) block copolymer and DOX⋅HCl in methanol was proceeded at 50 °C for 48 h, then the mixture was dialyzed against deionized water for 4 days (MWCO 1000) until no DOX could be detected in the dialyzate. The product was collected by lyophilization and gave a red residuum, with the content of DOX in the prodrug of 0.1560 mg/mg. Considering the Mn of the P(PEGMA-b-(MAO-co-Rh6GEAm)), it could calculated that each prodrug P(PEGMA-b-(MAH-DOX-co-Rh6GEAm)) molecule contained two DOX units. The CMC of the prodrug P(PEGMA-b-(MAH-DOX-co-Rh6GEAm)) was tested by the method of the fluorescent probe pyrene. A certain concentration of pyrene acetone solution was pipetted into the colorimetric tube with pipette, followed by removing the acetone. Then, a series of the P(PEGMA-b-(MAH-DOX-co-Rh6GEAm)) solutions with different concentrations were pipetted into each colorimetric tube. Each sample was dealt with ultrasonic concussion for 2 h, and incubated overnight. The concentration of pyrene in each sample was adjusted to 5×10-7 mol/L. The fluorescence of the solution was tested by using 335 nm as the excitation wavelength and the emission spectra ranged from 370 to 480 nm. The fluorescence intensities ratio of the emission at 372 nm (I1) and 384 nm (I3) were determined to calculate the plot of the intensity


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ratios (I1/I3) as a function of the prodrug concentration. The CMC of the prodrug P(PEGMA-b(MAH-DOX-co-Rh6GEAm)) was 1.0233×10-3 mol/L (Figure S3)38. Due to the hydrophilic part of PEGMA and PMAH and the hydrophobic part of PMAH-DOX, the P(PEGMA-b-(MAH-DOX-co-Rh6GEAm)) prodrug could easily form micelle in aqueous solution (5 mg/mL) with magnetic stirring without any organic solvent. It is very beneficial for the application as DDS. The regular sphere morphology of the P(PEGMA-b-(MAH-DOX-coRh6GEAm)) prodrug micelles was observed from the TEM with diameter of about 50 nm (Figure 3a). While in its aqueous dispersion, its hydrodynamic diameter (Dh) was in the range of 50-92 nm under different pH values (Figure 3b). The small diameter of the obtained prodrug micelle is favorable for the EPR effect.39 Therefore, it could accumulate in the tumor tissue and improve the therapeutic efficiency. Furthermore, its Dh decreased with the increase of the media pH value, due to the hydrolysis of the acid-cleavable acylhydrazone bonds in acidic condition.40 c

b 100

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Dh (nm)

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60 40 20

50 0 1

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pH

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Intensity

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Intensity

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Figure 3. TEM images of the P(PEGMA-b-(MAH-DOX-co-Rh6GEAm)) prodrug micelles formed in neutral solution (a); Variation of hydrodynamic diameter of the P(PEGMA-b-(MAHDOX-co-Rh6GEAm)) prodrug micelles as a function of media pH in 0.10 mg/mL aqueous solution after stirring for 24 h (b); DLS results of 0.10 mg/mL P(PEGMA-b-(MAH-coRh6GEAm)) aqueous solution (pH 6.0) (c), and the P(PEGMA-b-(MAH-DOX-co-Rh6GEAm)) prodrug micelles as a function of stirring time in 0.1 mg/mL aqueous solution (pH 6.0) for 0 h (d), 6 h (e) and 12 h (f). To reveal the drug release in acidic media, the aqueous solution of the P(PEGMA-b-(MAHco-Rh6GEAm)) copolymer (0.10 mg/mL) and the P(PEGMA-b-(MAH-DOX-co-Rh6GEAm)) prodrug micelles (0.10 mg/mL) at pH 6.0 were subjected to the DLS analysis. The Dh of the P(PEGMA-b-(MAH-co-Rh6GEAm)) copolymer was about 5 nm due to its water-solubility (Figure 3c). After conjugating the hydrophobic drug (DOX), an amphiphilic prodrug, the P(PEGMA-b-(MAH-DOX-co-Rh6GEAm)), was obtained, which formed mono-dispersed micelles with Dh of 67 nm (Figure 3d). After 6 h in the acidic media, its Dh was nearly same, indicating that the prodrug could keep its micellar shape within 6 h (Figure 3e). As for 12 h, the Dh decreased to 1.4 nm (Figure 3f). The result demonstrated that the hydrazone bonds between the copolymer and DOX had been hydrolyzed and the conjugated DOX had been release. Thus, the prodrug micelles transformed into water-soluble P(PEGMA-b-(MAH-co-Rh6GEAm)) copolymer. So it is expected that the P(PEGMA-b-(MAH-DOX-co-Rh6GEAm)) prodrug can release the conjugated DOX effectively in the acidic tumor microenvironment, and more importantly, the water-soluble copolymer with such small diameter produced after the drug release could be eliminated or metabolized easily.


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In vitro controlled release. The release of DOX from the P(PEGMA-b-(MAH-DOX-coRh6GEAm)) prodrug was researched at three various buffer solutions (pH 7.4, 6.0, and 5.0) at 37 °C, respectively. The results revealed that drug release in moderate acidic solution was dramatically faster than that in neutral condition (Figure 4). For instance, the cumulative release ratio was calculated to be 73.44% and 41.71% at pH 5.0 and pH 6.5 within 72 h, respectively. In contrast, only 13.12% of DOX was released in pH 7.4 solution. Obviously, the drug release ratio increased with the decrease of the pH value of the releasing media. The prodrug exhibited an ascendant release ratio in acidic condition; meanwhile, it was stable and had a low drug release in a neutral pH condition. This is due to the acid-cleavable acylhydrazone linkage was stable in neutral condition while it would be hydrolyzed in acid media.40 The result revealed that the P(PEGMA-b-(MAH-DOX-co-Rh6GEAm)) prodrug was adequately stable in physiological conditions such as in blood circulation, while rapidly activable in acidic environment, such as endo/lysosomal organelle.

70

Cumulative release/%

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pH7.4 pH6.5 pH5.0

60 50 40 30 20 10 0 0

1000

2000

3000

4000

Time/min

Figure 4. DOX release from the P(PEGMA-b-(MAH-DOX-co-Rh6GEAm)) prodrug in different media (pH=7.4, 6.5 and 5.0) at 37 °C.


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The Higuchi and Korsmeyer-Peppas equation models were used to simulate and analyze the DOX release behavior in different media (Figure S4). Parameters which obtained by these equation models were summarized in Table 1. It was easy to discover the linear coefficient which fitted by equation models were relative ideal. All the R2 values were higher than 0.9. And the k value at pH 5.0 solution was higher than 1. According to Higuchi theory, the DOX release behavior in this condition was diffusion-controlled release while the others were non-diffusioncontrolled release.41,42 From the Korsmeyer-Peppas models, the releasing at the other two solutions was nonFickian diffusion due to their n values were above 0.5.43 Table 1. DOX release data fitted in Higuchi and Korsmeyer-Peppas models. Models

Paramaters

5.0

6.5

Higuchi

7.4

R2

0.9636

0.9698

0.9313

k

1.2620

0.8172

0.2148

Korsmeyer-

R2

-

0.9769

0.9598

Peppas

n

-

0.8464

2.0629

Cytotoxicity. The cell toxicity of the P(PEGMA-b-(MAH-co-Rh6GEAm)) block copolymer and the P(PEGMA-b-(MAH-DOX-co-Rh6GEAm)) prodrug was investigated in HepG2 cells by using MTT assays. As shown in Figure 5a, the P(PEGMA-b-(MAH-co-Rh6GEAm)) block copolymer showed an excellent biocompatibility with the concentration ranged from 0.02 to 0.1 mg/mL, with a cell viability of 92.16-83.99% within 48 h. While the P(PEGMA-b-(MAH-DOXco-Rh6GEAm)) prodrug was added with the concentration up to 0.1 mg/mL, the cell viability ratio decreased to 22.62%. To evaluate the potency of the prodrug as appropriate drug carriers, a


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comparison of the prodrug and free DOX was investigated by the corresponding concentration of DOX. The anticancer activity of the P(PEGMA-b-(MAH-DOX-co-Rh6GEAm)) prodrug was close to the free DOX in HepG2 cells (Figure 5b), indicating that the P(PEGMA-b-(MAH-DOXco-Rh6GEAm)) prodrug can effectively inhibit the growth of cancer cells.44 These results suggested that such kind of prodrug had better cytocompatibility and higher anticancer activity.

a

b

P(PEGMA-b-(MAH-co-Rh6GEAm)) P(PEGMA-b-(MAH-DOX-co-Rh6GEAm))

100

60

Free DOX P(PEGMA)-b-PMAH-b-PR6GEM-DOX

50 80

Cell viability (%)

Cell viability (%)

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60 40 20

40 30 20 10 0

0 0

20

40

60

80

100

concentration (µg/mL)

120

0

2

4

6

8

10

12

14

16

concentration of DOX dosage (µg/mL)

Figure 5. Cell viability assay of the P(PEGMA-b-(MAH-co-Rh6GEAm)) copolymer and the P(PEGMA-b-(MAH-DOX-co-Rh6GEAm)) prodrug (a), and the P(PEGMA-b-(MAH-DOX-coRh6GEAm)) prodrug and free DOX by the corresponding concentration of DOX (b) in HepG2 cells by the MTT assay for 48 h. Cellular uptake. The cellular uptake of the P(PEGMA-b-(MAH-DOX-co-Rh6GEAm)) prodrug micelles was investigated by fluorescence microscope for HepG2 cells (Figure 6). The cell nucleus were dyed with Hoechst, the color was blue. After HepG2 cells were incubated in 24well plate (1 × 105 cells/well), and hatched for 12 h, the cells were incubated in 100 µL of 20.0 µg/mL PBS dispersion of the P(PEGMA-b-(MAH-DOX-co-Rh6GEAm)) prodrug micelles. After 12 h of incubation, the culture medium was discarded and the cells were rinsed two times with anterior PBS solution to clean the fluorescence residuum, dyed with Hoechst 33258 and fixed


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with 4% paraformaldehyde. Therefore, the location of intracellular fluorescence was confirmed by using by fluorescence microscope with excitation wavelengths of 480 nm for DOX and 405 nm for Hoechst. It is obvious that the strong DOX fluorescence was emerged in the cells after 12 h incubation with the P(PEGMA-b-(MAH-DOX-co-Rh6GEAm)) prodrug micelles. It was illustrated that the reversible acylhydrazone linkage was cleaved in acid medium and hydrolyzed in cell nucleus, so the DOX was rapidly released inside cells. From the fluorescence images, it is definite that DOX has been effectively released from the prodrug. Observably, the fluorescence microscope analysis showed that most of the released DOX had been conveyed into the cell nucleus after 12 h incubation (Figure 6c). In addition, the fluorescence microscope analysis proved that the the P(PEGMA-b-(MAH-DOX-co-Rh6GEAm)) prodrug micelles were successfully transported into HepG2 cells and the DOX was released from the prodrug and mainly accumulated in cell nucleus.45,46

Figure 6. Cellular uptake of HepG2 cells stained by Hoechst, the P(PEGMA-b-(MAH-DOX-coRh6GEAm)) prodrug micelles (20 µg/mL) using HepG2 cells after 12 h by fluorescence


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microscope. From left to right, cell nuclei images was bright filed (a), stained by Hoechst (b), DOX fluorescence in cells (c), and merged images (d).

CONCLUSIONS In summary, novel pH stimuli-responsive block copolymer of P(PEGMA-b-(MAH-coRh6GEAm))

was designed by two-step sequential RAFT polymerization by using MAO,

PEGMA and Rh6GEAm as monomers. Due to the existence of Rh6GEAm, the fluorescence behavior of the copolymer are different in acid or neutral condition, it was very strong in acid condition while quenched in neutral or alkaline solution. DOX was conjugated onto the copolymer via acid-cleavable acylhydrazone bond between the carbonyl group of DOX and abundant hydrazide functional group of the copolymer, with content of DOX in prodrug of 0.156 mg/mg. The P(PEGMA-b-(MAH-DOX-co-Rh6GEAm)) prodrug could easily form micelle in aqueous solution (5 mg/mL) by stirring without any organic solvent with CMC of 1.0233×10-3 mol/L. Its regular sphere morphology of the micelles was observed from the TEM with diameter of about 50 nm, and Dh was in the range of 50-92 nm under different pH media. It released 73.44% of the conjugated DOX at pH 5.0 within 72 h, while the release ratio of DOX was only 13.12% in neutral environment. The in vitro DOX release experiment revealed that the designed prodrug was a potential pH stimuli-responsive drug delivery system for anti-cancer drugs, with low drug leakage in normal tissues. After the pH-triggered intracellular release of DOX, the prodrug micelles transformed into water-soluble copolymer P(PEGMA-b-(MAH-co-Rh6GEAm)), making them eliminated or metabolized easily. The cytocompatibility indicated that the P(PEGMA-b-(MAH-co-Rh6GEAm)) didn’t have toxicity, while the P(PEGMA-b-(MAH-DOXco-Rh6GEAm)) prodrug can effectively inhibit the growth and proliferation of tumor tissues. The


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fluorescence microscope analysis also revealed the conjugated DOX could be released and mainly accumulated in cell nucleus. These results indicate that such block copolymer-conjugated prodrug has the potential application for the targeting delivery and tumor micro-environment responsive controlled release of anti-tumor drugs, as well as the real-time fluorescent imaging of tumor tissues.

ASSOCIATED CONTENT The FT-IR spectra of the P(PEGMA-b-(MAO-co-Rh6GEAm) and P(PEGMA-b-(MAH-coRh6GEAm)) block copolymers, GPC curve, determination of the CMC and drug release models. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author. * Corresponding Author. Tel./Fax: 86 0931 8912582. Email: [email protected]. Notes. The authors declare no competing financial interest.

ACKNOWLEDGMENTS This project was granted financial support from the National Nature Science Foundation of China (Grant No. 20904017) and the Program for New Century Excellent Talents in University (Grant No. NCET-09-0441).

REFERENCES


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(1) Minko, T.; Kopeckova, P.; Pozharov, V.; Kopecek, J. J. Control Release 1998, 54, 223233. (2) Colin de Verdibre, A.; Dubernet, C.; Nematil, E.; Poupon, M. F.; Puisieuxl, E.; Couvreurl, P. Cancer Chemoth. Pharm. 1994, 33, 504-508. (3) Prato, M.; Kostarelos, K.; Bianco, A. Acc. Chem. Res. 2008, 41, 60-68. (4) Paul, D. Curr. Pharm. Des. 2009, 15, 153-172. (5) Kita, K.; Dittrich, C. Exp. Opin. Drug Deliv. 2011, 8, 329-342. (6) Pan, Y.; Chen, Y.; Wang, D.; Wei, C.; Guo, J.; Lu, D.; Chu, C.; Wang, C. Biomaterials 2012, 33, 6570-6579. (7) Zhang, X.; Meng, L.; Lu, Q.; Fei, Z.; Dyson, P. J. Biomaterials 2009, 30, 6041-6047. (8) Yuan, W.; Zou, H.; Guo, W.; Shen, T.; Ren, J. Polym. Chem. 2013, 4, 2658-2661. (9) Ding, J.; Chen, J.; Li, D.; Xiao, C.; Zhang, J.; He, C.; Zhuang, X.; Chen, X. J. Mater. Chem. B 2013, 1, 69-81. (10) Bae, Y.; Nishiyama, N.; Fukushima, S.; Koyama, H.; Yasuhiro, M.; Kataoka, K. Bioconjug. Chem. 2005, 16, 122-130. (11) Hu, X.; Liu, S.; Huang, Y.; Chen, X.; Jing, X. Biomacromolecules 2010, 11, 2094-2102. (12) Bae, Y.; Fukushima, S.; Harada, A.; Kataoka, K. Angew. Chem. Int. Ed. 2003, 42, 46404643. (13) Zhou, K.; Liu, H.; Zhang, S.; Huang, X.; Wang, Y.; Huang, G.; Sumer, B. D.; Gao, J. J. Am. Chem. Soc. 2012, 134, 7803-7811. (14) Lv, H.; Liu, J.; Zhao, J.; Zhao, B.; Miao, J. Sensor Actuat. B 2013, 177, 956-963. (15) Bae, Y.; Nishiyama, N.; Kataoka, K. Bioconjug. Chem. 2007, 18, 1131-1139. (16) Yoo, H. S.; Lee, E. A.; Park, T. G. J. Control Release 2002, 82, 17-27.


22

Page 23 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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(17) Vetvicka, D.; Hruby, M.; Hovorka, O.; Etrych, T.; Vetrik, M.; Kovar, L.; Kovar, M.; Ulbrich, K.; Rihova, B. Bioconjug. Chem. 2009, 20, 2090-2097. (18) Hruby, M.; Konak, C.; Ulbrich, K. J. Control Release 2005, 103, 137-148. (19) Zhan, F.; Chen, W.; Wang, Z.; Lu, W.; Cheng, R.; Deng, C.; Meng, F.; Liu, H.; Zhong, Z. Biomacromolecules 2011, 12, 3612-3620. (20) Arruebo, M.; Fernández-Pacheco, R.; Ricardo Ibarra, M.; Santamaría, J. Nano Today 2007, 2, 22-32. (21) Chen, Y.; Ai, K.; Liu, J.; Sun, G.; Yin, Q.; Lu, L. Biomaterials 2015, 60, 111-120. (22) Zhao, X.; Liu, P. Mol. Pharmaceut. 2014, 11, 1599-1610. (23) Lin, J.; Li, Y.; Li, Y.; Wu, H.; Yu, F.; Zhou, S.; Xie, L.; Luo, F.; Lin, C.; Hou, Z. ACS Appl. Mater. Interface 2015, 7, 11908-11920. (24) Knop, K.; Hoogenboom, R.; Fischer, D.; Schubert, U. S. Angew. Chem. Int. Ed. 2010, 49, 6288-6308. (25) Hubbell, J. A.; Chilkoti, A. Science 2012, 337, 303-305. (26) Hu, J.; Zhang, X.; Wang, D.; Hu, X.; Liu, T.; Zhang, G.; Liu, S. J. Mater. Chem. 2011, 21, 19030-19038. (27) Yu, K.; Li, K.; Hou, J.; Yang, J.; Xie, Y.; Yu, X. Polym. Chem. 2014, 5, 5804-5812. (28) Hu, J.; Li, C.; Liu, S. Langmuir 2010, 26, 724-729. (29) He, G.; Guo, D.; He, C.; Zhang, X.; Zhao, X.; Duan, C. A Angew. Chem. Int. Ed. 2009, 48, 6132-6135. (30) Wang, Y.; Wu, H.; Luo, J.; Liu, X. React. Funct. Polym. 2012, 72, 169-175. (31) Marsh, A.; Khan, A.; Haddleton, D. M.; Hannon, M. J. Macromolecules 1999, 32, 87258731. (32) Lai, J. T.; Filla, D.; Shea, R. Macromolecules 2002, 35, 6754-6756.


23

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 25

(33) Wang, X.; Wang, L.; Yang, S.; Zhao, H.; Liu, L. Polym. Chem. 2014, 5, 4797-4804. (34) Grotzky, A.; Manaka, Y.; Kojima, T.; Walde, P. Biomacromolecules 2011, 12, 134-144. (35) Binauld, S.; Scarano, W.; Stenzel, M. H. Macromolecules 2012, 45, 6989-6999. (36) Deng, G.; Tang, C.; Li, F.; Jiang, H.; Chen, Y. Macromolecules 2010, 43, 1191-1194. (37) Wang, X.; Canary, J. W. Bioconjug. Chem. 2012, 23, 2329-2334. (38) Aguiar, J., Carpena, P., Molina-Bolívar, J., Carnero Ruiz, C. J. Colloid Interface Sci. 2003, 258, 116-122. (39) Fang, J.; Nakamura, H.; Maeda, H. Adv. Drug Deliv. Rev. 2011, 63, 136-151. (40) Zhu, L.; Tu, C.; Zhu, B.; Su, Y.; Pang, Y.; Yan, D.; Wu, J.; Zhu, X. Polym. Chem. 2011, 2, 1761-1768. (41) Siepmann, J., Peppas, N. Int. J. Pharm. 2011, 418, 6-12. (42) Kalia, Y., Guy, R. Adv. Drug Delivery Rev. 2001, 48, 159-172. (43) Costa, P., Sousa, L. Eur. J. Pharm. Sci. 2001, 13, 23-133. (44) Kim, E.; Jung, Y.; Choi, H.; Yang, J.; Suk, J.; Huh, Y.; Kim, K.; Haam, S. Biomaterials 2010, 31, 4592-4595. (45) Shi, M.; Ho, K.; Keating, A.; Shoichet, M. S. Adv. Funct. Mater. 2009, 19, 1689-1696. (46) Li, Y.; Zhu, L.; Liu, Z.; Cheng, R.; Meng, F.; Cui, J.; Ji, S.; Zhong, Z. Angew. Chem. Int. Ed. 2009, 48, 9914-9918.


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For Table of Contents Use Only

Fluorescent copolymer-based prodrug for pHtriggered intracellular release of DOX Xu Jia, Xubo Zhao, Kun Tian, Tingting Zhou, Jiagen Li, Ruinian Zhang, and Peng Liu*


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Fluorescent Copolymer-Based Prodrug for pH-Triggered Intracellular

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