From Invisible Structures of SWCNTs toward Fluorescent and

Aug 29, 2013 - Department of Biochemistry, Faculty of Science, Ege University, 35100 Bornova-Izmir, Turkey. ‡. Institute for Technical Chemistry, Leib...
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From Invisible Structures of SWCNTs toward Fluorescent and Targeting Architectures for Cell Imaging Didem Ag,† Muharrem Seleci,† Rebecca Bongartz,‡ Mustafa Can,§ Seda Yurteri,∥ Ioan Cianga,∥,⊥ Frank Stahl,‡ Suna Timur,*,† Thomas Scheper,‡ and Yusuf Yagci*,∥,# †

Department of Biochemistry, Faculty of Science, Ege University, 35100 Bornova-Izmir, Turkey Institute for Technical Chemistry, Leibniz University of Hannover, Callinstrasse 5, 30167 Hannover, Germany § Faculty of Engineering and Architecture, Engineering Science, Katip Celebi Izmir University, 35620 Balatcik- Izmir, Turkey ∥ Department of Chemistry, Istanbul Technical University, Maslak, Istanbul 34469, Turkey ⊥ Petru Poni Institute of Macromolecular Chemistry, Iasi 700487, Romania # Center of Excellence for Advanced Materials Research (CEAMR) and Chemistry Department, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia ‡

S Supporting Information *

ABSTRACT: Single-walled carbon nanotubes (SWNTs) are unique nanostructures used as cargo systems for variety of diagnostic and therapeutic agents. For taking advantage of these structures in biological processes, they should be visible. Therefore, fluorescence labeling of SWCNTs with various probes is a significant issue. Herein, we demonstrate a simple approach for cell specific imaging and diagnosis by combining SWCNTs with a copolymer poly(para-phenylene) (PPP) containing polystyrene (PSt) and poly(ε-caprolactone) (PCL) side chains (PPP-g-PSt-PCL). In this approach PPP-g-PSt-PCL is noncovalently attached on carboxyl functional SWCNTs. The obtained fluorescent probe is bound to folic acid (FA) for targeted imaging of folate receptor (FR) positive HeLa cells. In vitro studies demonstrate that this conjugate can specifically bind to HeLa cells and indicate great potential for targeting and imaging studies.

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INTRODUCTION Semiconducting polymers is a class of specialty soft materials that have undergone an unprecedented pace of development in the last four decades. Near to the intellectual curiosity of scientists that focuses on understanding the behavior of these systems, in particular on the mechanism of charge transfer and charge transport processes occurring in the course of redox reactions of conducting polymeric materials, the wide range of promising applications was the major reason stimulating the intense research in this polymer family.1 The knowledge accumulated during the last two decades has been a good guide concerning the opportunities of applications for their useful properties. Therefore, the variation of conductivity could be used in electronic devices including thin film transistors or insulated gate field effect transistors2 and in gas sensors as well.3 The color change could find application in electrochromic display devices or in smart windows,4,5 whereas the electroluminescence could be applied in light emitting devices,6,7 the swelling−deswelling accompanying the charging−discharging processes in artificial muscles8 or the charge storage capacity in energy technologies (organic photovoltaics).9,10 In this class of materials, poly(p-phenylene)s (PPP)s are by far the oldest investigated ones,11 and the history of the research developed for their synthesis and improvement of the © XXXX American Chemical Society

processing capability has been reported in a comprehensive review.12 Although these materials possess such interesting properties like electroluminescence, electrical conductivity (when doped), liquid crystallinity, high third-order optical nonlinearities, impressive mechanical properties, and the rigid, conjugated backbone that is generally responsible for these attributes also renders these polymers insoluble and infusible, and therefore, nonprocessable. So, during that time, the main driving force behind the new developed synthesis methods of PPPs was the aim to overcome the inherent insolubility and intractability, which these structures pose as a formidable obstacle in front of their properties accessing. Research has been devoted to circumventing this insolubility/intractability problem, largely through the development of two basic, divergent synthetic strategies. The “precursor routes” involved the synthesis of a precursor polymer containing a solubilizing functionality that can then be eliminated, generally by treatment with heat, to give the desired conjugated polymer.13−15 For a milder route to PPP, in comparison with the thermolysis of appropriate precursors the incorporation of Received: June 12, 2013 Revised: August 13, 2013

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long, flexible side chains that disrupt the crystallinity and exploit the entropy of solvent-side chain mixing was used in order to gain solubility, mediated by transition-metal-catalyzed aryl−aryl coupling.12 Yamamoto et al. used 1,4-dibromobenzene with solubilizing n-alkyl side chains in the 2- and 5-positions as a monomeric building block in conjunction with magnesium and various low-valent nickel catalysts.16 Schlüter, and Wegner et al. used the palladium-catalyzed heteroaryl−aryl coupling of various dialkyl-substituted dibromobenzenes with phenylboronic acids and the soluble, highest molecular weight accompanied by the highest defect free structure PPPs was reported, by translating Suzuki −Myiaura coupling reaction from organic chemistry to the polymer science.17−19 From this point, it was only one step toward macromolecules called “hairy-rods”. Wegner, stated that “shape persistence”, an important principle in the world of biomacromolecules, can be achieved by direct synthesis of “hairy-rods”.20 This concept is based on the introduction of conformationally mobile, relatively long, flexible and densely grafted side chains to a rigid backbone and was transferred to the conjugated polymers field.21−26 The resulting rod−coil copolymers, in which the rod sequence is a conjugated polymer, form domains that can be organized to give a plethora of different morphologies in solid and solventdispersed states.27,28 Unlike coil−coil copolymers, the rod−coil ones can form ordered structures even at low molecular weights due to the anisotropic molecular shape and orientational organization imparted by the stiff rod-like conformation of the rod blocks. The rise of the new polymerization methods, notably of controlled free radical polymerizations, inspired us to combine them with those specific for conjugated PPPs (Yamamoto and Suzuki polycondensation types), a strategy that facilitated the synthesis of various “hairy-rods”.29,30 In related studies31−35 from the authors’ laboratory, different monografted or heterografted types of “hairy-rods” were obtained through the combination of hydrophobic conjugated, rigid backbones with hydrophobic/hydrophilic flexible coils by taking advantage of the molecular design flexibility of “macromonomer technique”. The key point of the strategy was to synthesize low molecular weight intermediates which contain in their structure useful functionalities for both initiating controlled polymerization of conventional monomers (for example styrene) and subsequent Yamamoto or Suzuki polycondensation. This way, control over the molecular weight and dispersity of the flexible side chains as well as the nature of the rigid main chains together with the topology of the copolymers was achieved. Upon the type of the main chain, PPPs,31−35 poly(p-phenylene vinylene) (PPV)s,36 or different conjugated copolymers37−39 were synthesized. Generally, as grafted flexible coils, traditional polymers like polystyrene (PSt),40−42 poly(ε-caprolactone) (PCL),33 polyethylene glycol (PEG),34,35 and their combination,43−45 or block-copolymers46−48 were successfully used. The presence of the conjugated sequences allows the induction of special properties to these common polymeric materials (electro/ photo/piezo-activity) and also self-assembling capability in appropriate conditions. Nowadays, an impetuous research has been developed in semiconducting polymer field oriented toward theirs biomedical applications49−51 and the opportunity of their use in tissue engineering52,53 or as nanomedicines54,55 was recently demonstrated. Conversely, the use of PPPs in biomedical applications has been scarcely investigated. Several reported studies involve their use as fillers in an acrylic denture

and dental adhesive resin systems56−58 and as fluorescent labels in bioimaging.34,35 Cancer is a serious and widespread health threat that is investigated regarding its early diagnosis and therapy by most scientists. Early detection of tumor cells is a crucial need for an effective cure of diseases. Targeted molecules in cancer cells have a great potential for cell type specific detection in diagnosis. Therefore, development of targeting methods using new materials is still very important. The use of nanomaterials appears to be the most prominent approach to develop an efficient detection method. These nanoscale particles have large surface area and natural functionalities that allow easy structural modifications for altering their pharmacokinetics, improving their extravasations capacity, prolonging their vascular circulation lifetime, providing an enhanced biodistribution in vivo, and cause a continuous and controllable delivering efficiency as drug cargoes.59,60 Nanoparticles provide also an effective bridge between bulk materials and molecular structures to develop new multifunctional architectures for drug delivery systems, diagnostics, and therapy. Among various nanostructured materials, carbon nanotubes (CNTs) are very promising materials for diagnostic,35 gene61 and drug delivery62 applications due to their unique structural and mechanical properties. Based on their structure, they can be classified in two categories, namely single-wall carbon nanotubes (SWCNTs), containing one layer of cylinder graphene, and multiwalled CNTs (MWCNTs), which are formed from several concentric graphene sheets. Due to their high specific surface areas, SWCNTs can easily be modified with desired biomolecules via adsorption, encapsulation and chemical attachment processes. SWCNT-based bioconjugates have the ability to deliver bioactive molecules across cell membranes and also into the cell nuclei.63−65 It is claimed that nanotubes could release drugs into the cells without giving any damages to the healthy cells of the tissue. Hence, SWCNTs appear to be promising candidates as imaging-guided drug delivery systems. However, their insolubility arising from strong hydrophobic nature limits their wider use in bioapplications. To enhance their dispersibility in aqueous phase and provide the proper functional groups that can bind to the desired targeting molecules, their surface should be functionalized. Two main approaches have been used to incorporate functional groups onto a CNT surface. In the covalent functionalization, functional substances are chemically linked onto the CNT’s carbon scaffold through selected organic reactions.66 A typical example of this approach involves oxidation of CNTs under acidic conditions, resulting in the formation of carboxyl groups (−COOH) along their length.67 In the noncovalent coating, however, π−π stacking or hydrophobic interactions68,69 play a dominant role to decorate CNTs with surfactants, aromatic compounds, and polymers. It has been well established that conjugated polymers are effective dispersant agents for solubility of CNTs via conducting π−π interactions with CNT surfaces.70,71 As part of our continuing interest in developing complex macromolecular structures in combination with nanomaterials to create individual functional objects for bioapplications, we herein report the design and fabrication of a multicomponent system consisting of PPP conjugated polymer with PSt and PCL side chains, COOH functionalized SWCNT’s (f-SWCNT, and folic acid (FA) as a multifunctional fluorescent probe for targeted cell imaging through their collective behavior. As will be shown below, the conjugated polymer is coated onto B

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SWCNT’s side walls via π−π stacking, while bioconjugation is achieved by using carboxyl groups of SWCNT with FA via simple EDC/NHS reactions. The resulting bioconjugate was used as a fluorescent marker for targeted imaging of folate receptor (FR) positive HeLa cells.72 The cell-specific binding ability of the bioconjugates was tested in in vitro studies, and the cellular internalization was monitored in live cells via fluorescence microscope technique. Our results show that these conjugates can specifically bind to HeLa cells indicating that such materials with desired structural variations can be made available for the emergence of unprecedented biomedical sensing and diagnosis technologies.

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Noncovalent Attachment of PPP-g-PSt-PCL Copolymer to fSWCNTs. f-SWCNTs (0.3 mg) and 0.6 mg of PPP-g-PSt-PCL were dispersed in 3.0 mL of THF. The mixture was shaken overnight at 25 °C at 1000 rpm. π−π stacking interactions occurred between phenylene groups of the polymer and hexagonal rings of f-SWCNTs during the mixing time. Finally, the mixture was washed with distilled water to remove THF, and both unbonded f-SWCNTs and polymer. The resulting conjugate was solved in PBS. Covalent Modification of f-SWCNT/PPP-g-PSt-PCL with Folic Acid. EDC (2.0 M) and 0.5 M NHS were prepared in MES buffer (25 mM, pH 6.0). Two-hundred microliter f-SWCNT/PPP-g-PSt-PCL (1.2 mg/mL) conjugate were mixed with 25 μL EDC and 25 μL NHS at room temperature for 30 min to activate carboxyl groups of fSWCNT. After activation of carboxyl groups, 0.2 mg folic acid was added to the mixture and allowed to shake for 4 h at 25 °C with 1000 rpm. Finally, the bioconjugates were purified using 10 kDa membrane filters. Labeling of f-SWCNT/PPP-g-PSt-PCL/FA conjugates with FITC. To perform the flow cytometry analysis, f-SWCNT/PPP-g-PSt-PCL/FA conjugates were labeled with FITC. Three hundred microliters of conjugate was incubated with 12.5 μL (1.0 mg/mL) FITC overnight. To remove nonbound FITC, this mixture was dialyzed against PBS for 2 days. After dialyses, final FITC-labeled conjugate was obtained. Characterization Methods. The synthesized SWCNT, f-SWCNT, PPP-g-PSt-PCL copolymer, and conjugates were characterized with transmission electron microscopy (TEM), atomic force microscopy (AFM), and Fourier transform infrared (FT-IR) analysis. Optical characteristics of polymer dispersed in a DMSO−PBS mixture (1:4, v/ v) and conjugates in PBS with a concentration of 0.1 mg/mL were also examined with a spectrofluorometer (Varian Cary Eclipse, U.S.A). TEM analysis was performed by using JEOL type microscopy to obtain high-definition images of SWCNT-based samples. For analyses, a drop of SWCNT-based sample was placed onto a copper grid surface and dried at room temperature. The samples were placed onto the holder then given to the microscope, and images were obtained using a voltage of 200 kV. AFM. AFM measurements were carried out at ambient conditions by using an Ambios Q-Scope 250 instrument. The tapping mode was used to take topographic images. A 20 μm scanner equipped with silicon tips with 10 nm tip-curvature and plasma oxygen-treated indium tin oxide (ITO)-coated glass substrate was used for measurements.78 The system is covered with acoustic chamber to prevent electromagnetic noises that may affect the measurements. SWCNT, f-SWCNT, and f-SWCNT/PPP-g-PSt-PCL were spin coated (WS400B-6NPP-Lite spin processor by Laurel) onto the substrate at a spin speed of 1000 rpm for 60 s and 1500 rpm 15 s then dried at 80 °C for 5 min in vacuum. FT-IR Spectroscopy. FT-IR spectra were recorded by a PerkinElmer Spectrum BX-FT-IR spectrophotometer by using both the ATR system (powder form directly usable) and KBr pellets. IR spectra of SWCNTs and f-SWCNTs as 1.0% suspensions in KBr pellets were recorded with an FT-IR spectrophotometer. Conformation of FR Expression on the Cell Surface. The expression of FR in HeLa cells and the lack of expression in A-549 cells were confirmed by polymerase chain reaction (PCR) and flow cytometry analyses in a previous study.72 Briefly, trizol reagent (Invitrogen) and the manufacturer protocol were used for isolation of total cellular RNA. The primers were designed using the NCBI reference mRNA sequence for homosapiens folate receptor-1 (adult) (NM_016724.2). The PCR step for FR1 was carried out with a VWR Thermal Cycler. The PCR products were separated in 1.5% agarose gel in TAE buffer by a Thermo EC electrophoresis unit. For flow cytometry studies as positive staining cells were treated with antihuman FOLR1-phycoerythrin antibody, clone 548908 (R&D Systems GmbH, Wiesbaden-Nordenstadt). For negative control staining matched mouse IgG1, k PE isotype control antibody (BD Biosciences, Heidelberg) was used. Cytotoxicity. It was reported that insoluble formazan crystals that are yielded by MTT assay strongly interact with CNTs, which in turn cause false-positive results.79 Thus, we used alternative tetrazolium-

EXPERIMENTAL SECTION

Materials. FA, EDC), N-hydroxysulfosuccinimide (sulfo-NHS), 2(N-morpholino) ethanesulfonicacid (MES), tetrahydrofuran (THF), nitric acid (78%), sulfuric acid (98%), dimethyl sulfoxide (DMSO), 4,6-diamino-2-phenylindol (DAPI) and fluorescein isothiocyanate isomer I (FITC) were purchased from Sigma-Aldrich. Phosphate buffer saline (PBS) was prepared with 137 mM sodium chloride, 2.7 mM potassium chloride, 10.1 mM disodium hydrogen phosphate, and 1.8 mM potassium dihydrogen phosphate, pH 7.4; all chemicals were also provided from Sigma Aldrich. Sodium dodecyl sulfate (SDS) was ordered from Applichem. A 10 kDa membrane filter was purchased from (Sartorius Stedim Biotech). All other chemicals were of analytical grade. Methods. Cell line. A-549 (lung cancer) and HeLa cell lines were provided from German Collection of Microorganisms and Cell Cultures (DSMZ). Cell culture supplies including fetal calf serum (FCS) and penicillin/streptomycin (P/S) were purchased from PAA Laboratories GmbH. Both cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FCS and 1.0% P/ S. All cells were cultivated in medium and incubated with samples and reagents at 37 °C in humidified environment with 5.0% CO2. Synthesis of Modified PPP. The heterografted PPP containing both PSt and PCL side chains statistically distributed on the main chain was synthesized by Yamamoto polycondensation. The synthesis details together with structural characterization were previously reported.44,45 Synthesis of SWCNTs. The synthesis of powders for use as catalysts and SWCNTs was followed the procedure by Rashidi et al.73 Co−Mo/ MgO catalyst was synthesized by sol−gel method in the ratio of Co:Mo:MgO; 0.5:0.25:10. In order to synthesize SWCNTs, chemical vapor deposition (CVD) furnace (PTF 16/50/450, PROTHERM) was employed. Before the SWCNT growth, H2 gas with a flow rate of 200 cc/min was sent through powder at 850 °C for 1 h with the purpose of pretreatment of the catalyst. During the nanotube synthesis, a mixture of CH4:H2 gases with 50 cc/min:200 cc/min flow rate was sent through the catalyst powder at 1000 °C for 40 min at atmospheric pressure. Oxidation of SWCNTs. CNT surfaces need to be functionalized for biomedical applications, especially if their sidewalls serve as a platform for further modification steps. One of the general functionalization methods is the “oxidative purification” of nanotubes. Oxidative treatment has been carried out using boiling sulfuric acid,74 nitric acid75,76 or mixtures of both73 at high temperatures. The method involving acid mixtures was used for this work. In the first step, 5.0 mg of synthesized SWCNTs were incubated in a H2SO4 (98%) and HNO3 (68%) 3:1 (v:v) mixture for 2 h at 80 °C. One minute sonication treatments were applied two times during incubation, and then the mixture was diluted 1:1 (w/w) with bidistilled water.77 To remove amorphous carbon fragments, dissolved metal catalyst particles and excess acid in the supernatant, the mixture was washed with distilled water and centrifuged (5000g) until the pH value of the sample became neutral. Finally, resulting carboxyl-functionalized SWCNTs were dried overnight at 80 °C in a compartment dryer. This process allows the formation of carboxylic acid groups on the side walls of SWCNTs. Henceforth, this material is referred to as f-SWCNTs. C

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based cytotoxicity assay, WST-1, for determination the cytotoxic effects of f-SWCNT, PPP-g-PSt-PCL, f-SWCNT/PPP-g-PSt-PCL, and f-SWCNT/PPP-g-PSt-PCL/FA conjugates.35 A-549 and HeLa cells were seeded out in 96-well-tissue plates (Sarstedt, USA) in a volume of 200 μL and cultivated 3 days at 37 °C, 5.0% CO2, and 100% air humidity. After this cultivation time, the cells were treated with fSWCNT (0.1 and 50 μg/mL), PPP-g-PSt-PCL, f-SWCNT, fSWCNT/PPP-g-PSt-PCL, and f-SWCNT/PPP-g-PSt-PCL/FA conjugates at between 0.1 and 50 μg/mL polymer concentrations for 24 h. A 4-fold determination was carried out. Furthermore, a no-cell control served as the negative control to determine background intensities. Then the samples were transferred into a new 96-well flat bottom plate and stored for 30 min at room temperature. Cells were incubated with 110 μL/well, WST-1 solution (10 μL WST-1 reagent and 100 μL medium for 30 min. During this incubation time, an orange formazan complex was produced in the metabolic active cells. Caused by its solubility in water, the formazan is released into supernatant, and UV− vis absorption was measured at 450 nm with 630 nm as the reference wavelength using a microplate reader Model 680 (BioRad). To confirm the results of the WST-1 assay, the release of lactate dehydrogenase (LDH) was observed utilizing the CytoTox-One Homogeneous Membrane Integrity Assay (Promega). Here, the supernatants collected from the highest sample concentration used in cytotoxicity tests were applied. Even cells with intact membranes release small amounts of LDH. Therefore, as untreated cells control, supernatants of cells grown in medium were used. One hundred microliters of CytoTox-One Reagent (11 mL of Assay Buffer transferred into one vial of Substrate Mix) was added to 100 μL of sample per well. The plate was shaken for 30 s and then incubated at 22 °C for 10 min. The reaction of resazurin to the fluorescent resorufin was stopped by adding 50 μL of Stop Solution. After shaking the plate for 10 s, fluorescence was measured (Ex544 nm/Em590 nm) by the use of a Thermo Fluoroscan Ascent Plate Reader. Flow Cytometry Analysis. The cells were harvested by accutase and washed once in ice-cold incubation buffer consisting of PBS supplemented with 2.0% FCS. After that, 3 × 105 cells in incubation buffer were collected and centrifuged. FITC-labeled f-SWCNT/PPP-gPSt-PCL/FA conjugates were added, and the cell suspension was shaken at room temperature for 1 h at 450 rpm in the dark. To remove unbound conjugates, the cells were washed once in 500 μL incubation buffer. Before flow cytometric analysis, cells were suspended in 300 μL incubation buffer and then analyzed in a COULTER EPICS XL-MCL flow cytometer. More than 10 000 gated events were observed in total, and living cells were gated in a dot plot of forward versus side scatter signals. The software WinMDI 2.9 was used to draw dot plots and histograms. Fluorescence Microscopy. Ten thousand HeLa and A-549 cells were cultivated 2 days in a chamber slide in a volume of 200 μL medium. After cultivation, PPP-g-PSt-PCL copolymer, f-SWCN/PPPg-PSt-PCL, and f-SWCNT/PPP-g-PSt-PCL/FA were diluted with medium 1:1, and 160 μL of the samples (0.05 mg/mL) were added to the cells. The cells were incubated for 2 h at 37 °C with samples then were washed twice in PBS. After this step for control staining cell’s nuclei were stained by DAPI solution (1.0 μg/mL) for 15 min, and cells were washed twice in PBS. Labeled cells were imaged using Olympus BX53F fluorescence microscope equipped with a CCD camera (Olympus DP72). For polymer fluorescence, a U-MNB excitation filter, BP470-490 (exciter filter), and BA515 (barrier filter) and for DAPI fluorescence a U-MWU excitation filter, BP330-385 (exciter filter), and BA420 (barrier filter) were used.

Scheme 1. The Overall Reaction Pathway for the Synthesis of Heterografted PPP Containing Statistically Distributed Side Chains of PSt and PCL

Table 1. Molecular Weight Characteristics of the Macromonomers and Final PPP-g-PSt-PCL polymer

Mn,H NMRa

Mn,GPCb

Mw/Mn (GPC)

PCL content (mol %)a

PCL macromonomer PSt macromonomer PPP-PSt-PCL

3050

4270

1.12

-

2250

2550

1.25

-

-

23400

1.35

58

a

Calculated by 1H NMR analysis according to PSt standards

b

Determined by GPC analysis

shown in Table 1. Due to the presence of the oligomeric side chains, this “hairy rod” copolymer presented solubility in organic solvents that enabled its photophysical characterization in chloroform at the concentration of 0.025 g/L.44 The UV−vis spectrum showed two absorption maxima in the range 220− 265 nm, while the emission maxima appeared at 379 nm. It must be noticed that both absorption and emission spectra are asymmetric in shape, with vibronic fine structure. Also redshifted shoulders relative to the main peaks and long red tails appeared in both absorption and emission spectra. Such optical features are usually observed for the conjugated polymers forming nanoparticles.80 Taking into account the amphipolar character of the investigated PPP, its anisotropic molecular shape and the selectivity of chloroform toward PCL as well as the self-assembling in nanoparticles form could be very probable. The amphipolar character was previously confirmed by electrospining investigations of the blends of this copolymer with PSt and poly(methyl methacrylate) (PMMA).81 The selfalignment and bundling characteristics observed for the obtained fibers were ascribed to the unique molecular architecture of this PPP and its peculiar interactions with the solvents and polymer matrix used for electrospinning. Synthesis and Characterization of PPP-g-PSt-PCL Conjugates. In our study, we aimed to demonstrate the use of SWCNT and targeting ligand FA as carrier for PPP-g-PStPCL copolymer and imaging of HeLa cells, respectively. The first stage of the approach involves functionalization of the CNT component via acid treatment facilitating conjugation with biomolecules. Carboxyl functionalized CNTs were attached with PPP-g-PSt-PCL fluorescent conjugated polymer via π−π stacking interactions between phenyl groups of polymer and hexagonal rings of SWCNTs. Then the obtained

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RESULTS AND DISCUSSION Synthesis and Characterization of PPP Containing Both PSt and PCL Side Chains. The overall reaction pathway for the synthesis of heterografted PPP containing statistically distributed side chains of PSt and PCL is presented in Scheme 1. Details concerning its synthesis and structural characterization were previously reported.44 Molecular weight characteristics of the macromonomers and final PPP-g-PSt-PCL are D

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Scheme 2. Schematic Representation of Conjugation Process and Cell Specific Imaging

Figure 1. (A) FT-IR spectra of SWCNT and f-SWCNT with acid treatment. (B) f-SWCNT/PPP-g-PSt-PCL and f-SWCNT/PPP-g-PSt-PCL/FA.

fluorescent probe was coupled with FA via EDC/NHS reactions using amino groups of FA and carboxyl groups of SWCNTs. The overall process is depicted in Scheme 2. The intermediates obtained in various stages and the final multifunctional compound were characterized by FT-IR analyses. The O−H bonds of f-SWCNT resonate at around 3400 cm−1, which is believed to arise from either ambient

atmospheric moisture tightly bound to the SWCNTs or oxidation during purification of the pristine material.82 The peak at around 1720 cm−1 is based on the CO stretch of the carboxylic acid group.83 These two important peaks of fSWCNT (Figure 1A) proved that carboxyl groups are successfully integrated on SWCNT surfaces. The IR spectral changes of the nano conjugated polymer before and after FA E

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Figure 2. AFM images of SWCNT (A), f-SWCNT (B), f-SWCNT/PPP-g-PSt-PCL (inset: 2D images of f-SWCNT/PPP-g-PSt-PCL) (C), and fSWCNT/PPP-g-PSt-PCL/FA (inset: 2D images of f-SWCNT/PPP-g-PSt-PCL/FA) (D).

Figure 3. TEM images of SWCNT (A), f-SWCNT (B), and fSWCNT/PPP-g-PSt-PCL (C). Scale bars used in the images are 5 and 20 nm.

coupling was studied (Figure 1B). In the spectrum of the conjugated polymer, the peaks at 1720 and 960 cm−1 confirms the presence of PCL chains. Absorptions from 750 cm−1 were specific to the PSt component, while the peaks attributed to both side polymers were present at 1450 cm−1.21 The spectrum of the FA coupled product exhibits a carbonyl band of carboxylic acids and FA at 1686 cm−1. Also, the amide (−CONH−) bands around 3100 and 3400 cm−1, the conjugated aromatic ring at about 1600 cm−1, the bands of CN and CC conjugated groups at about 1600−1480 cm−1, and the fingerprint region of aromatic rings between 1225 and 950 cm−1 of FA are assigned in the structure (Figure

Figure 4. Fluorescence spectrum of PPP-g-PSt-PCL, f-SWCNT/PPPg-PSt-PCL, f-SWCNT/PPP-g-PSt-PCL/FA (polymer was suspended in DMSO-PBS mixture, conjugates were suspended in PBS buffer, pH 7.4, with a concentration 0.1 mg/mL and excited at 250 nm.).

1B). These data clearly confirm the expected structures at various stages. F

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Figure 5. Dose-dependent cytotoxic effects on viability of A-549 cells (A, B) and HeLa cells (C, D).

of nanotubes (Figure 2A). When f-SWCNTs were applied to the substrate, a more homogeneous surface as a self-assembled monolayer was obtained by hydrogen bonding interactions between carboxyl groups of f-SWCNT and hydroxyl of ITO (Figure 2 B).78 On the other hand, acid treatment caused deformation and consequently reduction in the size of SWCNT. The dimensions were then again increased by the incorporation of the polymer on the f-SWCNT surfaces (Figure 2C). It can also be claimed that when the polymer was introduced, the carboxyl groups of f-SWNT could interact with the ITO, and intermolecular interactions between the aromatic groups of the polymer may become effective essentially resulting in a more heterogeneous surface. Notably, covalent binding of FA molecules to the structure results in decrease in the dimensions (Figure 2 D) due to the more compact structure and stronger interaction with the ITO and surface homogeneity facilitated by the presence of hydrophilic FA residues. The observed dimensional changes clearly confirm the success of each stage in the approach. Further evidence for the formation of nanostructured material via described processes was obtained by TEM investigations. As can be seen from Figure 3A,B, after acid treatment, an increased sidewall defect and reduced dimension of f-SWCNT compared with pristine SWCNT were observed. Noncovalent attachment of polymer on f-SWCNT resulted in different surface morphology (Figure 3 C).

Figure 6. Histogram of specific binding of FITC-labeled f-SWCNT/ PPP-g-PSt-PCL/FA conjugates to FR-negative A-549 cells and FRpositive HeLa cells.

Surface morphologies of raw SWCNT, f-SWCNT, fSWCNT/PPP-g-PSt-PCL, and f-SWCNT/PPP-g-PSt-PCL/FA were analyzed with AFM. Figure 2A,B shows AFM images of pristine SWCNTs and f-SWCNTs, respectively. To observe microscopy images, plasma oxygen-treated ITO glasses were used as substrate prior to surface imaging. In the case of pristine SWCNTs, layer by layer formation could become dominant due to the π−π stacking interactions between aromatic groups G

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SWCNT conjugates were evaluated by using standard WST-1 and LDH assays. The results of cell viability after 24 h incubation with the samples at different concentrations are shown in Figure 5, for FR negative A-549 (A and B) and FR positive Hela (C and D). According to these results, f-SWNT (50 μg/mL) caused only 2.0 and 5.0% drops in viability of Hela and A-549 cells. In the case of polymer itself, 46% and 58% decreases were observed for these cell lines. On the other hand, toxicity of the polymer bound nanotube is higher for A-549 cells (35%) than Hela cells (16%). The presence of FA in the structure decreases cellular uptake of conjugates to FR negative A-549 cells and consequently, resulting in less toxicity (15%) compare to FR positive HeLa cells (18%). Additionally, cell viability during 24 h was also confirmed with LDH assay, which is a marker for membrane integrity. Approximately 100% viability for each sample at 50 μg/mL were observed (Supporting Information, Figure S1). This means that treatment of the cells with the SWNT conjugates indicated no membrane damage. Cellular Uptake and Targeting. One of the useful methods to improve cellular uptake and specific targeting efficiency of materials is to bind their surfaces to a specific ligand capable of recognizing receptors overexpressed in the cancer cells.89 Among targeted molecules, FA receives particular attention since it achieves cell specific internalization via receptor-mediated endocytosis as a targeting anticancer agent to avoid nonspecific attacks on healthy tissues as well as to increase their cellular uptake within cells.90−92 In a related work from our laboratory, a high expression level of FR in HeLa cells and a lack of protein expression in A-549 cells were demonstrated in both gene and protein levels using PCR and flow cytometry analysis.70 For flow cytometry analysis, initially, f-SWCNT/PPP-g-PSt-PCL/FA was labeled with FITC, which provides more proper excitation wavelength compare to the polymer itself during the analysis. Then, both FR positive HeLa and FR-negative A-549 cells were incubated with FITC-labeled f-SWCNT/PPP-g-PSt-PCL/FA conjugates to confirm the binding specificity of the conjugates. Fluorescence intensity of HeLa cells incubated with f-SWCNT/PPP-g-PSt-PCL/FA conjugates (Figure 6) show more than 2-fold increase compared to that of the control A-549 cells. These results suggest that f-SWCNT/PPP-g-PSt-PCL/FA conjugates bind to FR expressing HeLa cells more effectively than to A-549 cells. For further investigation, fluorescence microscopy images of internalized fluorescence polymer and conjugates in cells were obtained after 2 h incubation with samples. Figures 7A,B demonstrate that pristine polymer accumulates around HeLa cell’s nuclei. With the binding of f-SWCNTs to the polymer, cellular uptake was increased, and whole cytoplasm was labeled with the conjugates (Figure 7C,D). In this connection, it should be pointed out that nonspecific cell associations are also possible as observed prevously.93 Bioconjugate can bind to A549 cells nonspecifically but the fluorescence signal is very low in comparison to FA positive HeLa cells (Figure 8C). However, the presence of targeting ligand (FA) resulted in significant increase in binding efficiency of the structure. Moreover, these fluorescence microscope results are entirely consistent with the flow cytometry results.

Figure 7. Fluorescence microscopy images of HeLa cells. Cells were treated with PPP-g-PSt-PCL for 2 h at 37 °C (A). Overlap of two images, control nuclei staining with DAPI (B). Cells were treated with f-SWCNT/PPP-g-PSt-PCL for 2 h (C). Overlap of two images, control nuclei staining with DAPI (D). Cells were treated with fSWCNT/PPP-g-PSt-PCL/FA for 2 h (E). Overlap of two images, control nuclei staining with DAPI (F).

Figure 8. Fluorescence microscopy images of A549 cells. Cells were treated with PPP-g-PSt-PCL for 2 h at 37 °C (A);.Cells were treated with f-SWCNT/PPP-g-PSt-PCL for 2 h (B). Cells were treated with fSWCNT/PPP-g-PSt-PCL/FA for 2 h (C).

Florescence properties of polymer before and after conjugation were evaluated. Also, the polymer loading was evaluated using spectrofluorometric measurements after removing the unbound compounds during the stackingand each conjugation step. Since very low fluorescence signals have been observed in each steps, lost of polymer has been neglected. Fluorescence spectra (Figure 4) illustrated that the polymer which is dispersed in DMSO−PBS mixture (1:4, v/v) and conjugates in PBS has a maximum emission at 496 nm. Conjugation reactions caused a drop in fluorescence intensity of the polymer, but no shift in emission wavelength was observed. This result demonstrates that after conjugation, fluorescent properties of polymer did not exhibit any major changes. Also, another peak at 450 nm was observed in the spectra as a result the addition of FA into the structure. Cytotoxicity. For effective bioapplications of SWCNT such as imaging and drug delivery35,84,85 without undesired side effects, toxicity and solubility in aqueous phase are very important. Raw CNTs are more toxic than functional ones,86 and functionalization of CNTs improve their biocompatibility.87,88 In our case, dose-dependent cytotoxic effects of f-

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CONCLUSION SWCNTs possessing fluorescent polymer and FA conjugates were successfully used as a novel platform for targeted imaging of FR overexpressing cancer cells. In the described approach, H

dx.doi.org/10.1021/bm400862m | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

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the acid-treated carboxyl functional SWCNTs were furnished with PPP-g-PSt-PCL copolymer via π−π stacking, and then FA was covalently bound to the resulting conjugates. For the evaluation of the specific binding efficiency and cellular localization, SWCNTs conjugates were applied to A-549 and HeLa cells. Our results showed that f-SWCNT/PPP-g-PStPCL/FA conjugates bound to FR positive HeLa cells more effectively than to FR negative A-549 cells. Cellular localization of polymer and SWCNT conjugates were easily determined by means of nuclei staining, and the localization differences were clearly observed. The results presented here indicate the usage potential of functional SWCNTs with high water solubility and low toxicity for cell specific targeting and biomarker detection in diagnosis. Thus, the development of the three-component conjugate reported here may provide a platform for a new generation of targeting and labeling systems.

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ASSOCIATED CONTENT

S Supporting Information *

Cytotoxicity detection with lactate dehydrogenase assay. This material is available free of charge via Internet at http://pubs. acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*Mailing address: Ege University, Faculty of Science, Biochemistry Department 35100 Bornova-Izmir, Turkey. Phone: +902323112455; fax: +902323115485; e-mail: suna. [email protected]. * Mailing address: Department of Chemistry, Istanbul Technical University, Maslak, Istanbul 34469, Turkey Center of Excellence for Advanced Materials Research (CEAMR) and Chemistry Department, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia. Phone: +90 (212) 285 3241; fax: +90 (212) 285 6386; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by TUBITAK (Project No.109T573) and EBILTEM (Project No. 2010 BIL 004).

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