Dendrons and Multiarm Polymers with Thiol-Exchangeable Cores: A

Department of Chemistry, Bogazici University, Bebek 34342, Istanbul, Turkey. 2Center for Life Sciences and Technologies, Bogazici University, Istanbul...
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Dendrons and Multiarm Polymers with Thiol-Exchangeable Cores: A Reversible Conjugation Platform for Delivery Ozgul Gok,† Pelin Erturk,† Burcu Sumer Bolu,† Tugce Nihal Gevrek,† Rana Sanyal,†,‡ and Amitav Sanyal*,†,‡ †

Department of Chemistry, Bogazici University, Bebek 34342, Istanbul, Turkey Center for Life Sciences and Technologies, Bogazici University, Istanbul, Turkey

‡

S Supporting Information *

ABSTRACT: Disulfide exchange reaction has emerged as a powerful tool for reversible conjugation of proteins, peptides and thiol containing molecules to polymeric supports. In particular, the pyridyl disulfide group provides an efficient handle for the site-specific conjugation of therapeutic peptides and proteins bearing cysteine moieties. In this study, novel biodegradable dendritic platforms containing a pyridyl disulfide unit at their focal point were designed. Presence of hydroxyl groups at the periphery of these dendrons allows their elaboration to multivalent initiators that yield poly(ethylene glycol) based multiarm star polymers via controlled radical polymerization. The pyridyl disulfide unit at the core of these star polymers undergoes efficient reaction with thiol functional group containing molecules such as a hydrophobic dye, namely, BodipySH, glutathione, and KLAK sequence containing peptide. While conjugation of the hydrophobic fluorescent dye to the PEG-based multiarm polymer renders it water-soluble, it can be cleaved off the construct through thiol−disulfide exchange in the presence of an external thiol such as dithiothreitol. The multiarm polymer was conjugated with a thiol group containing apoptotic peptide to increase its solubility and cellular transport. In vitro cytotoxicity and apoptosis assays demonstrated that the resultant peptide−polymer conjugate had almost five times more apoptotic potential primarily through triggering apoptosis by disrupting mitochondrial membranes of human breast cancer cell line (MDA-MB-231) compared to naked peptide. The novel dendritic platform disclosed here offers an attractive template that can be modified to multiarm polymeric constructs bearing a “tag and release” characteristic.

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INTRODUCTION In recent years a lot of effort has been focused on developing novel polymer architectures for efficient conjugation of peptides, proteins and oligonucleotides to serve as polymeric nanomedicine platforms.1,2 Several studies have demonstrated that polymer based conjugates and complexes are innovative platforms for improving the stability and efficacy of therapeutic peptides and proteins.3,4 Conjugation of synthetic or biological therapeutic agents to polymeric constructs addresses main challenges such as poor bioavailability of drugs or tracer agents, their limited stability in the biological milieu, and their short residence time in body. In particular, one of the challenges of peptide based therapeutics is their rapid proteolytic degradation in the bloodstream, apart from their rapid elimination from kidneys. However, upon conjugation to high molecular weight polymers increased hydrodynamic volume of the resultant entity provides an extended half-life by reducing rate of renal clearance.5,6 Moreover, such conjugation enhances the stability of peptides since the polymer acts as a shield toward proteolytic degradation through the so-called “umbrella-like effect”.7 Conjugation of peptides to polymers is widely used in drug delivery either to improve or preserve the activity of therapeutically relevant peptides in addition to use these © 2017 American Chemical Society

amino acid sequences to achieve cell specific targeting. A wide variety of chemistry has been utilized to perform this conjugation in an efficient manner, the most common one being utilization of amine end to react with an activated ester on a polymeric scaffold. Oftentimes, thiol group emanating from a cysteine group on a peptide provides an attractive alternative since an active peptide sequence may contain amine groups necessary for specific activity. To this end, polymers containing vinyl sulfone, maleimide, and activated disulfide endgroups have been widely investigated in literature as popular thiol reactive scaffolds.8−13 Another aspect that needs consideration is whether the conjugation sought is reversible or irreversible; the former may be preferable if the therapeutic peptide requires a release mechanism to become active, while the latter may be preferred for robust attachments of peptides serving as targeting units. Most of the above-mentioned chemistry for thiol conjugation usually provides a nonreversible conjugation. For obtaining reversible conjugation, pyridyl disulfide based thiol-exchange reaction has been exploited to Received: April 29, 2017 Revised: June 21, 2017 Published: June 24, 2017 2463

DOI: 10.1021/acs.biomac.7b00619 Biomacromolecules 2017, 18, 2463−2477

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Biomacromolecules

Figure 1. Illustration of the reversible-conjugation to core reactive polymeric constructs for cellular delivery.

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attach and release thiol-containing payload onto polymeric materials. Over the past decade, based on this chemistry, extensive research has been focused on development of novel delivery platforms for nucleic acids,14 proteins,15 and chemotherapeutics16 based on multifunctionalizable copolymers,17 cross-linked and redox-sensitive micelles,18 and surface functionalizable polymer nanogels.19 Furthermore, disulfide exchange reaction is a powerful tool for fabricating such bioconjugates since the presence of degradable disulfide bonds would provide an enhanced drug release pathway in cancerous cells due to high glutathione concentrations, as well as a degradation pathway to allow clearance of these materials from the body after some time.20−22 Apart from the thiol-mediated degradation pathway, biodegradability of the polymeric construct is also important for clearance of the construct from the body. In light of this, several polymeric constructs including polycarbonates,23 polyesters,24 and poly(β-amino ester) based polymers25−30 have been explored in recent years. While the disulfide based reversible conjugation has been exploited with linear and cross-linked polymeric constructs, its combination with dendritic and star-like polymeric architectures remains to be explored. Materials obtained using dendritic constructs as building blocks benefit from attributes such as their precise multivalent peripheral functionality and welldefined architectures. To date, dendritic scaffolds have been extensively utilized to design multiarm polymers31−33 and hydrogels34−38 for various biomedical applications such as sensing and drug delivery.39 Herein, we report a novel biodegradable dendritic scaffold that contains a thiol-reactive pyridyl disulfide unit at its focal point, which enables reversible conjugation of thiol-containing molecules through the thiol−disulfide exchange reaction. The dendrons carry hydroxyl functional groups at their periphery and, thus, can be easily derivatized in a manner orthogonal to their core. In this study, the peripheral hydroxyl groups were utilized to install multiple free-radical polymerization initiators for obtaining water-soluble multiarm polymers containing the thiol-reactive functional group at the core. Thereafter, the pyridyl disulfide unit was used to conjugate a hydrophobic dye, Bodipy-SH, to render it soluble in aqueous media. Reversible nature of the conjugation was demonstrated by inducing reduction with dithiothreitol (DTT), whereby a decrease in fluorescence intensity of the solution was observed over time due to release of dye which is insoluble in aqueous media. Likewise, it is demonstrated that a thiol-containing tripeptide, glutathione, can be efficiently conjugated to the core. After successful conjugation of this model tripeptide, a pro-apoptotic KLAK-sequence containing peptide was attached to the core to investigate the apoptotic activity of polymer-conjugated form compared to the nonconjugated free peptide on MDA-MB-231 human adenocarcinoma cell line (Figure 1).

EXPERIMENTAL SECTION

Materials. Chemicals. All reagents were obtained from commercial sources and were used as received unless otherwise stated. 2-(2(Pyridin-2-yl)disulfanyl)ethanol (1),17 acetonide-2,2-bis(methoxy)propionic anhydride (4),40 and Bodipy-SH41 were synthesized according to literature procedures. Di(ethylene glycol) methyl ether methacrylate (DEGMEMA, 99%, Aldrich) was passed through basic alumina column to remove inhibitor prior to use 4,4′-dinonyl-2,2′bipyridine (99%, Aldrich). DMAP, EtN3, and DTT were obtained from Alfa Aesar and used as received. 2,2-Bis(methoxy)propionic (98%, Sigma-Aldrich), 2-bromo-2-methylpropionyl bromide (98%, Sigma-Aldrich), L-glutathione reduced (98%, Sigma-Aldrich), and copper(I)bromide (CuBr, 99.9%, Aldrich) were used as received. Anhydrous solvents (CH2Cl2 and THF) were obtained from ScimatCo Purification System. The KLAK peptide was purchased from GenicBio Limited with 94.19% purity. Centrifugal filtration tubes (Spectra/Por Amicon Ultra-4 Regenerated Cellulose Membranes MWCO 3000 Da) were purchased from Spectrum Laboratories. All other chemicals were used as received from manufacturer (Merck, Sigma-Aldrich and Alfa Aesar). Thin layer chromatography was performed using silica gel plates (Kiesel gel 60 F254, 0.2 mm, Merck). Cells. Human breast adenocarcinoma MDA-MB-231 cell line was obtained from ATCC (Wessel, Germany). Cells were kept in the logarithmic phase of cell growth for the duration of experiments. MDA-MB-231 cells were maintained in RPMI-1640 culture medium (Roswell Park Memorial Institute) [Gibco, Invitrogen] supplemented with 10% fetal bovine serum [FBS] [Lonza], 100 U/mL penicillin, and 100 g/mL streptomycin at 37 °C, 5% CO2, and 95% relative humidity. Cell counting kit-8 (CCK-8) was obtained from Sigma-Aldrich for cytotoxicity assays. Annexin V-FITC Apoptosis Kit was obtained from BioVision for detection of apoptosis and necrosis. CCCP and JC-1 dye for mitochondrial membrane potential assay were purchased from Alfa Aesar and Molecular Probes (ThermoFisher Scientific), respectively. Instrumentation. The NMR spectra were recorded using a 400 MHz Varian spectrometer at 25 °C. 1H and 13C NMR measurements were made at frequency of 400 MHz, and calibrated with respect to the solvent signal. The measurements were performed in deuterated chloroform (CDCl3), water (D2O), and dimethyl sulfoxide (DMSO). The molecular weight of the polymer was estimated by gel permeation chromatography (GPC) analysis using a PSS-SDV (length/ID 8 × 300 mm, 10 μm particle size) Linear M column calibrated with polystyrene standards (1−400 kDa) using a refractive-index detector. Tetrahydrofuran (THF) was used as eluent at a flow rate of 1 mL/min at 30 °C. UV−vis spectra were recorded using a Varian Cary-100 UV−vis spectrophotometer, centrally controlled by a single dispersion equipped with high performance R928 photomultiplier tube, tungsten halogen visible source with quartz window, deuterium arc ultravioletsource. Wavelength accuracy is ±0.2 nm. IR spectra of polyester dendrons were obtained by using Fourier transform infrared (FTIR) spectroscopy (Thermo Fisher Scientific Inc. Nicolet 380) and high resolution mass spectroscopy (HRMS) analysis were performed by mass spectroscopy (Agilent, 1200/6210). Cell viability values for the cytotoxicity experiments were determined by measuring the absorbance values of samples in 96-well plates at 450 nm by Multiscan FC Microplate Photometer from Thermo Scientific equipped with a quartz halogen light source of a precision CV ≤ 0.2% (0.3−3.0 Abs). It 2464

DOI: 10.1021/acs.biomac.7b00619 Biomacromolecules 2017, 18, 2463−2477

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Biomacromolecules

C(CH3)2), 1.33 (s, 6H, C(CH3)2), 1.28 (s, 3H, C(CH3)), 1.11 (s, 6H, C(CH3)). 13C NMR (CDCl3, δ, ppm) 173.5, 172.3, 159.4, 149.8, 137.1, 119.9, 120.0, 98.1, 66.0, 65.3, 62.8, 46.8, 42.0, 36.9, 25.2, 22.0, 18.5, 17.7. HRMS m/z: [M + H]+ Calcd for C28H41NO10S2H, 616.2250; Found, 616.2213; [M + 2H]+ Calcd for C12H17NO4S2H2, 617.2328; Found, 617.2245; [M + 3H]+ Calcd for C28H41NO10S2H3, 618.2407; Found, 618.2210. FTIR (cm−1): 2989.8, 1732.5. Compound 5 (1.33 g, 2.16 mmol) was dissolved in 30 mL of THF and then 30 mL of 1 M HCl was added into the mixture. The resulting mixture was stirred at room temperature until the consumption of compound 5 was observed via TLC. At the end of the reaction, THF was evaporated and the residue was diluted to 50 mL with ethyl acetate. The cure was extracted with 5% NaHCO3 solution (2 × 30 mL). The organic layer was dried over anhydrous Na2SO4 and all volatiles were evaporated. The pure product was dried under vacuo and obtained as a yellow viscous compound (6; 1.13 g, 98% yield). 1H NMR (CDCl3, δ, ppm) 8.47−8.45 (m, 1H, CHN), 7.65−7.64 (m, 2H, CHCH−CH N), 7.12−7.09 (m, 1H, N−C−CH), 4.44−4.26 (m, 6H, CH2 ester protons), 3.86−3.80 (m, 4H, OCH2), 3.72−3.66 (m, 4H, OCH2), 3.15−3.10 (m, 4H, OH), 3.04 (t, 2H, J = 6.4 Hz, SCH2), 1.30 (s, 3H, C(CH3)), 1.03 (s, 6H, C(CH3)). 13C NMR (CDCl3, δ, ppm) 175.1, 172.7, 159.2, 149.8, 137.2, 121.1, 120.2, 67.8, 64.8, 63.0, 49.7, 46.4, 36.9, 18.1, 17.1. HRMS m/z: [M + H]+ Calcd for C22H33NO10S2H, 536.1624; Found, 536.1594; [M + Na]+ Calcd for C22H33NO10S2Na, 558.1444; Found, 558.1404; FTIR (cm−1): 3370.1, 2937.6, 1725.0. Synthesis of Third-Generation PDS-Functionalized Dendron (8). Acetonide-2,2-bis(methoxy)propionic anhydride (4; 2.69 g, 7.46 mmol) and DMAP (0.182 g, 1.49 mmol) were dissolved in 40 mL of CH2Cl2 in a round-bottom flask. A solution of compound 6 (0.50 g, 0.93 mmol) in 10 mL of CH2Cl2 and pyridine (0.75 mL, 9.33 mmol) were also added into this mixture. After stirring 30 h at room temperature, 0.75 mL (same amount with pyridine) of water was added to the reaction mixture in order to get rid of the excess anhydride. The reaction crude was extracted with 1 M NaHSO4 solution (3 × 20 mL), then 10% Na2CO3 solution (3 × 20 mL), and with brine solution (20 mL). After extraction, the organic layer was dried over anhydrous Na2SO4, and all volatiles were evaporated. The crude product was then purified by column chromatograph using silica gel as stationary phase and mixture of ethyl acetate/hexane as eluent. The pure product was dried under vacuo and obtained as a yellow solid (7; 0.71 g, 82% yield). 1H NMR (CDCl3, δ, ppm) 8.47−8.45 (m, 1H, CHN), 7.66−7.64 (m, 2H, CHCH−CHN), 7.11−7.08 (m, 1H, N−C−CH), 4.39 (t, 2H, J = 6.4 Hz, SCH2CH2O), 4.32−4.21 (m, 12H, OCH2 ester protons), 4.13 (d, 8H, J = 12.0 Hz, OCH2), 3.60 (d, 8H, J = 12.0 Hz, OCH2), 3.04 (t, 2H, J = 6.4 Hz, SCH2), 1.39 (s, 12H, C(CH3)2), 1.33 (s, 12H, C(CH3)2), 1.26 (s, 3H, C(CH3)), 1.25 (s, 6H, C(CH3)), 1.12 (s, 12H, C(CH3)). 13C NMR (CDCl3, δ, ppm) 173.5, 171.85, 171.83, 159.3, 149.8, 137.1, 121.0, 120.0, 98.1, 66.96, 66.91, 64.9, 63.0, 46.9, 46.7, 42.0, 36.9, 25.2, 22.0, 18.5, 17.7, 17.6. HRMS m/z: [M + H]+ Calcd for C54H81NO22S2H, 1160.4770; Found, 1160.4756; [M + 2H]+ Calcd for C54H81NO22S2H2, 1161.4848; Found, 116.4887; [M + 3H]+ Calcd for C54H81NO22S2H3, 1162.4926; Found, 1162.4783. FTIR (cm−1): 2990.2, 1731.6. Compound 7 (0.10 g, 0.086 mmol) was dissolved in 65 mL of methanol and then 18 M H2SO4 (0.32 mL, 5.82 mmol) was added into the mixture. After stirring for 2 h at room temperature, 7 M NH3 in methanol was added until pH becomes 7.0. The salt, (NH4)2SO4, was filtered with filter paper and methanol was evaporated. To get rid of the remaining salt, the residue was washed with a minimum amount of water and pure product was obtained as a white solid (0.086 g, 62%). 1H NMR (CD3OD, δ, ppm) 8.43−8.41 (m, 1H, CHN), 7.86−7.84 (m, 2H, CHCH−CHN), 7.26−7.23 (m, 1H, N−C−CH), 4.42 (t, 2H, J = 6.4 Hz, SCH2CH2O), 4.34−4.22 (m, 12H, OCH2 ester protons), 3.69−3.58 (m, 16H, OCH2), 3.14 (t, 2H, J = 6.4 Hz, SCH2), 1.31 (s, 3H, C(CH3), 1.28 (s, 6H, C(CH3), 1.14 (s, 12H, C(CH3)). 13C NMR (CD3OD, δ, ppm) 174.5, 172.3, 172.2, 159.5, 149.1, 137.9, 121.2, 120.0, 65.8, 64.8, 64.4, 62.8, 50.4, 46.5, 36.9, 33.3, 16.8, 16.6, 15.9. FTIR (cm−1): 3283.1, 2938.7, 1729.7. Synthesis of 4-Arm Multiarm Star Polymer via G2PDS Initiator (P1). Synthesis of G2PDS Initiator (9). Pyridyl disulfide containing

has an excitation wavelength range 340 to 850 nm with excitation filters are installed at 405, 450, and 620 nm. Apoptosis assay was evaluated by flow cytometry analysis using Guava Easycyte Flow Cytometer (Merck Millipore). Simultaneously, stained cells were visualized by Zeiss Observer Z1 fluorescence microscope connected to Axiocam MRc5. Images were processed to evaluate cell nuclei and morphology using Zeiss AxioVision software. Synthesis of Pyridyl Disulfide (PDS) Functionalized Polyester Dendrons. Synthesis of First-Generation PDS-Functionalized Dendron (3). 2,2-Bis(methoxy)propionic acid (0.505 g, 2.90 mmol), EDCI (0.612 g, 3.19 mmol), and DMAP (0.106 g, 0.87 mmol) were added into a round-bottom flask and dissolved in 10 mL of CH2Cl2. A solution of 2-(2-(pyridin-2-yl)disulfanyl)ethanol (1; 1.63 g, 8.70 mmol) in 4 mL of CH2Cl2 was added to the clear mixture. After stirring for 20 h at room temperature, reaction mixture was diluted to 30 mL with CH2Cl2 and extracted with 7% NaHCO3 solution (3 × 10 mL). Organic layer was dried over anhydrous Na2SO4. The crude product was then purified by column chromatograph using silica gel as stationary phase and mixture of ethyl acetate/hexane as eluent. The pure product was dried under vacuo and obtained as a yellow viscous compound (2; 0.78 g, 78% yield). 1H NMR (CDCl3, δ, ppm) 8.45 (d, 1H, J = 4.0 Hz, CHN), 7.68−7.58 (m, 2H, CHCH−CHN), 7.09 (d, 1H, J = 16.0 Hz, N−C−CH), 4.39 (t, 2H, J = 6.4 Hz, SCH2CH2O), 4.17 (d, 2H, J = 12.0 Hz, OCH2), 3.62 (d, 2H, J = 12.0 Hz, OCH2), 3.05 (t, 2H, J = 6.4 Hz, SCH2), 1.41 (s, 3H, C(CH3)2), 1.37 (s, 3H, C(CH3)2), 1.18 (s, 3H, C(CH3)). 13C NMR (CDCl3, δ, ppm) 174.0, 159.6, 149.8, 137.1, 120.9, 119.8, 98.1, 66.0, 62.5, 41.9, 37.3, 24.7, 22.5, 18.6. HRMS m/z: [M + H] + Calcd for C15H21NO4S2H, 344.0990; Found, 344.0994; [M + Na]+ Calcd for C15H21NO4S2Na, 366.0810; Found, 366.0815. FTIR (cm−1): 2990.3, 1723.8. Compound 2 (2.08 g, 6.05 mmol) was dissolved in 20 mL of tetrahydrofuran (THF) and then 20 mL of 1 M HCl was added into the mixture as well. The resulting mixture was stirred at room temperature until the consumption of compound 2 was observed via TLC. At the end of the reaction, THF was evaporated and the residue was diluted to 50 mL with CH2Cl2. The crude was extracted with 10% Na2CO3 solution (2 × 30 mL). The organic layer was dried over anhydrous Na2SO4 and all volatiles were evaporated. The crude product was then purified by column chromatograph using silica gel as stationary phase and mixture of ethyl acetate/hexane as eluent. The pure product was dried under vacuo and obtained as a yellow viscous compound (3; 1.74 g, 92% yield). 1H NMR (CDCl3, δ, ppm) 8.46− 8.44 (m, 1H, CHN), 7.64−7.58 (m, 2H, CHCH−CHN), 7.11−7.08 (m, 1H, N−C−CH), 4.40 (t, 2H, J = 6.4 Hz, SCH2CH2O), 3.90 (d, 2H, J = 11.2 Hz, OCH2), 3.73 (d, 2H, J = 11.2 Hz, OCH2), 3.06 (t, 2H, J = 6.4, SCH2), 1.08 (s, 3H, C(CH3)). 13C NMR (CDCl3, δ, ppm) 175.5, 159.0, 149.8, 137.1, 121.1, 120.2, 68.4, 62.4, 49.4, 37.7, 17.1. HRMS m/z: [M + H]+ Calcd for C12H17NO4S2H, 304.0677; Found, 304.0657; [M + 2H]+ Calcd for C12H17NO4S2H2, 305.0755; Found, 305.0688. FTIR (cm−1): 3357.0, 2940.1, 1721.8. Synthesis of Second-Generation PDS-Functionalized Dendron (6). Acetonide-2,2-bis(methoxy)propionic anhydride (4; 4.63 g, 14.0 mmol) and DMAP (0.46 g, 3.79 mmol) were dissolved in 70 mL of CH2Cl2 in a round-bottom flask. A solution of compound 3 (1.44 g, 4.74 mmol) in 30 mL of CH2Cl2 and pyridine (2.26 mL, 28.0 mmol) were added into this mixture as well. After stirring 30 h at room temperature, 2.26 mL (same amount with pyridine) of water was added to the reaction mixture in order to get rid of excess anhydride. The reaction crude was extracted with 1 M NaHSO4 solution (3 × 40 mL), then 10% Na2CO3 solution (3 × 40 mL) and with brine solution (40 mL). After extraction, the organic layer was dried over anhydrous Na2SO4 and all volatiles were evaporated. The crude product was then purified by column chromatograph using silica gel as stationary phase and mixture of ethyl acetate/hexane as eluent. The pure product was dried under vacuo and obtained as a yellow viscous compound (5; 2.44 g, 84% yield). 1H NMR (CDCl3, δ, ppm) 8.47−8.46 (m, 1H, CHN), 7.65−7.64 (m, 2H, CHCH−CHN), 7.11−7.08 (m, 1H, N−C−CH), 4.38 (t, 2H, J = 6.4 Hz, SCH2CH2O), 4.31 (s, 4H, OCH2 ester protons), 4.12 (d, 4H, J = 12.0 Hz, OCH2), 3.59 (d, 4H, J = 12.0 Hz, OCH2), 3.02 (t, 2H, J = 6.4 Hz, SCH2), 1.39 (s, 6H, 2465

DOI: 10.1021/acs.biomac.7b00619 Biomacromolecules 2017, 18, 2463−2477

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Biomacromolecules second generation polyester dendron furnished with four hydroxyl groups at the periphery (6; 0.10 g, 0.19 mmol) and DMAP (13.7 mg, 0.11 mmol) were dissolved in THF (15.0 mL), and then triethylamine (0.18 mL, 1.31 mmol) was added into this solution under nitrogen. The mixture was cooled to 0 °C in an ice bath. On the other side, 2bromo-2-methylpropionyl bromide (115.0 μL, 0.93 mmol) was diluted in THF (2.0 mL) and added into the former mixture dropwise. Obtained white suspension was stirred for 30 min at 0 °C, then warmed to room temperature and stirred for 6 h. Formed ammonium salt was filtered off and the residue was concentrated in vacuo that was purified by column chromatography on silica (15% EtOAc in hexane), affording the initiator as a pure light yellow solid (9; 0.14 g, 67% yield). 1 H NMR (CDCl3, δ, ppm) 8.46 (br s, 1H, CHN), 7.65 (br s, 2H, CHCH−CHN), 7.10 (br s, 1H, N−C−CH), 4.42−4.26 (m, 14H, CH2 ester protons), 3.05 (t, 2H, J = 6.0 Hz, SCH2), 1.59 (s, 24H, CBr(CH3)), 1.30 (s, 6H, C(CH3)), 1.27 (s, 3H, C(CH3)). 13C NMR (CDCl3, δ, ppm) 171.6, 170.9, 149.8, 137.1, 121.0, 120.0, 83.7, 66.0, 63.1, 55.3, 46.8, 36.9, 30.6, 17.9. Synthesis of G2PDS-DEGMEMA (P1). G2PDS-DEGMEMA was prepared by ATRP of DEGMEMA. Pyridyl disulfide containing G2PDS-initiator (9; 10.0 mg, 8.84 μmol) dissolved in a minimum amount of degassed anisole was introduced into a flask containing Cu(I)Br (5.1 mg, 3.53 μmol), 4,4′-dinonyl-2,2′-bipyridine (28.9 mg, 70.7 μmol), and degassed DEGMEMA (0.65 mL, 3.53 mmol) dissolved in degassed anisole (5.05 mL) under stirring. The flask was then placed in a thermostated oil bath at 80 °C for 120 min. After polymerization, the reaction mixture was cooled down to room temperature, passed through a neutral alumina column to remove the catalyst, and precipitated in diethyl ether twice. P1 was filtered and obtained as a viscous solid (0.15 g). ([I]0/[M]0/[CuBr]/[ 4,4′Dinonyl-2,2′-bipyridine] = 1:400:4:8, conversion = 23%, Mn,theo = 17560 g mol−1, Mn,NMR = 64760 g mol−1, Mn,GPC = 27100 g mol−1, Mw/Mn= 1.13, relative to PS). 1H NMR (CDCl3, δ, ppm) 8.46 (br s, 1H, CHN), 7.67 (br s, 2H, CHCH−CHN), 6.95 (br s, 1H, N−C−CH), 4.34−3.90 (m, 16H, CH2 ester protons), 3.78−3.54 (m, 2H, OCH2 of DEGMEMA), 3.37 (br s, 3H, OCH3 of DEGMEMA), 1.89−0.87 (m, 38H, C(CH3), CBr(CH3), CH2 and CH3 along polymer backbone). Reduction of P1 with dithiothreitol (DTT). P1 (0.9 mg, 0.014 μmol) was dissolved in a minimal amount of methanol and then diluted with phosphate buffered saline (PBS, pH 7.3) to 5 mL. A total of 1 mL of this solution was mixed with 1 mL of a DTT solution (1.20 mM in PBS). Absorbance values at 379 nm were determined at several time points (0, 10, 30, 60, 90, 120, 180, and 240 min). The concentration of released pyridine 2-thione was calculated using reported molar extinction coefficient (8.08 × 103 M−1cm−1).11 Comparison with the total polymer concentration (molecular weight determined from 1H NMR) gave the end group percentage. Reversible Conjugation of Multiarm Star Polymers. Conjugation of Polymer P1 with Glutathione (P2). Polymer P1 (10.0 mg, 0.15 μmol) and glutathione (0.19 mg, 0.31 μmol) were dissolved in H2O (0.2 mL) and acetic acid (0.88 μL, 0.015 μmol) was added into the reaction mixture. The reaction was allowed to proceed at room temperature for 24 h. Unreacted tripeptide was removed by centrifugal filtration (MWCO: 3000 Da, 7000 rpm, 10 min × 4), and the concentrated solution was lyophilized. Glutathione-conjugated multiarm star polymer P2 was obtained as a viscous solid (8.30 mg, 82% yield). 1H NMR (CDCl3, δ, ppm) 4.34−3.92 (m, 16H, CH2 ester protons), 3.78−3.43 (m, 2H, OCH2 of DEGMEMA), 3.38 (br s, 3H, OCH3 of DEGMEMA), 2.03−0.86 (m, 38H, C(CH3), CBr(CH3), CH2, and CH3 along the polymer backbone). 1H NMR (D2O, δ, ppm) 8.41 (s, 2H, NH of glutathione), 4.17 (br s, 16H, CH2 ester protons), 3.76−3.62 (m, 2H, OCH2 of DEGMEMA), 3.39 (br s, 3H, OCH3), 2.96−2.90 (m, 2H, SCH2 of glutathione), 2.49 (br s, 4H, COCH2CH2 of glutathione), 2.13−0.86 (m, 38H, C(CH3), CBr(CH3), CH2, and CH3 along the polymer backbone). Conjugation of Polymer P1 with Bodipy-SH (P3). Polymer P1 (20.0 mg, 0.31 μmol) and Bodipy-SH (0.16 mg, 0.37 μmol) were dissolved in THF (0.5 mL) and acetic acid (1.76 μL, 0.03 μmol) was

added into the reaction mixture. The reaction was allowed to proceed at room temperature for 24 h. At several time points (0, 1, 2, 3, 4, 5, 6, 7, 8 and 24 h), samples were taken from the mixture and THF was removed by a vacuum line. Then samples were dissolved in 3 mL of water and analyzed with a UV−vis spectrophotometer to monitor the release of pyridine 2-thione and conjugation of Bodipy-SH with respect to time. At the end of 24 h, unreacted dye was removed by washing the conjugate with cold diethyl ether several times. Dye conjugated multiarm star polymer P3 was obtained as a fluorescent viscous solid (16.30 mg, 81% yield). 1H NMR (CDCl3, δ, ppm) 6.03 (s, 2H, HCCCH3 of Bodipy), 4.32−3.89 (m, 16H, CH2 ester protons), 3.77−3.53 (m, 2H, OCH2 of DEGMEMA), 3.37 (br s, 3H, OCH3 of DEGMEMA), 2.48 (s, 6H, NCCH3 of Bodipy), 2.40 (s, 6H, CH3CC of Bodipy), 2.30 (t, 2H, J = 8.0 Hz, CCH2 of Bodipy), 2.02−0.85 (m, 54H, C(CH3), CBr(CH3), CH2 of Bodipy, CH2, and CH3 along the polymer backbone). Release of Bodipy-SH from P3 with Dithiothreitol (DTT). The in vitro release of Bodipy dye from polymer P3 was evaluated using reducing agent, DTT. Polymer P3 (2.0 mg, 0.03 μmol) was dissolved in H2O (0.5 mL) and then DTT (0.047 mg, 0.30 μmol) was added into this solution. The reaction was allowed to proceed at room temperature for 24 h. At several time points, samples were taken from the mixture, diluted to 3 mL with water, and then analyzed with UV− vis spectrophotometer to monitor the release of Bodipy-SH by time. Conjugation of Polymer P1 with Apoptotic Peptide (P4). Polymer P1 (10.0 mg, 0.15 μmol) and KLAK peptide (0.36 mg, 0.23 μmol) were dissolved in THF (0.10 mL) and acetic acid (0.86 μL, 0.015 μmol) was added into the reaction mixture. The reaction was allowed to proceed at room temperature for 24 h. At several time points, samples were taken from the mixture and added into 3 mL of THF to be analyzed with UV−vis spectrophotometer to monitor the release of pyridine 2-thione over time. At the end of 24 h, unreacted peptide was removed by centrifugal filtration (MWCO: 3000 Da, 6000 rpm, 10 min × 4) over MeOH/H2O (1:1) mixture, and the concentrated solution was lyophilized to give the resultant peptide conjugated multiarm star copolymer as a viscous solid P4 (7.60 mg, 74% yield). 1 H NMR (CDCl3, δ, ppm) 7.68−6.95 (m, 33H, NH and NH3+ of KLAK), 5.55−5.62 (m, 2H, NH2 of KLAK), 4.33−3.90 (m, 17H, CH2 ester protons of DEGMEMA and OCCHN of KLAK), 3.78−3.49 (m, 2H, OCH2 of DEGMEMA), 3.38 (br s, 15H, OCH3 of DEGMEMA and CH2NH3+ of KLAK), 3.08−3.01 (m, 2H, SCH2), 2.86−2.79 (m, 2H, SCH2), 2.33 (t, 12H, J = 7.6 Hz, CH2CH2CH2 of KALK), 1.99− 0.86 (m, 110H, C(CH3), CBr(CH3), CH2, and CH3 along the polymer backbone, and CH, CH2, and CH3 of KLAK). 1 H NMR (DMSO, δ, ppm) 8.54−8.22 (m, 5H, NH of KLAK), 7.41−7.17 (m, 26H, NH and NH3+ of KLAK), 6.88 (br s, 1H, NH of KLAK), 6.70 (br s, 1H, NH of KLAK), 5.74−5.57 (m, 2H, NH2 of KLAK), 4.85 (br s, 8H, OCH2CH2S and OCCHN of KLAK), 4.61 (br s, 6H, CH2 ester protons of dendron and OCCHN of KLAK), 4.26− 3.91 (m, 2H, CH2 ester protons of DEGMEMA), 3.60−3.46 (m, 2H, OCH2 of DEGMEMA), 3.27 (br s, 15H, OCH3 of DEGMEMA and CH2NH3+ of KLAK), 2.75−2.67 (m, 2H, SCH2), 2.30−2.24 (m, 12H, CH2CH2CH2 of KALK), 1.92−0.80 (m, 110H, C(CH3), CBr(CH3), CH2, and CH3 along the polymer backbone, and CH, CH2, and CH3 of KLAK). Cytotoxicity Experiments. Cytotoxicity of apoptotic peptide KLAK, multiarm star polymer P1 and peptide conjugated multiarm polymer P4 were assessed by CCK-8 viability assay on adenocarcinoma MDAMB-231 human breast cell line. Cells (5000 cells/well) were seeded in a 96-well plate as quadruplet in 100 μL of culture medium and the plate was incubated at 37 °C for 24 h for cells to adhere completely. Samples were dissolved in DMSO at 1 × 10−2 M stock concentration and seven different concentrations (10−8 M to 10−2 M) of samples were prepared via serial dilutions. Cells were then treated with these samples and incubated at 37 °C for 72 h. After removal of sample solutions, wells were washed with 100 μL of PBS twice. The number of viable cells was determined by CCK-8 cell viability assay by adding 10 μL CCK-8 reagent in 100 μL of fresh medium onto wells and at the end of 1 h incubation absorbance values at 450 nm were measured via 2466

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Biomacromolecules Scheme 1. Synthesis of Pyridyl Disulfide Group Containing Polyester Dendrons

a microplate reader. EC50 values were calculated by GraphPad prism software using nonlinear regression mode. Apoptosis Analysis by Flow Cytometry. To determine apoptotic cell death, annexin, V-FITC kit was used according to manufacturer’s specifications and treated cells were analyzed by flow cytometry. Briefly, MDA-MB-231 cells (20000 cells/well) were seeded in a 24well plate as triplicate in 500 μL of culture medium, and the plate was incubated at 37 °C for 24 h for cells to adhere completely. Then the culture medium was aspirated and replaced with fresh medium containing either peptide itself, naked polymer P1, or peptide conjugated polymer P4 at a concentration of 1 × 10−5 M. After incubation at 37 °C for 72 h, sample solutions were removed. Wells were washed with 500 μL of PBS twice, and then cells were detached by trypsin and collected by centrifugation. After resuspending cells in 500 μL of 1× binding buffer, 5 μL of annexin V-FITC and 5 μL of propidium iodide (PI) were added and cells were incubated at room temperature for 5 min. Flow cytometry analysis was performed for all and both dot plots and histogram distributions of pooled cell populations (15000 events) were obtained. Mitochondrial Membrane Potential Assay. To investigate induction of apoptosis through mitochondrial membrane disruption by KLAK conjugated polymer, JC-1 probe was used according to manufacturer’s specifications with modifications and analyzed by fluorescence microscopy. Briefly, MDA-MB-231 cells (20000 cells/ well) were seeded in a 24-well plate as triplicate in 500 μL of culture medium, and the plate was incubated at 37 °C for 24 h for cells to

adhere completely. Then the culture medium was aspirated and replaced with fresh medium containing either peptide itself, naked polymer P1, or peptide conjugated polymer P4 at a concentration of 1 × 10−5 M. After incubation at 37 °C for 48 h, sample solutions were removed. For positive control wells, CCCP was added, with a final concentration of 50 μM in fresh RPMI and incubated for 5 min. Subsequently, cells were treated with JC-1, with a final concentration of 200 μM in fresh RPMI for another 20 min. After removal of media and washing with PBS, cells were fixed in 4% formalin solution and stained cells were visualized using Zeiss Observer Z1 fluorescence microscope at room temperature.

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RESULTS AND DISCUSSION Synthesis of Polyester Dendrons and Multiarm Star Polymer Containing Pyridyl Disulfide Group at Their Core. Three generations of biodegradable polyester dendrons possessing a pyridyl disulfide unit at their focal point were prepared by adaptation of synthesis of polyester dendrons using 2,2-bis(hydroxymethyl)propionic acid as a building block (Scheme 1). A pyridyl disulfide group containing alcohol (1) was utilized to install the desirable disulfide based reactive group at the focal point of dendrons. Treatment of alcohol (1) with bis(hydroxy)propionic acid yielded the acetal protected first generation dendron (2). Removal of acetal protecting 2467

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Biomacromolecules Scheme 2. Synthesis of a Pyridyl Disulfide Group Containing Multivalent Initiator

Figure 2. 1H NMR spectrum of pyridyl disulfide based initiator (9) in CDCl3.

Scheme 3. Synthesis of Multiarm Star Polymer P1 with a Pyridyl Disulfide Unit at the Core Using the Second Generation Initiator

groups in acidic media generated hydroxyl groups at the periphery of dendrons (3), which were treated with the acetalcontaining anhydride (4) to obtain second generation dendron (5). By iterative protocol, eight hydroxyl groups containing third generation polyester dendron (8) with pyridyl disulfide group at the focal point was prepared. The second generation hydroxyl containing dendron (6) was chosen as a multivalent building block for preparation of thiolreactive multiarm polymeric carrier. Hydroxyl groups at the periphery of the dendron were esterified using 2-bromo-2methylpropionyl bromide in the presence of triethylamine and 4-(dimethylamino)pyridine (DMAP) to decorate the dendron surface with four initiators to enable polymerization (Scheme 2).42

The pyridyl disulfide group containing initiator (9) was purified by column chromatography and characterized by 1H and 13C NMR spectroscopy. Presence of the pyridyl disulfide unit is clear due to proton resonances at 8.46, 7.65, and 7.10 ppm (peaks a, b, and c, respectively, in Figure 2), along with the methylene protons adjacent to the ester groups in dendron at 4.30 ppm. Complete derivatization of polyester dendron with initiator units was evident from the correlated integration values of singlet at 1.88 ppm belonging to the methyl groups on the initiator fragments with the methyl groups located on tertiary carbon atoms of dendron at 1.30 and 1.27 ppm (peaks m and n respectively, in Figure 2). Additionally, 13C NMR was used to verify the structure of the multiarm initiator (Figure S25). 2468

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Figure 3. 1H NMR spectrum of G2PDS-DEGMEMA (P1) in CDCl3.

Scheme 4. Conjugation of Multiarm Star Polymer P1 with Gluthatione via Disulfide Exchange

homogeneity of the arms. Ascertaining individual arm molecular weights is a challenging issue for such constructs. Chemical composition of the multiarm star polymer P1 was verified using 1H NMR spectroscopy to confirm the intactness of the pyridyl disulfide unit during polymerization. Proton resonances from the pyridyl unit at 8.46, 7.67, and 6.95 ppm (peaks a, b, and c, respectively, in Figure 3) are clearly seen with the expected splitting patterns. Additionally, the presence of the resonances from the methylene units adjacent to the ester functional group at 4.08 ppm and methoxy groups at 3.37 ppm confirm successful polymerization of DEGMEMA. Presence of the pyridyl disulfide (PDS) group at the core of this multiarm star polymer was also quantified via reduction of

Thereafter, dendritic initiator (9) was utilized to polymerize the hydrophilic monomer, diethylene glycol methacrylate (DEGMEMA), to yield a water-soluble multiarm star polymer (P1) containing a pyridyl disulfide unit at the core. Suitable with the nature of the initiator groups, atomic transfer radical polymerization (ATRP), mediated by a catalyst complex of Cu(I)Br with 4,4′-dinonyl-2,2′-bipyridine as a ligand, was employed for polymerization of this monomer (Scheme 3). Polymerization at 80 °C resulted in a water-soluble multiarm polymer P1 bearing a pyridyl disulfide group at the core with Mn = 27000 g mol−1 and a distribution of 1.13, as determined using size exclusion chromatography. It should be noted that molecular weight distribution obtained here does not reflect the 2469

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Figure 4. 1H NMR spectra of (a) P1 in CDCl3 and conjugate P2 in (b) CDCl3 and (c) D2O.

the disulfide group with a reducing agent, DTT, and the release of pyridine-2-thione was monitored by UV−vis spectroscopy. Concentration of the liberated pyridine-2-thione from the polymer was determined by measuring absorbance values at 379 nm (SI, Figure S27). According to the absorbance value obtained after release reached stationary phase, percent of polymers containing the activated disulfide group was calculated as 85%, which clearly suggests that the majority of multiarm star polymers were functionalized at their core with a pyridyl disulfide unit. Conjugation of Multiarm Star Copolymer with Thiol Bearing Molecules. With the thiol-reactive pyridyl sulfide unit containing multiarm polymer at hand, its functionalization was investigated by employing a hydrophobic dye (Bodipy-SH)

and two different thiol containing peptides, namely, glutathione and a KLAK-sequence bearing peptide. First, functionalization of this multiarm star polymer P1 was assessed by conjugation of the tripeptide “glutathione”. This free-thiol group containing tripeptide was conjugated to the core of the multiarm polymer via thiol−disulfide exchange reaction in the presence of catalytic amount of acetic acid (Scheme 4). Peptide conjugated multiarm star polymer P2 was characterized by 1H NMR spectroscopy. Efficiency of disulfide exchange reaction seems to be quite high upon comparison of the proton spectra of original polymer P1 with its peptide conjugated version P2. Complete disappearance of proton resonances at 8.46, 7.67, and 6.95 ppm (peaks a, b, and c, respectively, in Figure 4a) points out to complete removal of 2470

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Biomacromolecules Scheme 5. Conjugation of Copolymer P1 with Bodipy-SH Using Thiol−Disulfide Exchange

Figure 5. 1H NMR spectra of polymer P1 (a) and Bodipy-SH conjugate P3 (b) in CDCl3.

pyridyl disulfide unit from P1. While, due to hydrophilic nature of glutathione, its characteristic peaks are almost not visible in proton NMR spectrum obtained in CDCl3, but appearance of new peaks at 2.85 and 2.35 ppm (peaks x and y, respectively, in Figure 4c) for the spectrum obtained in D2O confirms successful attachment of the peptide to the polymer. Peptide conjugation to the core of multiarm star polymer was also verified by monitoring release of pyridine 2-thione from polymer as a byproduct during the disulfide exchange reaction

(SI, Figure S30). The absorption peak detected after conjugation reaction was the characteristic peak of the generated thione at 353 nm. Appearence of this absorbance peak in the UV−vis spectrum clearly indicates the successful attachment of glutathione to the core of polymer scaffold which does not show any absorption at this wavelenght. Additionally, conjugation of a hydrophobic fluorescent dye, namely, Bodipy-SH, was performed to demonstrate efficient and reversible conjugation of a highly hydrophobic molecule. 2471

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the aqueous media, and as a result the solution losses its fluorescence to a large extent. During disulfide exchange reaction performed to conjugate Bodipy-SH dye to the multiarm star polymer, UV−vis spectroscopy was used to monitor the absorbance spectra in aqueous medium, where only the polymer bound dye would give absorbance signal in the reaction mixture. Increase in amount of conjugated dye was deduced by monitoring absorbance of the aqueous solutions of residues obtained upon solvent removal of the aliquots collected from the reaction mixture. As expected, increasing absorbance of the peak at 497 nm belonging to Bodipy moiety over time demonstrated that the dye molecule is gradually conjugated to the water-soluble polymer (SI, Figure S32). Likewise, reversibility of disulfide exchange reaction was examined by reducing agent driven release of the Bodipy-dye from the conjugate. Upon reduction of disulfide bond between dye and polymer core, concentration of the dye molecule bound to polymer scaffold decreased, and the absorbance intensity observed at 497 nm in aqueous medium diminished accordingly, which is evident in UV−vis spectra of the samples taken from the reaction mixture at certain time points (SI, Figure S33). After 24 h, from UV−vis analysis it was deduced that more than 95% of the dye conjugated to the core of the multiarm star polymer was released. As a model application, we conjugated a thiol-containing apoptotic peptide to the core of the water-soluble multiarm polymer. Conjugation of a well-known KLAK sequence based apoptotic peptide to the core of multiarm star polymer P1 was undertaken. This therapeutic peptide has an α-helical cationic peptide sequence which can interact strongly with lipid membranes. It is often conjugated to cell targeting and penetrating peptides for better receptor-mediated internalization and subsequently inducing mitochondrial-dependent apoptosis.43 Cysteine terminated KLAK peptide was reacted with the pyridyl disulfide bearing polymer and in the presence of a catalytic amount of acetic acid, thus, it was attached to the polymer core (Scheme 6). After removal of the unreacted peptide from synthesized conjugate P4 using dialysis, structural characterizations were done using 1HNMR spectroscopy. Compared with parent multiarm star polymer P1, peptide conjugated polymer possesses new peaks between 7.0 and 8.0 ppm due to presence of labile protons (amine and amide protons, together with NH3+ protons of lysine units; Figure 7a,b). Moreover, signals appearing as broad singlets around 4.80 and 4.60 ppm belong to the protons attached to the chiral carbons in amino acid units of the peptide. While the methylene and methoxy proton resonances belonging to the polymer part of the conjugate remain intact, disappearance of peaks for the pyridyl disulfide unit was not obvious to conclude from the comparison of proton NMR spectra. Instead, UV−vis spectroscopy was utilized to demonstrate the liberation of pyridyl-2-thione from the polymer upon disulfide exchange reaction with KLAK peptide. Based on the increase in the intensity of the signal appearing at 372 nm, UV spectra of the samples taken at different time points from the conjugation reaction clearly show the release of pyridine 2-thione from the polymer (SI, Figure S36). Based on the results of 1H NMR and UV−vis spectroscopy, one can conclude that the apoptotic peptide KLAK was attached to the core of the star polymer via thiolresponsive disulfide exchange reaction.

Disulfide exchange reaction takes place between the free thiol group on the dye and the pyridyl disulfide group at the core of multiarm star polymer P1 resulting in attachment of the hydrophobic fluorescent dye to water-soluble polymer. Due to insolubility of the dye in aqueous media, this reaction was conducted in THF. However, resultant conjugate solubilizes the dye in aqueous media. Notably, such efficient functionalization was ensured by utilization of the dye with only a slightly higher stoichiometric ratio (1:1.2 equiv; Scheme 5). Both NMR and UV analysis were performed to confirm complete conjugation of the hydrophobic dye to the core of the multiarm star polymer. It is clear from the 1H NMR spectrum of the conjugate obtained in CDCl3 that upon disulfide exchange reaction, peaks at 8.46, 7.67, and 6.95 ppm (peaks a, b, and c, respectively, Figure 5a) belonging to the pyridyl disulfide unit at the core disappeared and new peaks were observed at 6.03 ppm (peak z, Figure 5b), 2.48 and 2.40 ppm (peaks p and r, respectively, Figure 5b) belong to characteristic peaks of conjugated dye molecule from the pyrrole ring and methyl groups, respectively. So it can be concluded that the hydrophobic dye was efficiently conjugated to the core of multiarm star polymer P1 via disulfide exchange reaction, accompanied by quantitative removal of pyridyl disulfide moiety located at core of the multiarm polymer. Conjugation of Bodipy dye molecule to ethylene glycol based polymeric scaffold provides expected water solubility to this hydrophobic dye. It is clearly visible from the photograph displayed in Figure 6 that the nonconjugated dye molecule is

Figure 6. Aqueous solutions of free Bodipy-SH (a), polymer conjugated Bodipy-SH (b), and liberated Bodipy-SH from the polymer upon DTT treatment (c) under UV illumination.

insoluble in 5% methanol containing aqueous medium, whereas polymer conjugated dye is completely soluble, as evident from the strong green fluorescence of the aqueous solution. Cleavage of disulfide linked Bodipy dye at the polymeric core was achieved upon reduction by DTT. Thus, liberated free dye molecule was observed as a precipitate due to its insolubility in 2472

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Scheme 6. Conjugation of Multiarm Star Polymer P1 with Apoptotic Peptide KLAK via the Thiol−Disulfide Exchange Reaction

Figure 7. 1H NMR spectra of P1 (a) in CDCl3 and conjugate P4 (b) in CDCl3 and (c) in DMSO.

Cytotoxicity of the peptide−protein conjugate (P4), KLAK peptide itself and naked polymer (P1) was investigated on MDA-MB-231 human breast adenocarcinoma cell line. Cell viability evaluated by CCK-8 assay indicates the nontoxic behavior of polymer P1 since EC50 value for this polymer is more than 100 μM. While free KLAK peptide did not exhibit a

significant toxicity, peptide conjugated polymer P4 showed its toxic behavior during 72 h incubation time period with an EC50 value ranging from 6.90 × 10−6 to 1.65 × 10−5 M (Figure 8). To investigate the apoptotic potential of KLAK-peptide conjugated polymer P4, an annexin-V based apoptosis assay was employed. Cells were classified as early apoptotic when 2473

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flow cytometry (Figure 9). A significant increase in the density of late apoptotic cells from 0.9% to 12.9% in comparison to control was observed by KLAK conjugated construct whereas both KLAK peptide and naked polymer failed to induce such a dramatic difference. In terms of total apoptotic cells, KLAK conjugated polymer (P4) showed a 5-fold increase (15.6%) compared to the control population (2.8%), whereas increase in apoptotic portion of the cell population with KLAK peptide and naked polymer (P1) was 6.8% and 5.8%, respectively. These results indicate that neither KLAK peptide itself nor naked polymer was able to improve the rate of apoptosis induction, whereas a notable portion of cells treated with KLAK-polymer conjugate (P4) was observed to shift from live region to apoptotic one (Figure 9). It is known that the KLAK peptide which is poorly internalized into cells when introduced by itself can induce mitochondrial-dependent apoptosis due to its interaction with lipid membranes.44,45 The disruption in mitochondrial membrane was monitored via JC-1 assay.46,47 In normal cells, cationic JC-1 dyes form red aggregates in mitochondria (Jaggregates). However, upon induction of apoptosis, mitochondrial depolarization of JC-1 results in a red to green shift due to dissolution of red J-aggregates to green JC-1 monomers (J-

Figure 8. Inhibition of MDA-MB-231 cells proliferation by P1 (G2PDS-DEGMEMA), KLAK peptide, and P4 (peptide conjugated polymer) for 72 h. Cell viabilities were determined by CCK-8 assay. Results are expressed as mean ± SD (n = 3).

they were annexin-V positive, PI negative, and late apoptotic when they were annexin-V positive, PI positive. Cells were classified as not apoptotic when they were both annexin-V and PI negative. Flow cytometry and fluorescence microscopy analysis were used to assess apoptosis level. MDA-MB-231 cells treated with the KLAK peptide, naked polymer (P1), and KLAK conjugated polymer (P4) for 72 h were analyzed using

Figure 9. Flow cytometry results of apoptosis assay using Annexin-V-FITC on MDA-MB-231 cells following treatments with (a) control, (b) P1 (G2PDS-DEGMEMA), (c) KLAK peptide, (d) P4 (peptide conjugated polymer). Cells were incubated at 37 °C for 72 h. 2474

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Figure 10. Fluorescence microscopy results of mitochondrial membrane potential assay (JC1) on MDA-MB-231 cells following treatments with control, CCCP (positive control), KLAK peptide only, P1 (G2PDS-DEGMEMA), and P4 (peptide conjugated polymer). Cells were incubated at 37 °C for 48 h. Scale bar is 20 μm.

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monomer). Carbonyl cyanide 3-chlorophenylhydrazone (CCCP), which is a known protonophore and uncoupler of oxidative phosphorylation on mitochondrial membrane was employed in the assay as a positive control. Similar to control samples, images of cells treated with the KLAK peptide alone and naked polymer (P1) clearly showed the formation of red JC-1 aggregates (Figure 10). In contrast, for cells treated with KLAK conjugated polymer (P4), a decrease in red and an increase in green fluorescence were observed which was notably similar to the case with CCCP positive control. This results highlight that the KLAK conjugated polymer was able to induce apoptosis through mitochondrial pathway owing to the KLAK peptide in its structure, with much higher efficiency than the free KLAK peptide, as well the parent polymer.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.7b00619. NMR and UV−vis analysis results and GPC trace of the synthesized polymer (PDF).

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +90-212-3597613. Fax: +90-212-287-2467. ORCID

Rana Sanyal: 0000-0003-4803-5811 Amitav Sanyal: 0000-0001-5122-8329 Author Contributions

CONCLUSIONS

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Overall, this study discloses the synthesis of a promising class of thiol-reactive dendrons and a multiarm polymeric platform for redox responsive conjugation of thiol containing small molecules and peptides. The pyridyl disulfide functional group placed at the core of these structures enables the reversible conjugation through thiol−disulfide exchange reaction. First through third generation of thiol-reactive biodegradable dendrons were obtained using a divergent synthetic strategy. Hydroxyl groups at the periphery of a second generation dendron were modified by initiator groups to obtain a hydrophilic four-armed star polymer. Successful conjugation to the pyridyl group at the core was demonstrated by attachment of a hydrophobic dye Bodipy-SH, and thiol bearing tripeptide glutathione and a KLAK-sequence containing apoptosis inducing peptide. Both in vitro cytotoxicity and apoptosis studies point out a clear increase in the ability of this peptide to induce apoptosis with increased effectiveness upon conjugation to the multiarm polymeric scaffold. The facile synthesis and the versatile use of these thiol-reactive redoxsensitive dendrons warrant their potential to serve as an attractive building blocks for construction of useful polymeric scaffolds for various biomedical applications.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the Bogazici University Research Fund (Project No. 13B05D4) and Ministry of Development of Turkey (Grant No. 2009K120520 and 2012K120480) for providing financial support for this research.

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