Aptamer-Functionalized and Backbone Redox-Responsive

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Aptamer-Functionalized and Backbone Redox-Responsive Hyperbranched Polymer for Targeted Drug Delivery in Cancer Therapy Yuanyuan Zhuang, Hongping Deng, Yue Su, Lin He, Ruibin Wang, Gangsheng Tong, Dannong He, and Xinyuan Zhu Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00262 • Publication Date (Web): 25 Apr 2016 Downloaded from http://pubs.acs.org on May 2, 2016

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Aptamer-Functionalized and Backbone RedoxResponsive Hyperbranched Polymer for Targeted Drug Delivery in Cancer Therapy Yuanyuan Zhuang,† Hongping Deng,† Yue Su,†,* Lin He,‡ Ruibin Wang,‡ Gangsheng Tong,‡ Dannong He,§,* Xinyuan Zhu†,*

†

School of Chemistry and Chemical Engineering, Shanghai Key Lab of Electrical

Insulation and Thermal Aging, Shanghai Jiao Tong University,800 Dongchuan Road, Shanghai 200240, People’s Republic of China ‡

Instrumental Analysis Center, Shanghai Jiao Tong University, 800 Dongchuan Road,

Shanghai 200240, People’s Republic of China §

National Engineering Research Center for Nanotechnology, 28 East Jiang Chuan Road,

Shanghai 200241, People’s Republic of China

* Corresponding authors. E-mail: [email protected] (Y.S.); [email protected] (D.H.); [email protected] (X.Z.). Telephone: +86-21-54746215. Fax: +86-21-54741297.

ACS Paragon Plus Environment

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ABSTRACT: A novel type of backbone redox-responsive hyperbranched poly(2-((2(acryloyloxy)ethyl)disulfanyl)ethyl

4-cyano-4-(((propylthio)carbonothioyl)-thio)-

pentanoate-co-poly(ethylene glycol) methacrylate) (HPAEG) has been designed and prepared successfully via the combination of reversible addition-fragmentation chaintransfer (RAFT) polymerization and self-condensing vinyl polymerization (SCVP). Owing to the existence of surface vinyl groups, HPAEG could be efficiently functionalized by DNA aptamer AS1411 via Michael addition reaction to obtain an active tumor targeting drug delivery carrier (HPAEG-AS1411). The amphiphilic HPAEG-AS1411 could form nanoparticles by macromolecular self-assembly strategy. Cell Counting Kit-8 (CCK-8) assay illustrated that HPAEG-AS1411 nanoparticles had low cytotoxicity to normal cell line. Flow cytometry and confocal laser scanning microscopy (CLSM) results demonstrated that HPAEG-AS1411 nanoparticles could be internalized into tumor cells via aptamer-mediated endocytosis. Compared with pure HPAEG nanoparticles, HPAEG-AS1411 nanoparticles displayed enhanced tumor cell uptake. When the HPAEG-AS1411 nanoparticles loaded with anticancer drug doxorubicin (DOX) were internalized into tumor cells, the disulfide bonds in the backbone of HPAEG-AS1411 were cleaved by glutathione (GSH) in the cytoplasm, so that DOX was released rapidly. Therefore, DOX-loaded HPAEG-AS1411 nanoparticles exhibited a high tumor cellular proliferation inhibition rate and low cytotoxicity to normal

cells.

This

aptamer-functionalized

and

backbone

redox-responsive

hyperbranched polymer provides a promising platform for targeted drug delivery in cancer therapy.


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INTRODUCTION Over past decades, the drug delivery systems have been widely utilized for cancer chemotherapy. As a promising drug delivery vehicle, polymeric nanoparticles exhibit unique advantages, such as improved water solubility of hydrophobic anticancer drug, prolonged circulation time, and enhanced accumulation in tumor tissues by enhanced permeation and retention (EPR) effect.1-5 Although polymeric nanoparticles possess a number of advantages, there are still several important challenges to face, such as low blood circulation stability, inefficient cell internalization and poor intracellular drug release behavior.6-8 To address the issues mentioned above, considerable efforts have been made to improve the therapy effect. In recent years, in order to enhance the cell internalization efficiently, the design and development of active tumor targeting polymeric drug delivery systems have received tremendous attention.4,9-11 Various ligands, such as antibodies, peptides, aptamers, folic acid, and small molecules, have been used for constructing active targeting ligand-decorated polymeric drug delivery system.12-15 These ligands could bind to specific receptors which are overexpressed on cancer cells, leading to receptor-mediated endocytosis and improve their cell internalization. Among these ligands, aptamers have attracted more and more attention because of their small size, remarkable affinity, high in vivo stability and easy synthesis.16-18 For example, Tan et al. have reported aptamer-drug conjugates, which could be efficiently internalized into target cancer cells.19,20 Langer et al. have reported that aptamer-modified and drugloaded block copolymer PEG-PLGA nanoparticles have specific binding to the


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membrane proteins on the tumor cells, selectively killing tumor cells.21,22 When the polymeric nanoparticles were internalized by receptor-mediated endocytosis, it is more desirable to accomplish rapid drug release, to enhance the therapy effect and reduce drug resistance. In this regard, tremendous efforts have been directed to explore various stimuli-responsive polymeric nanoparticles for efficient anticancer drug release in tumor cells. These polymeric nanoparticles will disassemble in respond to a range of external stimuli such as reduction/oxidation, pH, and temperature change, and realize a controlled drug release in the tumor cells.23-25 Due to the dramatic concentration gradient of reducing glutathione (GSH) between cytoplasm (2-10 mM) and blood plasma (2-20 μM),26,27 it is efficient to design and fabricate disulfide-contained redox-responsive polymer for anticancer drug delivery. Under low concentration of GSH in the blood plasma, the disulfide-contained polymer is stable. Once in the tumor sites, this disulfide bonds of the polymer will be broken rapidly by the high level of GSH, resulting in the burst drug release.28,29 Therefore, an active targeting aptamer-functionalized polymeric drug carrier with disulfide bonds in its backbone could disassemble in reducing environment to achieve a redox-triggered release of anticancer drugs. Up to now, most of aptamer-functionalized and disulfidecontained polymeric nanoparticles are prepared by linear polymers. However, the conventional linear polymers only possess limited terminal groups, which will limit their drug loading ability and further functionalization. Compared with conventional linear polymers, hyperbranched polymers possess many inner cavities for encapsulating small molecules and a large number of the terminal groups for further


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functionalization.30-32 Therefore, it can be imagined if the polymeric nanoparticles for anticancer drug delivery were prepared by hyperbranched polymers with disulfide bonds in their backbones and aptamer-functionalized terminal groups, an active tumor targeting backbone redox-responsive polymeric drug delivery system with high drug payload could be generated. In this work, we developed a facile way to obtain aptamer-functionalized backbone redox-responsive hyperbranched polymers (HPAEG-AS1411) by a combination of reversible addition-fragmentation chain-transfer (RAFT) polymerization and selfcondensing vinyl polymerization (SCVP) followed by Michael addition reaction. Here, we designed and synthesized a vinyl and disulfide contained RAFT chain transfer monomer

2-((2-(acryloyloxy)ethyl)disulfanyl)ethyl

(((propylthio)carbonothioyl)thio)-pentanoate

(ACPP),

which

4-cyano-4was

further

copolymerized with poly(ethylene glycol) methacrylate (PEGMA) by one-pot RAFT reaction. Correspondingly, the redox-responsive hyperbranched polymers (HPAEGs) were successfully synthesized. After the terminal vinyl groups were functionalized with AS1411 aptamer, the active tumor targeting redox-responsive drug delivery carrier HPAEG-AS1411 was prepared. AS1411 is a 26-mer DNA oligonucleotides, which could bind specifically to the overexpressed nucleolin receptors on the cancer cells.3335

Such an amphiphilic structure enabled the HPAEG-AS1411 to self-assemble into

polymeric nanoparticles and encapsulate anticancer drug DOX efficiently. When the nanoparticles were internalized into tumor cells, the disulfide bonds in the backbone of HPAEG-AS1411 were cleaved by GSH in the cytoplasm to release DOX rapidly. The


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DOX-loaded HPAEG-AS1411 nanoparticles exhibited a high anticancer efficiency. EXPERIMENTAL SECTION Materials. PEGMA (~360 g/mol) was purchased from Sigma-Aldrich and purified by passing through a column of basic aluminum oxide. 1-Propanethiol (99%) was obtained from TCI Development Co., Ltd (Shanghai). 2,2’-Azobis(isobutyronitrile) (AIBN) was recrystallized twice from ethanol. 4,4’-Azobis(4-cyanovaleric acid) (98%) and acryloyl chloride(96%) were purchased from Aladdin Industrial Inc. Bis(2hydroxyethyl) disulfide (90%), dicyclohexylcarbodiimide (DCC) (99%) and 4dimethylaminopyridine (DMAP) (99%) were purchased from Alfa Aesar. Dimethyl sulfoxide (DMSO), N,N-dimethyl-formamide (DMF), triethylamine (TEA) and dichloromethane (CH2Cl2) (A.R. grade, Shanghai Chemical Reagent Co. Ltd.) were dried over calcium hydride and then purified by vacuum distillation. Aptamer AS1411 (5’-GGT GGT GGTGGT TGT GGT GGTGGTGGT TT-C3-SH-3’; Mw = 9339 Da) was purchased from Shanghai Sangon Bioengineering Technology and Services Co. Ltd. GSH, glutathione monoester (GSH-OEt), and buthionine sulfoximin (BSO) were obtained from Sigma. Human endostatin was purchased from PeproTech (USA). Trizma hydrochloric acid (Tris-HCl) (pH8.0) and CCK-8 were received from Beyotime Biotechnology Corporation. Unless mentioned, all other materials and solvents were commercially purchased and used as received. Characterization. Nuclear Magnetic Resonance (NMR). The 1H NMR,

13

C NMR

and 13C,1H-HSQC spectra were recorded using Bruker AVANCEIII 400 spectrometer with CDCl3 as solvents.


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Gel Permeation Chromatography (GPC). The number-average molecular weights (Mn) and the polydispersity indices (Mw/Mn) were determined by gel permeation chromatography/multi-angle laser light-scattering (GPC-MALLS). The GPC system consisted of a Waters degasser, a Waters 515 HPLC pump, a 717 automatic sample injector, a Wyatt Optilab DSP differential refractometer detector, and a Wyatt miniDAWN multi-angle laser light-scattering detector. Three chromatographic columns (styragel HR3, HR4, and HR5) were used in series. THF was used as the mobile phase at a flow rate of 1 mL/min at 30 °C. The refractive index increment dn/dc was determined with a Wyatt Optilab DSP differential refractometer at 690 nm. Data analysis was performed with Astra software (Wyatt Technology). Fourier Transform Infrared Spectroscopy (FTIR). FTIR spectra were measured by a Perkin-Elmer Paragon 1000instrument in the range of 4000-400 cm-1. All the samples were prepared by KBr holder method. Dynamic Light Scattering (DLS). The average diameter, size distribution and zeta potential of the nanoparticles were performed with a Zetasizer Nano-ZS90 (Malvern Instrument Ltd.). All the samples were tested at a scattering angle of 90°, laser operating at 633 nm. Transmission Electron Microscopy (TEM). TEM studies were performed with a JEOL JEM-100CX-II instrument at a voltage of 200 kV. A drop of aqueous sample solution was spread onto carbon-coated copper grids. The grids were allowed to air-dry at room temperature before tests. Synthesis of 4-Cyano-4-(propylsulfanylthiocarbonyl) Sulfanylpentanoic Acid,


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CPP (1). CPP was synthesized according to the literature.36 1H NMR was shown in the Supporting Information, Figure S1. CPP: 1H NMR (400 MHz, CDCl3) (ppm) = 3.31 (t, 2H, -CH2-S-), 2.70-2.30 (m, 4H,-CH2-CH2-), 1.89 (s, 3H, CH3-), 1.74 (m, 2H, CH3CH2-CH2-), 1.03 (t, 3H, CH3-). Synthesis of 4-Cyano-4-(propylsulfanylthiocarbonyl) Sulfanylpentanoic Acid Hydroxyethyl Disulfide Ester, CPPE (2). In a 100 mL one-neck round-bottom flask equipped with a magnetic stirring bar, bis(2-hydroxyethyl) disulfide (1.67 g, 10.8 mmol), DCC (0.891 g, 4.32 mmol) and DMAP (0.040 g, 0.36 mmol) were dissolved in 40 mL of CH2Cl2. With stirring, the solution became homogenized. Then the flask was placed into an ice bath. CPP (1.0 g, 3.6 mmol) was added dropwise through a syringe with vigorous stirring. After 24 h of stirring, the precipitate was filtered off. The solution was dried by rotary evaporation after being filtered. The crude product was purified by column chromatography on silica gel with hexane/ethyl acetate (1:1) to obtain CPPE (2) as a yellow solid (0.91 g, 88.6%). 1H NMR was shown in the Supporting Information, Figure S2. CPPE: 1H NMR (400 MHz, CDCl3) (ppm) = 4.40 (t, 2H, -CH2-O-), 3.90 (t, 2H, -CH2-OH), 3.31 (t, 2H, -CH2-S-), 2.94 (t,2H, HO-CH2CH2-S-), 2.89 (t,2H, -O-CH2-CH2-S-), 2.70-2.30 (m, 4H, -CH2-CH2-), 1.89 (s, 3H,CH3-), 1.74 (m, 2H, CH3-CH2-CH2-), 1.03 (t,3H,CH3-). Synthesis

of

Multifunctional

RAFT

Agent

2-((2-(Acryloyloxy)ethyl)-

disulfanyl)ethyl 4-cyano-4-(((propylthio)carbonothioyl)thio)pentanoate (ACPP) (3). ACPP was synthesized by three steps chemical reaction, the synthetic route was shown in Scheme 1. CPPE (0.83 g, 2 mmol) was dissolved in 15 mL CH2Cl2 and cooled


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to 0 °C. Then, dry TEA (0.30 g, 3mmol) was added. Acryloyl chloride (0.27 g, 3 mmol) was added dropwise through a syringe with vigorous stirring and the mixture was kept overnight. Upon completion, the mixture was filtered. After filtration, the solution was concentrated by a rotary evaporator. The crude product was purified by column chromatography on silica gel with hexane/ethyl acetate (3:1) to obtain ACPP as yellow solid (0.88 g, 80.3%). 1H NMR was shown in the Supporting Information, Figure S3. ACPP: 1H NMR (400 MHz, CDCl3)  (ppm) = 6.45 (d, 2H, cis CH2=CH), 5.90 (d, 2H, trans CH2=CH),6.14 (dd, 1H, ROOCCH=CH2), 4.43-4.40 (m, 4H, RCOO-CH2-CH2S-), 3.31 (t, 2H, -CH2-S-), 2.94 (t, 2H, HO-CH2-CH2-S-), 2.89 (t, 2H, -O-CH2-CH2-S-), 2.70-2.30 (m, 4H, -CH2-CH2-), 1.89 (s, 3H,CH3-), 1.74 (m,2H, CH3-CH2-CH2-), 1.03 (t, 3H,CH3-). Synthesis of Hyperbranched Poly(ACPP-co-PEG), HPAEG. HPAEG was prepared by the combination of RAFT polymerization and SCVP. The typical polymerization procedures were as following: PEGMA (1.80 g, 5 mmol), ACPP (0.47 g, 1 mmol), and AIBN (3.2 mg, 0.02 mmol) were added to a round bottom flask with a rubber stopper. DMF (10 mL) was used as solvent. The solution was bubbled with nitrogen for 30 min. The RAFT polymerization was conducted at 70 °C for 20 h. The polymerization was quenched by liquid nitrogen. The residue was precipitated into cold diethyl ether for three times and then the product was purified and dried under vacuum overnight. The purified yellow products were obtained (yield: 78%). HPAEG: 1H NMR (400 MHz, CDCl3, 298 K)  (ppm) = 4.52-3.90 (COO-CH2-CH2-, ACPP and PEGMA units), 3.90-3.36 (CH2, PEGMA unit), 3.29-2.88 (-CH2-CH(CH2)-COO-, ACPP unit),


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2.78-2.70 (CH3CH2CH2S, terminal trithiocarbonate functionality; -CH2-S-S-CH2-, ACPP unit), 2.58-2.32 (-CH2-COO-CH2-, ACPP unit), 0.5-2.2 (CH3, CH2, ACPP and PEGMA units). FTIR (KBr, cm-1): 3449, 2953, 2922, 2863, 1727, 1635, 1452, 1387, 1355, 1251, 1105, 952, 845, 746. Preparation of HPAEG Nanoparticles and Their Critical Micelle Concentration (CMC). Dry and purified HPAEG (10 mg) dissolved in DMF (2 mL) was added dropwise into the deionized water (10 mL). The resulting dispersions were stirred. Then, the solution was purified by dialyzed against deionized water for 24 h (MWCO = 1 kDa). The formation of nanoparticles was confirmed by partitioning of hydrophobic dye 1,6diphenyl-1,3,5-hexatriene (DPH) in the presence of HPAEG. The HPAEG solutions were prepared with different concentrations from 1.0×10-4 mg/mL to 0.8 mg/mL. DPH solution in methanol (5.0 × 10-4 mol/L) was injected into the HPAEG solutions. At the fixed concentration, the absorbance at 313 nm increased with the polymer solution concentration. The CMC value of HPAEG was determined by the inflection point. Preparation of Aptamer-Functionalized HPAEG-AS1411. HPAEG (0.05 g, ~3.6×10-5 mol of OH group) and a little amount of TEA were dissolved in CH2Cl2 (10 mL) and cooled down to 0 oC. Then, acryloyl chloride (AC, 0.001 g, 1.1×10-5 mol) was added with stirring. The solution was kept for 12 h. After the reaction finished, the mixture was filtered to remove precipitates and the solution was concentrated by a rotatory evaporator. The crude products were dialyzed against deionized water, and the purified products (HPAEG-AC) were obtained by a freeze-dryer system. 3’-Thiol aptamer AS1411 (180 μL, 100 μM, 1.0 equiv) and HPAEG-AC (~1.4×10-5 mmol of AC


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group, 0.75 equiv) were added in 10 mM Tris-HCl (pH 8.0) and stirred for 12 h. The resulting products were washed with 10 mM PBS (pH 7.4) using an Amicon Ultra-4 centrifugal filter (3 K) to obtained HPAEG-AS1411. Preparation of DOX-Loaded HPAEG-AS1411 Nanoparticles and In Vitro Drug Release. 5 mg DOX·HCl was dissolved in 2 mL DMF solution, and the equimolar amount of TEA was added. After the mixture was stirred for 2 h, the resulting solution was added to DMF solution of HPAEG-AS1411 (10 mg/mL) with stirring. The mixture was stirred for 2 h at room temperature. Thereafter, the solution was added dropwise to ultrapure water (10 mL) with stirring and then transferred into the dialysis membrane (MWCO = 1,000 Da). The sample was dialysis against ultrapure water for 48 h. Followed by lyophilization, DOX-loaded HPAEG-AS1411 nanoparticles were obtained. The drug loading contents (DLC) were determined by UV-Vis spectroscopy at 485 nm. The DLC and drug loading efficiency (DLE) were calculated according to the following formulas: DLC (wt%) = (weight of loaded drug / weight of polymer) × 100% DLE (wt%) = (weight of loaded drug / weight of feed) × 100% The DLC and DLE of DOX-loaded HPAEG-AS1411 nanoparticles were determined to be 3.96% and 49.30%, respectively. The DOX-loaded HPAEG-AS1411 nanoparticles were dispersed in PBS (50 mM, pH = 7.4), and then transferred into dialysis membrane tubing (MWCO = 1 kDa). The dialysis membrane tubes were immersed into PBS solutions with or without GSH (10 mM) in a 37 °C shaking table at 120 rpm. The PBS buffer was refreshed at fixed time


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intervals. The released amount of DOX was calculated according to the UV-Vis spectroscopy at 485 nm. The Stability of HPAEG-AS1411 Nanoparticles in Reductive Condition. The stability of HPAEG-AS1411 nanoparticles in reductive condition was investigated in PBS buffer (50 mM, pH = 7.4) with 10 mM GSH. The change of nanoparticle size was determined by DLS after 6 h and 12 h. Cell Culture. L929 cell lines (a mousefibroblast cell line) and MCF-7 (a breast cancer cell line) were cultured in DMEM (Dulbecco’s modified Eagle’s medium) supplied with 10% fetal bovine serum (FBS) and antibiotics (50 units/mL penicillin and 50 units/mL streptomycin) in a 5% CO2 atmosphere at 37 °C. Cytotoxicity Measurements of Nanoparticles. The relative cytotoxicity of HPAEG and HPAEG-AS1411 against cultured L929 and MCF-7 cells was evaluated in vitro by CCK-8 assay. L929 and MCF-7 cells were seeded at a density of 1.0×104 cells/well in 96-well plates incubated with 200 μL culture medium. After incubation for 24 h, the culture medium was removed and then 200 μL medium of a serial dilutions of samples was added. After 48 h incubation, 20 μL of CCK-8 was added to each well. After incubating the cells for another 4 h, the absorbance at 450 nm was recorded in a BioTek Elx800. Cellular Uptake Behavior. The experiments of cellular uptake of DOX-loaded HPAEG and HPAEG-AS1411 nanoparticles were performed by flow cytometry and confocal laser scanning microscopy.


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Flow Cytometry. MCF-7 and L929 cells were seeded in 6-well plates at 8.0×105 cells per well in complete DMEM culture medium. After 24 h incubation, the medium was removed. The cells were treated with DOX-loaded HPAEG and HPAEG-AS1411 nanoparticles at a final concentration equivalent to 10 μg/mL of DOX and incubated at 37 °C. The cells incubated for 15 min, 1 h and 4 h, respectively. Thereafter, culture medium was removed and cells were washed in PBS. After trypsin treatment, the cells were collected and resuspended in PBS. Data for 1.0×104 gated events were recorded and analysis by a BD FACSCalibur flow cytometer. Confocal Laser Scanning Microscopy (CLSM). MCF-7 and L929 cells were seeded in 6-well plates at 2.0×105 cells per well. DOX-loaded HPAEG and HPAEG-AS1411 nanoparticles dissolved in DMEM culture medium at a final DOX concentration of 10 μg/mL were added to the wells and the cells were incubated at 37 °C for 15 min, 1 h and 4 h. After PBS treatment, the cells were fixed with 4% formaldehyde for 30 min. After rinsing with PBS for three times, the cells were stained with Hoechst 33342. Finally, the slides were observed by a LSM510 META. Mechanism of HPAEG-AS1411 Nanoparticles Cell Internalization. In order to investigate the cell internalization mechanism of HPAEG-AS1411 nanoparticles, the MCF-7 cells were treated with 0.1 mg/mL human endostatin and PBS for 1 h, respectively and then incubated with HPAEG-DOX and HPAEG-AS1411-DOX nanoparticles for another 0.5 h at fixed DOX concentration 5 μg/mL. The cells were stained with Hoechst 33342. Finally, the slides were observed by a LSM510 META.


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Cell Viability. The cell viability of MCF-7 and L929 cells incubated with DOXloaded HPAEG and HPAEG-AS1411 nanoparticles was investigated in vitro by CCK8 assay according to the prescribed instructions. MCF-7 and L929 cells were seeded in 96-well plates 1.0×104 cells per well in 200 μL of RPMI 1640 culture medium and incubated for 24 h. GSH-OEt or BSO was used to pretreat MCF-7 cells, respectively. The cells without pretreatment were used as control. After removal of GSH-OEt or BSO, DOX-loaded HPAEG and HPAEG-AS1411 nanoparticles were diluted in PBS (pH = 7.4, 50 mM) (DOX final concentration: 0.1~10 μg/mL). 48 h later, after removal of the cultured medium, the cells were washed with PBS. Then 20 μL of CCK-8 reagent was added to each well. After another 4 h incubation, the absorbance was measured at 450 nmin a BioTek Elx800. Statistics. The results of six individual experiments were expressed as mean ± standard deviation by SPSS 10.0 software using A paired t-test (Student’s t-test). Statistically significant means a p-value less than 0.05. RESULTS AND DISCUSSION Synthesis and Characterization of ACPP. Chain-transfer monomer ACPP was synthesized as shown in Scheme 1. Firstly, CPP (1) was converted into CPPE by reaction with bis(2-hydroxyethyl) disulfide to afford redox-responsive disulfide bonds in the structure of CPPE (2). Then, CPPE was reacted with acryloyl chloride to obtain ACPP (3) as yellow solid. In the 1H NMR spectrum of ACPP (Supporting Information, Figure S3), the signals appear at 6.45 ppm, 6.14 ppm and 5.90 ppm can be assigned to the protons from acryloyl in ACPP.


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Synthesis and Characterization of HPAEG. Since the existence of two different reactive groups in the structure of ACPP, namely, a vinyl group and a trithiocarbonate group, ACPP can be used as both chain-transfer agent and monomer. HPAEG was synthesized by combination of RAFT polymerization and SCVP between the monomer PEGMA and chain transfer monomer ACPP (Scheme 1). Correspondingly, the responsive disulfide bonds were introduced into the backbone of HPAEG. A series of HPAEGs with different feed molar ratios were synthesized (Table 1), and all the possible structural units of HPAEG are listed in Figure 1. The chemical structure of HPAEG was characterized by 1H NMR, 13C NMR and 2D NMR techniques. In the 1H NMR spectrum (Figure 2), the signals at 4.52-3.90 ppm assigned to the protons of CH2 come from ACPP and PEGMA units, which are attached to the ester groups. The signals from 3.90-3.36 ppm are attributed to the other protons of CH2 in PEGMA units. The signals at 2.78-2.70 ppm are ascribed to the protons of CH2 attached to -S-S- groups in ACPP units. Quantitative

13

C NMR and DEPT-135 spectra are shown in Figure 3.

Methylene, methine or methyl carbons of HPAEG could be distinguished by DEPT-135 spectrum. According to the DEPT-135 spectrum, the peaks 1 and 5 are assigned to the methyl groups from ACPP units and the peak 17 is assigned to the methyl groups from PEGMA units. The peaks 14 and 15 are attributed to the methyl and methylene groups which come from double-bond addition reactions of ACPP units. Peaks 19, 13, 28, 8 and 16 are attributed to quaternary carbon atoms, which disappear in the corresponding DEPT-135 spectrum. Besides, peak 16 is assigned to quaternary carbon atoms attached to methyl in PEGMA units.


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2D-NMR technique is used to analyze the topology of polymerized product.37,38 The molecular structure is characterized by

13

C,1H-HSQC spectrum. The assignment of

each structural unit for HPAEG is performed and Figure 4 shows the details. By using quantitative

13

C NMR spectrum, the degree of branching (DB) for HPAEG can be

calculated according to the following equation:39-41 DB = (D + T)/(D + T + L) where D, T, and L represent the fractions of the branched, terminal, and linear units, respectively. Table 1 summarizes the reaction conditions and the DBs of all samples. The DBs of the samples change from 0.35 to 0.06. It demonstrates HPAEGs with different branched architectures have been synthesized successfully.

Scheme 1. Synthetic Route of HPAEG


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Figure 1. Various structure units of HPAEG.


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Figure2. 1HNMR spectrum of HPAEG1 in CDCl3.

Figure3. (a) Quantitative 13C NMR and (b) 13C DEPT-135 NMR spectra of HPAEG1 in CDCl3, the insert spectra are an enlarged view of the spectra in the range of 10 to 75 ppm.


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Figure4. 13C,1H-HSQC spectrum of HPAEG1 in CDCl3.

Table 1. Reaction Conditions and Results of RAFT Polymerization and SCVP of ACPP and PEGMA

a

Samples a

A/Pb

Mn (× 104)c

Mw (× 104) c

PDI

DBd

HPAEG1

1/5

1.4

2.1

1.53

0.35

HPAEG2

1/10

2.2

2.8

1.27

0.14

HPAEG3

1/20

3.4

3.8

1.12

0.06

Polymerization conditions: [ACPP]:[AIBN] = 1:0.02, in DMF at 70 °C, 20 h. b Molar

ratio of monomer ACPP to PEGMA. determined by GPC-MALLS.

d

c

Molecular weights and polydispersity were

Degree of branching (DB) was calculated from

quantitative 13C NMR analysis. The chemical structure information of HPAEG is shown in Figure 5. The broad peak


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around 3449 cm-1 is hydroxyl stretching vibration, while the peaks at 1387, 1355, 1251 cm-1 are hydroxyl in-plane bending vibration from PEGMA units. The bands at 2953, 2922and 2863 cm-1 ascribe to the C-H stretching vibration. A strong carbonyl bands at 1727 and 1635 cm-1 confirm the presence of ester bonds. An intense vibration band at 1105 cm-1 can be assigned to C-O-C stretching vibration. According to GPC measurement, the weight-average molecular weights of HPAEGs increase from 2.1×104 g/mol to 3.8×104 g/mol with the increase of PEGMA/ACPP molar feeding ratio.

Figure5. FTIR spectra of HPAEG. Preparation of HPAEG-AS1411. Due to the existence of hydrophobic disulfide bonds segments and hydrophilic PEGMA segments, HPAEG could self-assemble into nanoparticles in an aqueous solution. DPH was used as hydrophobic probe to investigate the CMC of HPAEG aqueous solutions. The results in Figure S4


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(Supporting Information) show HPAEGs with different architectures self-assemble into nanoparticles and the CMC values are different. The CMC values of HPAEGs increase from 0.041 mg/mL to 0.046 mg/mL. Since HPAEG1 is the most easy to form nanoparticles in an aqueous solution, we selected HPAEG1 to synthesize HPAEGAS1411. The HPAEG-AS1411 was synthesized by end-group modification of HPAEG. HPAEG was first reacted with acryloyl chloride to obtain HPAEG-AC, and then HPAEG-AC was modified with AS1411 aptamer by Michael addition reaction. The 1H NMR spectrum of HPAEG-AC is shown in Figure S5 (Supporting Information). After HPAEG was modified with AS1411 aptamer, the product HPAEG-AS1411 was characterized by UV-Vis spectroscopy. Compared with the UV-Vis spectroscopy of HPAEG-AC, there is a new absorption peak at around 260 nm in Figure 6, which is attributed to the absorption of base pairs, appeared in the UV-Vis spectroscopy of HPAEG-AS1411. That indicates the AS1411 aptamer has been conjugated to HPAEG successfully. According to the standard absorption curve of pure AS1411 aptamer at 260 nm, we could calculate AS1411-conjugated ratio is 15 wt% in HPAEG-AS1411. AS1411 aptamer conjugation to HPAEG-AC was also confirmed by zeta potential measurement. The zeta potential of HPAEG-AC before AS1411 aptamer modification is 4.99 ± 0.4 mV. After AS1411 aptamer conjugation, the zeta potential of HPAEGAS1411becomes -17.50 ±0.6 mV (Figure S6). Due to the negatively charged phosphate groups of DNA aptamer, the zeta potential of HPAEG-AS1411 shifts to negativity. This result also indicates negatively charged AS1411 aptamer has been conjugated to HPAEG successfully.


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Figure. 6 Normalized UV-Vis spectra of HPAEG-AC, AS1411 and HPAEG-AS1411.

HPAEG-AS1411 and DOX-Loaded HPAEG-AS1411 Nanoparticles and Their Redox Responsiveness. After AS1411 modification, HPAEG-AS1411 could selfassemble into nanoparticles in an aqueous solution. The size of the HPAEGAS1411nanoparticles was characterized by DLS and TEM (Figure 7). DLS result reveals that the hydrodynamic diameter of HPAEG-AS1411 nanoparticles is 80.6 nm, which is in accordance with the TEM result. TEM measurement shows the HPAEGAS1411 aggregates into spherical nanoparticles. Compared with the size of HPAEG nanoparticles, the size of HPAEG-AS1411 nanoparticles become smaller after aptamer modification, which is attributed to the hydrophilicity of aptamer. Both the sizes of HPAEG and HPAEG-AS1411 nanoparticles are smaller than 200 nm, favoring for keeping low reticuloendothelial system (RES) uptake and minimal renal excretion. After loading anticancer drug DOX, the hydrodynamic diameter of DOX-loaded


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HPAEG-AS1411 nanoparticles is 93.7 nm, which was in accordance with the TEM result. The UV-Vis spectroscopy of DOX-loaded HPAEG-AS1411 was shown in Figure S7.

Figure. 7 Characterization of HPAEG and HPAEG-AS1411 nanoparticles: (a) TEM image of HPAEG nanoparticles; (b) The size distribution of HPAEG nanoparticles determined by DLS; (c) TEM image of HPAEG-AS1411 nanoparticles; (d) The size distribution of HPAEG-AS1411nanoparticles determined by DLS; (e) TEM image of


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DOX-loaded HPAEG-AS1411 nanoparticles; (f) The size distribution of DOX-loaded HPAEG-AS1411 nanoparticles determined by DLS.

Due to the existence of disulfide bonds in the structure of HPAEG-AS1411, HPAEGAS1411 nanoparticles could be reductively breakable with responsive to reducing agent. In order to study the responsiveness of HPAEG-AS1411, HPAEG-AS1411 and DOXloaded nanoparticles were treated with 10 mM GSH, respectively. The size of nanoparticles was measured by DLS at determined time. As shown in Figure 8 (a, b), after the HPAEG-AS1411 nanoparticles were treated with 10 mM GSH, the size of nanoparticles gradually decreases from 80 nm to 17 nm with the increase of time, while the DOX-loaded HPAEG-AS1411 nanoparticles exhibited multiplicity distribution after treated with 10 mM GSH for 12 h. In contrast, the size of nanoparticles without GSH treatment had no obvious change. This phenomenon suggests that the nanoparticle disintegration can be attributed to the cleavage of disulfide bonds in the backbone of polymer which lead to the drug release immediately. Since HPAEG-AS1411 nanoparticles could be breakable in reductive environment, it is beneficial to a drug carrier for anticancer drug releases in tumor.


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Figure. 8 DLS plots of HPAEG-AS1411 (a) and DOX-loaded HPAEG-AS1411 (b) nanoparticles with 10 mM GSH in PBS (pH = 7.4, 50 mM) at 37 oC over time.

In Vitro Drug Release. The release behavior of DOX from HPAEG-AS1411 nanoparticles was evaluated under GSH condition (Figure 9). DOX-loaded HPAEGAS1411 nanoparticles were treated with 5 mM and 10 mM GSH in PBS at 37 °C respectively. As a control, the drug release was also carried out in PBS without GSH. As shown in Figure 9, the cumulative release of DOX from HPAEG-AS1411 nanoparticles without GSH treatment is only 10% after 60 h, while the cumulative release of DOX from HPAEG-AS1411 nanoparticles with 10 mM GSH treatment is up to 90%. On the other hand, compared with the drug release behavior of HPAEGAS1411 nanoparticles with 5 mM GSH treatment, the drug releases from HPAEGAS1411 nanoparticles with 10 mM GSH treatment is obviously accelerated. This might be attributed to the cleavage of the disulfide bonds in the structure of HPAEG-AS1411 triggered by GSH, causing the nanoparticle disintegration, speeding up the drug releases rate.


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Figure 9. Cumulative release curve of DOX from HPAEG-AS1411 nanoparticles after incubation with 5 mM and 10 mM GSH in PBS (pH = 7.4, 50 mM) at 37 °C over time (nanoparticles without treatment of GSH were used as the control). Cell Cytotoxicity. The cytotoxicity of HPAEG and HPAEG-AS1411 against L929 and MCF-7 cells were studied by CCK-8 test. Tetrazolium compound (reagent, WST8) could be reduced by live cells into a colored formazan product. The number of live cells in the culture was directly proportional to the quantity of formazan product measured at 450 nm.42,43 L929 and MCF-7 cells were incubated with HPAEG and HPAEG-AS1411 for 48 h, respectively. As shown in Figure 10, compared with the untreated cells, HPAEG and HPAEG-AS1411 exhibit low cytotoxicity to L929 and MCF-7 cells. Especially, when the concentration of polymer is up to 2.0 mg/mL, most of L929 and MCF-7 cells are still alive after 48 h incubation. These results indicate HPAEG and HPAEG-AS1411 display a low cytotoxicity to L929 and MCF-7 cells and thus are appropriate as drug carriers. The cytotoxicity of the degraded components of


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HPAEG and HPAEG-AS1411 nanoparticles treated with 10 mM GSH were shown in Figure S8. The results demonstrated the degraded components of HPAEG and HPAEGAS1411 nanoparticles were low cytotoxicity.

Figure 10. Cytotoxicity of HPAEG and HPAEG-AS1411 against L929 cells (A) and MCF-7 cells (B) incubation for 48 h with different micelle concentrations. Cell Internalization. The cell internalization of DOX-loaded HPAEG and HPAEGAS1411 nanoparticles by MCF-7 and L929 cells were investigated by flow cytometry analysis. MCF-7 cells are breast cancer cells, which overexpress nucleolin on cell membranes. Nucleolin could provide a target point for drug delivery. L929 cells are normal cells, therefore, they don’t overexpress nucleolin receptor on their cell membranes. As shown in Figure 11, the cell internalization was evaluated by the increase of cell fluorescent intensity. After 15 min incubation with DOX-loaded HPAEG and HPAEG-AS1411 nanoparticles, MCF-7 cells shown red fluorescence of DOX in the cytoplasm. After 4 h incubation, MCF-7 cells incubated with DOX-loaded HPAEG-AS1411 nanoparticles show a two-fold increase in the relative geometrical mean fluorescence intensity compared to the cells incubated with DOX-loaded HPAEG. On the other hand, for L929 cells, the fluorescence of DOX can be observed after 15


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min incubation with DOX-loaded HPAEG and HPAEG-AS1411 nanoparticles. However, after 4 h incubation, the mean fluorescence intensity incubated with DOXloaded HPAEG-AS1411 nanoparticles does not show a multiple increase compared to that of the DOX-loaded HPAEG nanoparticles. This study clearly indicates the highly efficient internalization of HPAEG-AS1411 nanoparticles is probably induced by receptor-mediated endocytosis originating from the targeting ability of AS1411. Confocal laser scanning microscopy (CLSM) was used for further investigating the cellular uptake behaviors of DOX-loaded HPAEG and HPAEG-AS1411 nanoparticles. MCF-7 and L929 cells were cultured with DOX-loaded HPAEG and HPAEG-AS1411 nanoparticles for 15 min, 1 h and 4 h at 37 °C, respectively. Then, the nucleus was stained with Hoechst 33342, and the cellular uptake behavior was observed by CLSM. As shown in Figure 12A, the cell nucleus stained with Hoechst 33342 is in blue color. The red fluorescence of DOX can be observed in the cytoplasm after DOX-loaded HPAEG and HPAEG-AS1411 nanoparticles 15 min incubation. When MCF-7 cells are treated with HPAEG nanoparticles, the image shows that the fluorescence is lower than that of HPAEG-AS1411 nanoparticles. Four hours later, the red fluorescence gradually locates in the perinuclear region of cells. These results suggest that both HPAEG and HPAEG-AS1411 nanoparticles could be internalized into MCF-7 cells. Due to the specific binding between aptamer AS1411 and nucleolin, the fluorescence is significantly enhanced when MCF-7 cells are treated with DOX-loaded HPAEGAS1411 nanoparticles. Normal cell line L929 cell was also studied in the cellular uptake study. As shown in


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Figure 12B, after the cells are incubated with DOX-loaded HPAEG and HPAEGAS1411 nanoparticles for 15 min, the fluorescence of DOX could be observed in the cells. When incubation time is prolonged, the fluorescence is increased. However, the red fluorescence intensity of L929 cells incubated with DOX-loaded HAPAEG-AS1411 nanoparticles is lower than that of DOX-loaded HAPAEG nanoparticles. This phenomenon could be attributed to the repulsion between normal cell membrane and HPAEG-AS1411. Since the cell membrane presents negative charge and the zeta potential of HPAEG-AS1411 is -17.50 ±0.6 mV, HPAEG-AS1411 may be repulsed by cell membrane, which results in the lower fluorescence. Mechanism of HPAEG-AS1411 Nanoparticles Cell Internalization. The different cellular uptake behaviors of DOX-loaded HPAEG and HPAEG-AS1411 nanoparticles might be attributed to the different mechanism of nanoparticles transportation across the cell membrane. Since there is specific binding between aptamer AS1411 and nucleolin, which is overexpressed by MCF-7 cells, we selected human endostatin as a nucleolin blocker to investigate the mechanism of HPAEG-AS1411 nanoparticles across cell membrane. Nucleolin has been reported as a receptor of endostatin. MCF-7 cells were treated with endostatin for 1 h before incubated with DOX-loaded HPAEG and DOX-loaded HPAEG-AS1411 nanoparticles. As shown in Figure 13A, for the PBS treated MCF-7 cells, the fluorescence of cell incubated with DOX-loaded HPAEGAS1411 was higher than that of cells incubated with DOX-loaded HPAEG. For the endostatin treated MCF-7 cells (Figure 13B), the fluorescence of cells incubated with DOX-loaded HPAEG-AS1411 decreased significantly, while the fluorescence of cell


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incubated with DOX-loaded HPAEG nanoparticles were similar with that of endostatin absence. Therefore, we could suppose the main mechanism of HPAEG-AS1411 across cell membrane was nucleolin-mediated cell internalization.

Figure 11. Time-dependent curves of HPAEG-DOX and HPAEG-AS1411-DOX nanoparticles fluorescence intensity in the MCF-7 cells (A), and L929 cells (B) by flow cytometry analysis. Insert: representative flow cytometry histogram profiles of MCF-7 cells and L929 cells incubated with HPAEG-DOX and HPAEG-AS1411-DOX


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nanoparticles for 4 h, theuntreated cells are used as a control.

Figure 12. Confocal laser scanning microscopy (CLSM) images of MCF-7 cells (A) incubated with DOX-loaded HPAEG-AS1411 (a) and HPAEG (b) nanoparticles; L929 cells (B) incubated with DOX-loaded HPAEG-AS1411 (a) and HPAEG (b)


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nanoparticles. The incubation time is 15min, 1 h, and 4 h. Cell nuclei were stained with Hoechest 33342. All the images are merged images.

Figure 13. Confocal laser scanning microscopy images of nucleolin-expressed MCF-7 cells treated with PBS (A), and human endostatin (B) for 1 h, respectively and then incubated with DOX-loaded HPAEG and DOX-loaded HPAEG-AS1411 nanoparticles for another 0.5 h. Cell nuclei were stained with Hoechest 33342. All the images are merged images. Cell Viability Analysis. The cell proliferation inhibitions of DOX-loaded HPAEG and HPAEG-AS1411nanoparticles were determined in the MCF-7 and L929 cell lines


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using CCK-8 assay. The MCF-7 cells and L929 cells (cancerous and noncancerous cell lines, respectively) were incubated with the DOX-loaded HPAEG and HPAEG-AS1411 nanoparticles and free DOX at different DOX concentrations from 0.1 to 10 μg/mL for 48 h. The cell viability is shown in Figure 14. In the case of the L929 noncancerous mouse fibroblast cell line (Figure 14a), CCK-8 assay reveals that the DOX-loaded HPAEG-AS1411 nanoparticles have the weakest effect. The IC50 value of HPAEGAS1411 nanoparticles (3.52 μg/mL) is higher than that of HPAEG nanoparticles (2.27 μg/mL). The difference of IC50 value could be attributed to the different zeta potential value. The HPAEG-AS1411 nanoparticles with negative charge are apt to be repulsed by cell membrane of noncancerous cell lines. Hence, HPAEG-AS1411 nanoparticles exhibit lower cell cytotoxicity. Besides, compared to the free DOX, HPAEG-AS1411 and HPAEG nanoparticles show low cell cytotoxicity to L929 cells. These results indicate polymeric nanoparticles as drug delivery carriers could reduce the side effects of anticancer drugs. In the case of the MCF-7 breast cancer cell line (Figure 14b), the IC50 value of HPAEG-AS1411 nanoparticles (1.33 μg/mL) is lower than that of HPAEG nanoparticles (2.30 μg/mL). Due to more cell internalization via aptamer-mediated endocytosis, DOX-load HPAEG-AS1411 nanoparticles have the better tumor cellular proliferation inhibition effect. Moreover, the IC50 value of HPAEG-AS1411 nanoparticles is closed to the IC50 value of free DOX (1.06 μg/mL). Hence, DOXloaded HPAEG-AS1411 nanoparticles have a similar therapeutic effect with free DOX and low cell cytotoxicity to normal cells.


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To investigate the effect of intracellular GSH levels on therapeutic effect, we pretreated MCF-7 cells with 0.1 mM BSO for 12 h or 10 mM GSH-OEt for 2 h, respectively. The cells pretreated with BSO have a lower GSH level, since BSO is an inhibitor of the rate-limiting enzyme for GSH biosynthesis.44,45 The cells pretreated with GSH-OEt have a higher GSH level, since GSH-OEt is able to generate GSH by ethyl ester hydrolyzationin cytoplasm.46-49 The cells without pretreatment, which possess normal GSH level, were used as control. As shown in Figure 15, after the MCF7 cells pretreated with GSH-OEt are incubated with DOX-loaded HPAEG-AS1411 nanoparticles for 48 h, the cell viability is lower than that of the MCF-7 cells without pretreatment or with BSO pretreatment at the same drug concentration. The difference of cell viability could be attributed to the different drug release performance. The GSH concentration in the cytoplasm has influence on the cleavage of disulfide bonds in the backbone of HPAEG-AS1411, which has relationship with the rate of DOX release (Scheme 2). Hence, the higher intracellular GSH concentration can accelerate DOX release, which will enhance the therapeutic effect.


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Figure 14. Cytotoxicity of free DOX, DOX-loaded HPAEG-AS1411 and HPAEG against (a) L929 cells and (b) MCF-7 cells at different concentrations by CCK-8 assay. The average ± standard deviation (n = 6) is presented for each data. 0.01 < p ≤ 0.05 is statistically significantand denoted as “*”; while p ≤ 0.01 is highly significant and denoted as “**”.


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Scheme 2. Illustration of redox-responsive HPAEG-AS1411 nanoparticles for intracellular drug release triggered by glutathione (GSH).

Figure 15. Cytotoxicity of DOX-loaded HPAEG-AS1411 against MCF-7 cells at different concentrations by CCK-8 assay. The cells were pretreated with 10 mM of


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GSH-OEt or 0.1 mM of BSO compred to the control of nonpretreated cells. The average ± standard deviation (n = 6) is presented for each data. 0.01 < p ≤ 0.05 is statistically significantand denoted as “*”; while p ≤ 0.01 is highly significant and denoted as “**”. CONCLUSIONS A novel backbone redox-responsive hyperbranched polymer HPAEG was designed and prepared. The HPAEG was synthesized by combination of RAFT polymerization and SCVP. Vinyl disulfide-contained trithiocarbonate ACPP was used as both chaintransfer agent and monomer. ACPP was copolymerized with PEGMA to obtain HPAEG, which contained disulfide bonds in its backbone. Then, the HPAEG was modified with aptamer to fabricate targeted drug delivery carrier HPAEG-AS1411. The DOX-loaded HPAEG-AS1411 nanoparticles were prepared by self-assembled method due to the amphiphilic structure of HPAEG-AS1411. Aptamer AS1411 had high affinity to nucleolin, which is overexpressed by MCF-7 cells. DOX-loaded HPAEG-AS1411 nanoparticles could efficiently target to MCF-7 cells. Besides, when the nanoparticles were internalized into tumor cells, the disulfide bonds in the backbone of HPAEGAS1411 were cleaved by GSH to release DOX rapidly in the cytoplasm. Hence, DOXloaded HPAEG-AS1411 nanoparticles had a high tumor cellular proliferation inhibition rate. This aptamer-functionalized backbone redox-responsive hyperbranched polymer provides a promising platform for targeted drug delivery in cancer therapy.

ASSOCIATED CONTENT Supporting Information


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1

H NMR spectra of CPP, CPPE, ACPP and HPAEG-AC; CMC of HPAEG1, HPAEG2

and HPAEG3; Zeta potential distribution of HPAEG-AC and HPAEG-AS1411; UVVis spectra of DOX-loaded HPAEG-AS1411; Cytotoxicity of degraded component of HPAEG and HPAEG-AS1411. This materials available free of charge via the Internet athttp://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (Y.S.); [email protected] (D.H.); [email protected] (X.Z.). Tel.: +86-21-54746215. Fax: +86-21-54741297. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was financially supported by the National Basic Research Program of China (2015CB931801) and National Natural Science Foundation of China (91527304, 51473093).

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For Table of Contents use only Aptamer-Functionalized and Backbone Redox-Responsive Hyperbranched Polymer for Targeted Drug Delivery in Cancer Therapy Yuanyuan Zhuang, Hongping Deng, Yue Su,* Lin He, Ruibin Wang, Gangsheng Tong, Dannong He,* Xinyuan Zhu*


Aptamer-Functionalized and Backbone Redox-Responsive

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