Complex Structured Fluorescent Polythiophene Graft Copolymer as a

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COMPLEX STRUCTURED FLUORESCENT POLYTHIOPHENE GRAFT COPOLYMER AS A VERSATILE TOOL FOR IMAGING, TARGETED DELIVERY OF PACLITAXEL, AND RADIOTHERAPY Emine Guler, Huseyin Akbulut, Caner Geyik, Tulay Yilmaz, Z. Pinar GUMUS, F. Baris Barlas, Recep Erdem Ahan, Dilek Odaci Demirkol, Shuhei Yamada, Takeshi Endo, Suna Timur, and Yusuf Yagci Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00491 • Publication Date (Web): 15 Jun 2016 Downloaded from http://pubs.acs.org on June 16, 2016

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COMPLEX STRUCTURED FLUORESCENT POLYTHIOPHENE GRAFT COPOLYMER AS A VERSATILE TOOL FOR IMAGING, TARGETED DELIVERY OF PACLITAXEL, AND RADIOTHERAPY Emine Guler1,2, Huseyin Akbulut3, Caner Geyik2, Tulay Yilmaz2, Z. Pinar Gumus2, F. Baris Barlas1, Recep Erdem Ahan3, Dilek Odaci Demirkol1, Shuhei Yamada4, Takeshi Endo4, Suna Timur1,2, Yusuf Yagci3 1

Ege University, Faculty of Science, Department of Biochemistry, 35100-Bornova,

Izmir/Turkey. 2

Ege University, Institute on Drug Abuse, Toxicology & Pharmaceutical Sciences, 35100-

Bornova, Izmir/Turkey. 3

Department of Chemistry, Faculty of Science and Letters, Istanbul Technical University, 34467-

Istanbul, Turkey. 4

Molecular Engineering Institute, Kinki University, 11-6 Kayanomori, Iizuka, Fukuoka, 820-

8555, Japan.

ACS Paragon Plus Environment

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ABSTRACT

Advances in polymer chemistry resulted in substantial interest to utilize their diverse intrinsic advantages for biomedical research. Especially, studies on drug delivery for tumors have increased to a great extent. In this study, a novel fluorescent graft copolymer has been modified by drug and targeting moiety and the resulted structure has been characterized by alterations in fluorescent intensity. The polythiophene based hybrid graft copolymer was synthesized by successive organic reactions and combination of in situ N-carboxy anhydride (NCA) ring opening and Suzuki coupling polymerization processes. Initially, targeted delivery of the graft copolymer was investigated by introducing a tumor specific ligand, anti-HER2/neu antibody, on the structure. The functionalized polymer was able to differentially indicate HER2-expressing A549 human lung carcinoma cells, whereas no signal was obtained for Vero, monkey kidney epithelial cells, and HeLa, human cervix adenocarcinoma cells. After integrating paclitaxel to the structure, cell viability, cell cycle progression, and radiosensitivity studies demonstrate HER2/neu targeting polymers were most effective to inhibit cell proliferation. Importantly, the graft copolymer used had no cytotoxic effects to cells as evidenced by cell viability and cell cycle analysis. This work clearly confirms that specially designed and fabricated graft copolymer with highly complex structure is a promising theranostic agent capable of targeting tumor cells for diagnostic and therapeutic purposes.

KEYWORDS: Polythiophene, L-Lysine, Graft copolymer, HER2/neu, Bioconjugation, Theranostic


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INTRODUCTION Advances in the synthetic polymer field enable researchers to design smart molecules to be used in biological sciences1–3. Especially in cancer research, developments not only in drug carrier systems but also in imaging technology are strongly governed by the use of flexible and biocompatible polymers4,5. Prominent advantages that are provided by the specially designed polymers include but not limited to enhanced permeability and retention (EPR) effect6,7, sitespecific targeting 5,8,9 and enhanced solubility of hydrophobic drugs10,11. Given the side effects of conventional chemotherapeutic agents to healthy cells, there is an evidential need for these drugs to act at the site of tumors12. It is possible to alter the backbone structure of the polymers owing to the functional groups that are capable of incorporating different molecules via covalent bonds. Thus, targeting agents such as antibodies or aptamers13 can be used along with polymers for smart delivery of drug. Additionally, it is possible to use responsive bonds to internal or external stimuli, providing release of the drug at the site of interest14,15. Increased activity of receptor tyrosine-protein kinase erbB-2, widely known as HER2/neu, is an important tumor biomarker especially in breast cancer. It has been shown that, expression of HER2/neu may also be increased in lung cancer16,17. It functions as an enhancer of proliferative and anti-apoptotic signals which play an important role in cancer cell survival18. Thus, therapeutic strategies such as silencing the HER2/neu gene19,20 or using monoclonal HER2/neu antibodies21 were emerged. Increased expression of this receptor in tumor tissue also permits transport of conventional chemotherapeutics to cancer cells by using anti-HER2/neu antibodies22,23 or aptamers24 as targeting agents.


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Along with the chemotherapeutic agents, radiotherapy is also being used in conventional treatments of cancer. This method relies on inducing the oxidative stress in tumor cells

25,26

.

Several tumor cell types were found to resist radiotherapeutic approach and thus, enhancing the radiosensitivity of these cells also holds importance27,28. Previous studies demonstrate an additional advantage of polymeric drug carriers in terms of increasing radiosensitivity of tumor cells29,30. Fluorescent polymers serve as versatile tools for molecular and cellular imaging, flow cytometry, and a wide variety of applications in biology and biotechnology31.

Like other

various electro-active conjugated polymers, polyenes and polyaromatics like polyaniline (PAn), polypyrrole (PPy), polythiophene (PT), poly(p-phenylene), poly-(phenylenevinylene) (PPV) classes have been extensively investigated in chemical sensing applications32–37. Advances in this field have focused on the development of fluorescence-based water soluble organic conjugated polymers relying on π-electron delocalization features. In this regard, PTs and its derivatives have become as most promising conjugated polymers in terms of relatively good electrochemical, electrochromic, luminescent, and shielding properties3. Moreover, additional properties can be imparted to PTs by combining thiophene polymerization with controlled polymerization methods38. It should be pointed out that such conjugated polymers can be prepared in a more controlled manner by recently developed “chain-growth condensation polymerization” in which polymer propagating ends are more reactive than the monomers due to resonance or inductive effects between the functional groups of the terminal monomer units. However, these systems were applied only for the monomers with low molecular weight side chains39.


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Recent novel studies in the field of bio-imaging and sensing have profoundly impacted the use of water-soluble biopolymers containing polypeptide moiety or side chains5. To obtain polypeptide various synthetic strategies have been developed. For synthesis of high molecular weight polypeptides, living ring-opening polymerizations of N-carboxyanhydride (NCA) of αamino acid are widely used. However, the susceptive nature of NCA for moisture and heating is major drawbacks for its production toward practical use. Thus, newly synthetic route for production of NCA through intramolecular cyclization of N-(phenyloxycarbonyl) amino acids under mild heating condition is recently most used method for the synthesis of polypeptides40. Although polymers bearing bound polypeptides provide unique properties and advantage as regards biofunctionalization, the use of soluble support is a more essential requirement in biological application. Water solubility can be achieved by the incorporation of polyethers, such as poly(ethylene glycol) (PEG). Polypeptide-PEG combinations as well as glycopeptides with various topological structures receive great attention due to their excellent solubility in both water and organic solvents, and their biocompatibility41,42. In this study, a novel fluorescent polymer consisting of PT, poly-L-lysine (PLL) and PEG side chains, PT-g-PLL-PEG, was investigated for its potential as a drug carrier for targeted cancer therapy. While polypeptide introduction imparts biofunctionalization to the polymer, PEG incorporation makes it water-soluble and biocompatible. Because of the good combination of these segments together with the fluorescent conjugated backbone, it was foreseen that the synthesized polymer would be a promising agent for biological applications. The hybrid graft copolymer was synthesized by a multistep approach involving several organic reactions combined with NCA in situ ring opening and conventional Suzuki coupling polymerization processes. The intermediates at various stages and final graft copolymer were characterized by


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spectral analyses and molecular weight measurements. For targeting studies, PT-g-PLL-PEG was conjugated to anti-HER2/neu antibody (PT-g-PLL-PEG/anti-HER2) and paclitaxel (PTX), which was

chosen

as

a

model

chemotherapeutic

agent

(PTX/PT-g-PLL-PEG/anti-HER2).

Bioconjugates were characterized by alterations in fluorescence signal of PT-g-PLL-PEG. Three cell lines used for in vitro studies. Human cervix adenocarcinoma (HeLa) and monkey kidney epithelial cell line (Vero) were selected as control cell lines that have basal expression of HER2/neu and human lung carcinoma (A549) was employed for higher HER2/neu expression. Fluorescence intensity studies were carried out to compare cellular polymer uptake among cell lines to show the imaging potential of the fabricated structure. Furthermore, the effects of PTX/PT-g-PLL-PEG/anti-HER2 on cell viability, radiosensitivity and cell cycle progression of A549 were evaluated. MATERIALS AND METHODS Reagents. Tetrakis (triphenylphosphine) palladium(0) (Pd(PPh3)4), thiophene-3-carboxylic acid, thiophen-3-ylmethanol, phosphorus tribromide(PBr3), N-bromosuccinimide (NBS), LLysine, copper(II) sulfate pentahydrate (CuSO4.5H2O), di-tert-butyl dicarbonate (Boc2O), tetrabutylammonium

hydroxide,

4-(N,N'-dimethyl)

amino

pyridine

(DMAP),

dicyclohexylcarbodiimide (DCC), poly(ethylene glycol) mono methyl ether (PEG2000), Phthalimide potassium salt and hydrazine monohydrate (98%), 2,5-thiophenediboronic acid are from Sigma-Aldrich and used without any further purification. Dulbecco's modified Eagle medium (DMEM), penicillin/streptomycin (10,000 UI/mL), L-Glutamine (200 mM). trypsin/EDTA (0.05% trypsin; 0.20 g/L EDTA), and phosphate buffered saline (PBS) used in cell culture experiments were purchased from Lonza. Fetal bovine serum (FBS) was obtained from Biowest. Carbonyldiimidazole (CDI), 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium


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bromide (MTT), hydrochloric acid (HCl), 4',6-diamidino-2-phenylindole (DAPI) and sodium dodecyl sulfate (SDS) were purchased from Sigma. Anti-HER2 antibody was obtained from Abcam. Cycletest™ Plus DNA Reagent Kit (BD BIOSCIENCES) was used for cell cycle analysis by flow cytometry. All solvents were analytical grade. The precursors and intermediates, namely 2,5-dibromothiophene-3-carboxylic acid (1), dibromothiophene-functionalized PEG macromonomers (DBT-PEG), (2,5-dibromothiophen-3yl)methanol

(2),

2,5-dibromo-3-(bromomethyl)thiophene

yl)methyl)isoindoline-1,3-dione

(4),

N6-Boc-L-lysine

(3),

2-((2,5-dibromothiophen-3-

(LL(boc))

and

N6-Boc-N2-

(phenoxycarbonyl)-L-lysine (NCA-LL(boc)) were synthesized according to the previously described procedures43, (See supporting information). Synthesis of Polypeptide Macromonomer (DBT-PLL(boc)). To the solution of NCALL(boc) (1.0 g, in 1.0 mL) dry DMAc in a flame-dried Schlenk tube, the amine initiator, MADBT, (15.6 mg, (0.23 mmol) in 0.5 mL) dry DMAc was added. The reaction mixture was stirred at 60 °C for 48 h under argon atmosphere. After polymerization, the mixture was cooled to room temperature and poured into diethyl ether. The precipitates were filtrated, and then dried under vacuum to yield DBT-PLL(boc) (490 mg, 80%) (Mw, LS = 54720 g.mol-1). 1H-NMR (d-DMSO, 500 MHz): δ 1.69 – 0.86 (broad), 2.93 – 2.76 (broad), 3.49 – 3.18 (broad), 6.74 (broad), 7.00 (s, 1H), 8.69–7.60 (broad). Synthesis of PT Bearing poly-L-lysine(boc) and PEG (PT-g-PLL(boc)-PEG). The reaction solution was prepared with 30 mL THF and 20 mL of aqueous 2.0 M K2CO3 solution under nitrogen. Prior to use, this solution was degassed by bubbling nitrogen over a period of 30 min. DBT-PLL(boc) (450 mg, 0.0082 mmol), DBT-PEG (100 mg, 0.016 mmol), 2,5-


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thiophenediboronic acid (10 mg, 0.058 mmol) and 2.0 mL reaction solution were placed in a Schlenk tube under nitrogen atmosphere. Pd(PPh3)4 (5.0 mg, 0.0043 mmol) was added under nitrogen and the reaction mixture was degassed by free-thaw technique. After stirring at 70 oC for 4 days the reaction mixture extracted with CH2Cl2 and small amount of water. Organic phase was concentrated and precipitated in excess diethyl ether to yield PT-g-PLL(boc)-PEG (300 mg, white powder). 1H-NMR (d-DMSO, 500 MHz): δ 2.02 – 1.05 (broad), 2.98 – 2.70 (broad), 3.44 – 3.10 (broad), 4.30 – 3.45 (broad), 6.70 (broad), 8.28 – 7.45 (broad). Synthesis of PT Bearing poly-L-lysine and PEG (PT-g-PLL-PEG). To the solution of PPPg-PLL(boc)-PLL (150 mg) in 20 mL of CH2Cl2 at 0 oC, TFA (2.0 mL) was added. The reaction solution was stirred at room temperature for 1 h. Then, the solvent was evaporated in vacuo. And the remaining material was precipitated in diethyl ether to yield PPP-g-PLL-PLL (80 mg). 1HNMR (d-DMSO, 500 MHz): δ 1.81 – 1.16 (broad), 2.98 – 2.70 (broad), 3.44 – 3.10 (broad), 4.30 – 3.45 (broad), 8.74 – 7.39 (broad). Bioconjugation and Characterization. In order to evaluate the effect of each ligand, we synthesized various conjugates with different combinations. For this purpose, PT-g-PLLPEG/anti-HER2, PTX/PT-g-PLL-PEG and PTX/PT-g-PLL-PEG/anti-HER2 bioconjugates were designed and prepared by using both carbonyldiimidazole (CDI) chemistry44 and protein adsorption onto the polymer scaffold. In the first step of bioconjugation, 5.0 µg/mL PTX and 50 µg/mL CDI were dissolved in 250 µL of dimethyl sulfoxide (DMSO) and then the obtained mixture was kept at 37 °C for 2 h at 1000 rpm to activate functional hydroxyl groups of PTX. Afterwards, 250 µL of PT-g-PLL-PEG (2.5 mg/mL dissolved in DMSO) was added and the reaction solution was maintained under shaking with 1000 rpm at 25 °C overnight. At the end of the reaction time, unconjugated molecules, excess CDI and reaction by-products were removed


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by dialysis against PBS (pH 7.4, 10 mM) at +4 °C. Then 300 µL of the obtained PTX/PT-g-PLLPEG conjugate was diluted in 1200 µL PBS (pH 7.4, 10 mM) in order to reach desired concentrations of both drug and polymer for cell culture experiments. On the other hand, 200 µL of the PTX/PT-g-PLL-PEG conjugate solution was used for the second synthesis step including anti-HER2 linking. For this purpose, 250 µL anti-HER2 antibody was added to the solution containing PTX/PT-g-PLL-PEG conjugate and allowed for interaction at room temperature at 1000 rpm mixing in a thermal shaker overnight. Then the obtained final bioconjugate PTX/PT-gPLL-PEG/anti-HER2 was purified from unbounded molecules by dialysis method. The synthesis of PT-g-PLL-PEG/anti-HER2 was carried out according to the same procedure including second step of bioconjugation by using of PT-g-PLL-PEG instead of PTX/PT-g-PLL-PEG conjugate. In order to evaluate the success of bioconjugation, fluorometric characterization was applied to all of the bioconjugates. Fluorescence spectra of step by step modified bioconjugates were recorded by using a Varioskan™ Flash Multimode Reader (Thermo Scientific, USA). In addition, the amount of the drug linked to polymer and the antibody content of the conjugates were determined by HPLC analysis and Bradford assay, respectively. Conjugation Efficiency. To assess the drug binding efficiency, High Performance Liquid Chromatograpy (HPLC) method was applied.

As we mentioned before, the synthesized

conjugates were dialyzed to remove unbounded drug. The free drug in dialysis solution as analyzed by HPLC was used to determine drug binding efficiency. Hence, the percentage of drug binding was calculated according to the following equation: Drug conjugation efficiency (%) = [(Total PTX added - Free PTX in dialysis solution)/Total PTX added] x 100


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The following analysis conditions were used for this work: The chromatographic separation was performed using an Agilent HPLC-DAD system and Eclipse XDB-C18 column (4.6 mm, 150 mm, 5.0 µm). Flow rate of the mobile phase, consisting of a mixture of (A) 0.1% (v/v) aqueous phosphoric acid and (B) acetonitrile (40:60, v:v) was 1.20 mL min-1. The detector wavelength was set at 227 nm. The injection volume was 20.0 µL and the column temperature was maintained at 25 ˚C. Cell Culture. Human cervix adenocarcinoma (HeLa), human lung carcinoma (A549) and monkey kidney epithelial cell line (Vero) were maintained in DMEM supplemented with 10% FBS, 100 UI/mL Penicillin/Streptomycin, and 2.0 mM L-Glutamine at 37 °C in a humidified incubator with 5.0% CO2. Cells were subcultured at 80% confluency by trypsinization. PT-gPLL-PEG polymer and bioconjugates were dissolved in DMEM in all experiments. Cell Viability. Colorimetric MTT assay was used to assess relative cell viability. Briefly, 10,000 cells/well were incubated in 96-well cell culture plate for 24 h at standard culture conditions. After the incubation, cells were treated either with PT-g-PLL-PEG, PT-g-PLLPEG/anti-HER2, PTX/PT-g-PLL-PEG, and PTX/PT-g-PLL-PEG/anti-HER2 for 24, 48, and 72 h. All solutions were diluted with DMEM with final PT-g-PLL-PEG concentrations of 0.005– 0.250 mg/mL. The dose range of PTX in bioconjugates was 1.5–75 ng/mL. After desired incubation time with the polymer and bioconjugates, MTT solution (0.5 mg/mL in DMEM) was added to each well and incubated for 4 h. Intracellular formazan crystals produced by the enzymatic activity of living cells were dissolved in 10% SDS (in 0.01 M HCl) and quantified by reading the absorbance at 570 nm. Absorbance at 620 nm was used as reference wavelength.


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DMEM without any sample was used as control and considered as 100% viable. Relative cell viability was plotted as the percent absorbance of sample treated cells. Fluorescence Microscopy. HeLa, A549, and Vero cells were plated on chamber slides (6,000 cells/well) and incubated for 48 h before sample application. Then, medium was refreshed with 0.125 mg/mL PT-g-PLL-PEG or PT-g-PLL-PEG/anti-HER2 (equivalent polymer concentration, 0.125 mg/mL) containing growth media and incubated for 2 h. Images were obtained with (Olympus BX53 F) equipped with a CCD camera (Olympus DP72). Cell Cycle Analysis. 100,000 A549 cells/well were incubated in 6-well cell culture plate for 24 h. Cells were pre-incubated for 24 h with FBS-free DMEM to synchronize cell cycle. Following serum starvation, cells were incubated with PT-g-PLL-PEG/anti-HER2 (0.125 mg/mL), PTX/PT-g-PLL-PEG, PTX/PT-g-PLL-PEG/anti-HER2 and PTX alone (equivalent PTX concentration of 75 ng/mL), for 24 h. Cells were prepared to flow cytometry analysis as recommended in manufacturer’s protocol. 20,000 cells were analyzed for propidium iodide fluorescence signal in flow cytometer (FACSAria, BD BIOSCIENCES). Data were plotted by using Flowing Software 2.5.1, (Cell Imaging Core, Turku Centre for Biotechnology). Radiosensitivity. A549 cells were exposed to ionized radiation in presence of PT-g-PLL-PEG, PT-g-PLL-PEG/anti-HER2, PTX/PT-g-PLL-PEG, and PTX/PT-g-PLL-PEG/anti-HER2 to investigate the effect of the polymer on radiosensitivity. 4,000 cells/well were incubated in 96well cell culture plate for 24 h under standard culture conditions. Then, medium was removed and PT-g-PLL-PEG, PT-g-PLL-PEG/anti-HER2 (equivalent of 0.125 mg/mL polymer), PTX/PT-g-PLL-PEG, and PTX/PT-g-PLL-PEG/anti-HER2 (equivalent of 1.0 ng/mL PTX) were applied for 2 h. Cells were exposed to irradiation of 2.5 and 5.0 Gray (Gy) with photon beams generated by 6 MV linear accelerator system (LINAC, Siemens Primus, Germany). No radiation


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(0 Gy) was used as control. After radiation treatment, cells were incubated for 72 h and cell viability was assessed via MTT method (vide ante). RESULTS AND DISCUSSION The synthetic approach described here is based on the use of functional macromonomers in coupling polymerization processes that was successfully used for the other conjugated polymer such as polyphenylenes45–47 and poly(phenylene vinylene)s5,48,49. In the present study, thiophene functional macromonomers and their precursors were synthesized and characterized according to the previously established methods. The details of the preparation of 2,5-dibromothiophene-3-carboxylic acid , dibromothiophene-functionalized PEG macromonomer

(DBT-PEG),

(2,5-dibromothiophen-3-yl)methanol,

2,5-dibromo-3-

(bromomethyl)thiophene, 2-((2,5-dibromothiophen-3-yl)methyl)isoindoline-1,3-dione, N6-BocL-lysine (LL(boc)) and N6-Boc-N2-(phenoxycarbonyl)-L-lysine (NCA-LL(boc)) are given in the supporting information as Schemes S1, S2, and S3 and Figures S1, S2, and S3). Dibromotiophene

Functionalized

Polypeptide

Macromonomer.

Dibromothiophene

functionalized poly(L-lysine(boc)) (DBT-PLL(boc)) was synthesized via in situ NCA ROP in presence of MA-DBT as amine initiator. The NCA precursor forms NCA ring in the reaction pot via elimination of phenol in its structure. The carbonyl carbon of NCA is attacked by the primary amine group of initiator which yields amide bond between initiator and monomer with formation of CO2. Polypeptide chain growth is established by nucleophilic of activated amine side of opened NCA ring to another carbonyl carbon of NCA molecule, (Scheme 1). Scheme 1. Synthesis of DBT-PLL(boc) by in situ NCA ring opening polymerization.


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O O

H N

HO R

NH2

O O

O R

MA-DBT Br

DMAc, 60 ºC

NCA-LL(boc)

S

Br

N H

HN

O

R

p

H

Br O

-PhOH

H N

-CO2

Br S DBT-PLL(boc)

R = -(CH 2 ) 4NHC(=O)OC(CH 3) 3

The structure of 2,5-dibromothiophene-end-group functionalized poly(L-lysine(boc)) (DBTPLL(boc)) was confirmed with 1H-NMR analysis, (Figure 1). Protons of characteristic terminal group and repeating units are annotated in Figure 1.

Figure 1. 1H-NMR spectrum of DBT-PLL(boc) Synthesis of Polythiophenes Bearing Polypeptide and PEG side chains. Suzuki condensation polymerization was used to incorporate polypeptide and PEG groups onto polythiophene backbone. Coupling partners, DBT-PEG and DBT-PLL(boc) are reacted and coupled by palladium catalysis in presence 2-5 thiophenediboronic acid (Scheme 2). To obtain sufficient molecular weight, the reaction time was prolonged to 4 days. PEG macromonomer was deliberately taken in excess amount compare to the polypeptide macroonomer (mol: mol, 2:1) so as to achieve adequate solubility. Scheme 2. Copolymerization of DBT-PLL(boc) and DBT-PEG via Suzuki Coupling.


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O N H Br

H N

O p

R

H HO B HO

Br

S DBT-PLL(boc)

Pd(PPh3 )4 H H N

O

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NH

S

O

OH B OH

Br

O n

Br S DBT-PEG

THF / H 2 O K 2CO3

O

O O

p N H

O

S S

O n

S m

S

k

PT-g-PLL(boc)-PEG 1

H-NMR analysis was performed to reveal the structure of PT-g-PLL(boc)-PEG. As can be

seen from Figure 2, the spectrum exhibits the characteristic signals corresponding to the protons of the PT backbone, and PEG and PLL(boc) side chains.

Figure 2. 1H-NMR spectrum of PT-g-PLL(boc)-PEG The elimination of boc protecting groups from the polypeptide side chains of PT-g-PLL(boc)PEG in presence of TFA lead to formation of water soluble conjugated polymer (PT-g-PLLPEG), (Scheme 3) facilitated with the formation of free primary amine groups in the side chain.


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Scheme 3. Synthesis of PT-g-PLL-PEG by deprotection of PLL(boc) side chains by TFA.

H

NH

O

H N

O

O O

p N H

O

S S

O n

S S

m

k

PT-g-PLL(boc)-PEG

H

NH

TFA

O

CH2Cl2 O

H 2N

O

p N H S S

O n

S m

S

k

PT-g-PLL-PEG

Successful deprotection process was confirmed by 1H-NMR, (Figure 3). Notably, the signals corresponding to the amide protons disappeared completely.

Figure 3. 1H-NMR spectrum of PT-g-PLL-PEG The chemical structure of PT-g-PLL-PEG was further established by FTIR spectroscopy (Figure S4). The broad band at 3306 cm-1 is assigned to N-H asymmetric stretching vibrations of amide


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groups. The band at 2923 cm-1 was attributed to asymetric C-H stretching vibrations. The presence of a band at about 1116 cm-1 was related to C-O-C stretching vibration. The bands at around 1645 cm-1 and 1436 cm-1 show C=O (amide I) and N-H bending (amide II), respectively. The appearance of characteristic absorption peaks at 1116 and 1318 cm−1 corresponding to C-OC stretching and asymmetric stretching, respectively. In addition, the appearance of peaks at 2923 and 953 cm−1 for -CH2 stretching vibration and -CH out-of-plane bending vibration confirms the presence of PEG in the PT-g-PLL-PEG polymer. For the PT-g-PLL-PEG the bands were observed at 713 cm−1 (thienylene C-Hα tri-substituted ring bend) and 1010 cm−1 (thienylene C-Hα in plane bend). In addition, the peak monitored at 904 cm−1 indicated cis C-H wagging of the thiophene ring33a,37. The molecular weight characteristics of the precursor macromonomers and the graft copolymer after Suzuki coupling process were investigated by GPC (Table 1). Due to hybrid nature of the graft copolymer and difference in the hydrodynamic volumes of the structurally different segments, the expected molecular weight increase is limited. In this connection, it should also be pointed out that due to the possible degradation during synthesis, DBT ratio was deliberately kept in excess. Thus, graft copolymer with short thiophene repeating units was obtained. However, the graft copolymer has a monomodal distribution and clear shift to higher molecular weight region indicates successful coupling process (Figure S5). The observed high polydispersity is typical for the step-growth polymerization processes. Table 1. Molecular weight characteristics of precursor macromonomers and the final graft copolymer, DBT-PLL(boc), DBT-PEG, and PT-g-PLL(boc)-PEG Polymer

Mwa [g mol-1]

Mw/Mna


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a

DBT-PEG

6900

1.24

DBT-PLL(boc)

54700

2.98

PT-g-PLL(boc)-PEG

83200

4.58

Determined GPC by using light scattering detector according to polystyrene standards.

The UV-Vis absorption spectra and photophysical characteristics of the precursors and the final graft copolymer are shown in Figure S6 and Table S1, respectively. Notably, the graft copolymer has an absorption in 325- 400 nm region where the precursors are transparent. The relatively low quantum yield overserved in water may be due to the aggregations taking place during the nanoparticles formation through self-assembling of PTs backbones in solution. In fact, this aggregation deprive from strong interactions between hydrophobic backbones and aromatic π-π stacking of conjugated backbone in water that severally decreses their water solubility and photoluminescent quantum efficiency. Characterizations of Bioconjugates. The influence of each ligand was investigated comparatively by designing different conjugates including PT-g-PLL-PEG/anti-HER2, PTX/PTg-PLL-PEG and PTX/PT-g-PLL-PEG/anti-HER2. In addition, all results were compared with the bare PT-g-PLL-PEG. For the synthesis strategy, fluorescent copolymer PT-g-PLL-PEG was used as the scaffold for the covalent attachment of chemotherapeutic chemotherapeutic agent PTX and adsorption of targeting ligand anti-HER2. The bioconjugation steps include: i. covalent binding between hydroxyl group of PTX and amino group of PT-g-PLL-PEG (Scheme 4A), ii. adsorption via interaction between PT-g-PLL-PEG and anti-HER2 (Scheme 4B). While the wellknown CDI chemistry was applied for the first conjugation step, physical adsorption technique was used for the second step of bioconjugation. Attachment of antibody to the PT-g-PLL-PEG can attribute to the possible interactions between PLL chain of polymer and antibody. This


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interaction can be assumed that non-covalent electrostatic attraction forms between negatively charged –COO- groups of antibody and positively charged –NH3+ groups of PLL50. For the synthesis of PT-g-PLL-PEG/anti-HER2, only the second step was applied as PTX is not present in the bioconjugate structure, (Scheme 4C). Scheme 4. Synthesis steps of PT-g-PLL-PEG bioconjugates including A) PTX/PT-g-PLL-PEG, B) PTX/PT-g-PLL-PEG/anti-HER2 and C) PT-g-PLL-PEG/anti-HER2.

Spectroscophic characterization was performed for each step of bioconjugation by fluorescence measurements. Fluorescence signals of PT-g-PLL-PEG polymer before and after conjugation with different ligands are presented in Figure 4. In accordance with emission behavior of polymer and conjugates, fluorescence signals were centered at ~460 nm. The decrease in the fluorescence intensities with slight shifts after conjugation processes clearly


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confirms successful binding of the ligands, namely PTX and anti-HER2 into PT-g-PLL-PEG structure.

Figure 4. Florescence spectra of PT-g-PLL-PEG, PT-g-PLL-PEG/anti-HER2, PTX/PT-g-PLLPEG and PTX/PT-g-PLL-PEG/anti-HER2 conjugates (in PBS buffer using 395 nm for the excitation). In addition to the fluorescence characterization, anti-HER2 binding was confirmed by Bradford assay. For this aim, both anti-HER2 containing conjugate and PT-g-PLL-PEG polymer alone were applied to Bradford assay. In Bradford assay, a signal detection for PT-g-PLL-PEG is possible as it possesses polypeptide side groups. Hence, the amount of conjugated anti-HER2 was calculated by using the signal difference between PT-g-PLL-PEG and polymer/anti-HER2 conjugates and found to be 60.76 µg/mL. Thus, anti-HER2 conjugation to the polymeric structure was confirmed by a well-known protein assay.


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Conjugation Efficiency. The conjugation efficiency was calculated as the percentage of the amount of drug linked to dialyzed conjugates with reference to the total amount which was added in the initial step of the conjugate preparation. According to this calculation, drug conjugation efficiency of the newly synthesized polymer, PT-g-PLL-PEG, was determined to be 59% corresponding to the HPLC method. Limit of detection (LOD) and limit of quantification (LOQ) values were calculated as 1.91 ng/mL and 6.36 ng/mL, respectively by HPLC using DAD. These results indicate that the most of the PTX is effectively attached to the polymer through CDI coupling. Effects of Bioconjugates on A549 Cell Viability. Effects of the drug carrier polymers on cell viability should be examined to ensure they are not cytotoxic to cells alone. Thus, PT-g-PLLPEG, and PT-g-PLL-PEG/anti-HER2 were introduced to A549 cells to assess non cytotoxic concentrations for 24, 48, and 72 h (Figure 5). There is no significant decrease on cell viabilities up to 0.125 mg/mL dose whereas cell viability was decreased at 0.25 mg/mL. The appropriate highest dose was found to be 0.125 mg/mL up to which no significant decrease on cell viability was observed and used for fluorescent microscopy and PTX binding studies. Notably, the cell viability was substantially decreased at 0.25 mg/mL.


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Figure 5. The effects of polymer bioconjugates without PTX on A549 cell viability (PT-g-PLLPEG on the left, and PT-g-PLL-PEG/anti-HER2 on the right). Bioconjugates without PTX were plotted against equivalent polymer concentrations. Non-treated cells considered as 100% viable. Bars represent ±SD, (n=4). Thereafter, the effect of chemotherapeutic agent PTX was studied by comparison of the targeting ligand polymer before (PTX/PT-g-PLL-PEG) and after conjugation (PTX/PT-g-PLLPEG/anti-HER2). Concerning the cytotoxicity data, the samples (PTX/PT-g-PLL-PEG and PTX/PT-g-PLL-PEG/anti-HER2) applied are selected by considering the PTX amount in the conjugates. (Which equals 0.6 µg PTX/ 1.0 mg polymer). Figure 6 shows that both structures displayed similar effects up to 7.5 ng/mL PTX concentration. However, PTX/PT-g-PLL-PEG showed inhibition on cell viability for higher doses which was not dose dependent. It is


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characteristic for PTX to inhibit cell proliferation at constant rate for a wide dose range 51. On the other hand, higher concentrations of PTX/PT-g-PLL-PEG/anti-HER2 showed a dose dependent decrease in cell viability. This can be attributed either increased HER2-receptor mediated delivery of PTX52,53 or the synergistic effect caused by the fact that anti-HER2 antibody has therapeutic benefits alone21,54. To further investigate the cellular effects of the designed PTX/PTg-PLL-PEG/anti-HER2, cell cycle analyses were conducted.

Figure 6. The effects of polymer bioconjugates bearing PTX on A549 cell viability (PTX/PT-gPLL-PEG on the left, and PTX/PT-g-PLL-PEG/anti-HER2 on the right). Bioconjugates with PTX were plotted against equivalent PTX concentrations (Which equals 0.6 µg PTX/1.0 mg polymer). Non-treated cells considered as 100% viable. Bars represent ±SD, (n=4).

Cell Imaging via Fluorescence Microscopy. It is important to decrease side effects caused by chemotherapeutic drugs. Therefore, PT-g-PLL-PEG is modified with anti-HER2 antibody for


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selective targeting of HER2 positive cells. Cellular polymer uptake of healthy cells (Vero) and adenocarcinoma cells (HeLa) that are negative for HER2 expression were compared to A549 cells which has higher HER2 expression. Owing to intrinsic fluorescence properties of PT-gPLL-PEG polymer, cellular uptake was monitored by fluorescence microscopy (Figure 7). Without anti-HER2 conjugation, PT-g-PLL-PEG signal was low in all of three cell lines used. However, by introducing the anti-HER2 antibody to the structure, PT-g-PLL-PEG polymer was able to readily taken to A549 cells whereas there were no observable difference for Vero and HeLa cells. These results suggest that modification of the PT-g-PLL-PEG with targeting ligand, anti-HER2 antibody, effectively differentiates the fluorescent signal obtained from healthy cells or HER2 non-expressing carcinoma cells and HER2 positive cells, indicating feasible use of PTg-PLL-PEG/anti-HER2 bioconjugate for tumor imaging studies. While our fluorescent polymer has maximum emission peak at 460 nm after excitation at 395 nm, it enables us to obtain red fluorescence images by using green excitation filter under the fluorescent microscope. Emission light at 460 nm corresponds to blue images and DAPI as a well-known fluorescent dye that stains cell nuclei also emits blue fluorescence (Emission: 461 nm). For this reason, we preferred to capture red fluorescence images of cells treated with PT-gPLL-PEG polymer to avoid overlapping of two blue images obtained by using both our polymer and DAPI. Thus we have eliminated the limitations of excitation in the ultraviolet by using green excitation filter during fluorescence monitoring. In addition, our design to target fluorescent polymer to the desired cell type via conjugation a targeting ligand, can contribute to potential practical applications of the developed fluorescent probe. In the case of clinical applications, fluorescent agent can direct to a specific environment which can lead to high target-to-


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background ratio, enabling both visualization of small tumors against a dark background and minimization of negative effects on healthy cells.

Figure 7. Fluorescent images of the cells treated with HER2 targeted and non-targeted fluorescent PT-g-PLL-PEG polymer. Scale bar represents 10 µM. (Red emission; 604-644 nm, blue emission; 435–485 nm) Cell Cycle Analysis. Cell viability studies showed that PTX/PT-g-PLL-PEG/anti-HER2 showed a dose dependent decrease in cell viability. Cell cycle analyses were carried out to further investigate the effects of PTX compared to PTX-conjugated polymers.


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Figure 8. Flow cytometric analyses for cell cycle progression. Apoptotic cell population is shown as the sub-G1 peak. Arrow indicates G2/M phase of the cells. A) Non-treated cells, B) PT-g-PLL-PEG, C) PTX/PT-g-PLL-PEG/anti-HER2, and D)PTX. PT-g-PLL-PEG showed similar cell cycle progression compared to the non-treated controls (Figure 8 A,B). On the other hand, PTX alone resulted in an increase in G2/M peak (Figure 8 D). PTX exerts its effects by inhibition of the spindle formation as a consequence of disruption of the microtubule depolymerization55,56. Thus, cells treated with PTX were arrested in G2/M phase of the cell cycle. This effect is highly dependent on the concentration of PTX where lower doses induce apoptosis instead of G2/M arrest11,57. The concentration of PTX used in this study was high enough to induce arrest. Interestingly, PTX/PT-g-PLL-PEG/anti-HER2 lead to an


25

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accumulation of sub-G1 cell population which indicates apoptosis in A549 cells (Figure 8 C). This could be arisen from synergistic effect of anti-HER2 and PTX since anti-HER2 also disrupts cell cycle via G1/G0 arrest and stimulation of pro-apoptotic pathways58. Radiosensitivity. Introducing radiosensitizers to tumor cells enhances the effectiveness of radiotherapy59. Possible enhancement of the radiosensitivity of the A549 cells by PT-g-PLLPEG, PT-g-PLL-PEG/anti-HER2, PTX/PT-g-PLL-PEG, and PTX/PT-g-PLL-PEG/anti-HER2 was investigated (Figure 9). Without any radiation treatment (i.e. 0 Gy), there was no difference between the experimental groups. PT-g-PLL-PEG polymer alone showed higher inhibition on cell viability for both 2.5 and 5.0 Gy radiation compared to control cells. Additionally, this effect was higher when it was conjugated with anti-HER2 antibody. PTX containing bioconjugates PTX/PT-g-PLL-PEG and PTX/PT-g-PLL-PEG/anti-HER2 further enhanced the radiotherapeutic effect at 2.5 Gy and PTX/PT-g-PLL-PEG/anti-HER2 was most effective at 5 Gy. It is known that PTX have radiosensitizing effect on tumor cells60,61. Thus, PTX targeting with anti-HER2 antibody resulted efficient inhibition on cell proliferation.


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Figure 9. Radiosensitivity of PT-g-PLL-PEG, PT-g-PLL-PEG/anti-HER2, PTX/PT-g-PLL-PEG, and PTX/PT-g-PLL-PEG/anti-HER2 was determined via cell viability after irradiation of the cells. Non-treated cells considered as 100% viable. Bars represent ±SD (n=4). CONCLUSIONS In this study, a specially designed fluorescent PT-g-PLL-PEG polymer with functional groups was successfully modified with dual ligands; i) chemotherapeutic agent, PTX, and ii) targeting agent, anti-HER2 antibody. Binding efficiency of PTX was calculated to be 59% according to HPLC method. PT-g-PLL-PEG/anti-HER2 conjugate was tested in three model cell lines of healthy cells, HER2/neu non-expressing carcinoma and HER2/neu expressing carcinoma, and found to identify A549 cells that express HER2/neu selectively. This indicates that, it is possible to use PT-g-PLL-PEG/anti-HER2 for diagnostic purposes. Furthermore, investigations of PT-gPLL-PEG as a drug carrier were carried out. Cell viability studies showed that when it is targeted with HER2 ligand, inhibitory effect of PTX was increased. Cell cycle analysis revealed that


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combining anti-HER2 antibody with PTX alters the cell progression from G2/M arrest to induction of apoptosis. Additionally, radiotherapy studies demonstrated that, PT-g-PLL-PEG polymer have radiosensitizer effect which enhances with the introduction of PTX and anti-HER2 antibody. Considering the combination of chemotherapy and radiotherapy, the described approach provides possibility to use lower dosage of drug and/or irradiation intensity which would result lesser side effects and better prognosis of the illness. In summary, flexible nature of the PT-g-PLL-PEG polymer resulted in a theranostic agent that can target tumor cells specifically for visualization and suppression. ASSOCIATED CONTENT Supporting Information. Synthesis steps of the graft copolymer and relevant FT-IR, NMR, UV-Vis and Fluoroscence spectra and GPC chromatograms. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] *E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes


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The authors declare no competing financial interest. ACKNOWLEDGMENT Authors thank Research and Education Laboratory of Ege University School of Medicine (AREL) for flow cytometry analyses. We thanked to Dr. A. Murat Senisik for the fruitful support in the radiotherapy experiments. REFERENCES (1)

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Complex Structured Fluorescent Polythiophene Graft Copolymer as a

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