DielsâAlder âClickableâ Biodegradable Nanofibers: Benign Tailoring of

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Diels-Alder ‘Clickable’ Biodegradable Nanofibers: Benign Tailoring of Scaffolds for Biomolecular Immobilization and Cell Growth Ozlem I. Kalaoglu-Altan, Azize Kirac-Aydin, Burcu Sumer Bolu, Rana Sanyal, and Amitav Sanyal Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00411 • Publication Date (Web): 28 Aug 2017 Downloaded from http://pubs.acs.org on August 30, 2017

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Bioconjugate Chemistry

Diels-Alder ‘Clickable’ Biodegradable Nanofibers: Benign Tailoring of Scaffolds for Biomolecular Immobilization and Cell Growth Ozlem Ipek Kalaoglu-Altan,1 Azize Kirac-Aydin,1 Burcu Sumer Bolu,1 Rana Sanyal,1,2 and Amitav Sanyal1,2* 1 2

Department of Chemistry, Bogazici University, Bebek 34342, Istanbul, Turkey

Center for Life Sciences and Technologies, Bogazici University, 34342, Istanbul, Turkey

*Author to whom correspondence should be addressed; E-Mail: [email protected]

1

ACS Paragon Plus Environment


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ABSTRACT Biodegradable polymeric nanofibers have emerged as promising candidates for several biomedical applications such as tissue engineering and regenerative medicine. Many of these applications require modification of these nanofibers with small ligands or biomolecules such as peptides and other growth factors, which necessitates functionalization of these materials in mild and benign fashion. This study reports the design, synthesis and functionalization of such nanofibers and evaluates their application as a cell culture scaffold. Polylactide based copolymers containing furan groups and triethylene glycol (TEG) units as side chains were synthesized using organocatalyzed ring opening polymerization. The furan moiety, an electron rich diene, provides ‘clickable’ handles required for modification of nanofibers since they undergo facile cycloaddition reactions with maleimide-containing small molecules and ligands. The TEG units provide these fibers with hydrophilicity, enhanced biodegradability and anti-biofouling characteristics to minimize non-specific adsorption. A series of copolymers with varying amount of TEG units in their side chains were evaluated for fiber formation and anti-biofouling characteristics to reveal that an incorporation of 7.5 mol % TEG-based monomer was optimal for nanofibers containing 20 mol % furan units. Facile functionalization of these nanofibers in a selective manner was demonstrated through attachment of a dienophile containing fluorophore, namely, fluorescein maleimide. To show efficient ligand-mediated bioconjugation, nanofibers were functionalized with a maleimide appended biotin, which enabled efficient attachment of the protein, Streptavidin. Importantly, the crucial role played by the TEG-based side chains was evident due to lack of any nonspecific attachment of protein to these nanofibers in absence of biotin ligand. Furthermore, these nanofibers were conjugated with a cell adhesive cyclic peptide, cRGDfK-maleimide, at room temperature without the need of any additional catalyst. Importantly, comparison of the cell attachment onto nanofibers with and without the peptide, demonstrated that fibers appended with the peptides promoted cells to spread nicely and protrude actin filaments for enhanced attachment to the support, whereas the cells on non-functionalized nanofibers showed a rounded up morphology with limited cellular spreading.

INTRODUCTION Over the past decade, polymeric nanofibers have been investigated widely for biological applications such as fabrication of sensing platforms, tissue engineering scaffolds and drug delivery systems.1,2 The three dimensional, porous scaffold with high surface area 2


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provides them with excellent attributes desirable in many sensing platforms.3 In addition to the aforementioned properties, their fibrous morphology makes them attractive materials as synthetic tissue engineering scaffolds that can be tuned to closely mimic the native extracellular matrix (ECM). Such artificial ECMs provide the necessary physical support as well as chemical cues for cells to thrive by enhancing their adhesion, migration and proliferation.4 To access such fibrous polymeric scaffolds, electrospinning has emerged as one of the most widely employed method due to the low equipment cost, simplicity of the operation, and the feasibility in scaling-up the production.5-8 The method uses electrical potential to produce polymeric fibers with diameters ranging from tens of nanometers to several micrometers. Synthetic polymers such as poly(ε-caprolactone) (PCL),9 polylactide,10 poly(lactide-coglycolide),11 poly(lactide-co-ε-caprolactone)12 have been widely electrospun into nanofibers for various biological applications. However, a survey of literature reveals that most of the synthetic polymers used for electrospinning are not ideal substrates for applications involving cell attachment and proliferation due to insufficient biocompatibility resulting from their hydrophobicity and the lack of ability to incorporate bioactive sites for cellular recognition.1315

Modification of electrospun nanofibers through incorporation of biological cues such as

bioactive ligands, peptides or growth factors onto the nanofibrous scaffolds is crucial to obtain an effective biofunctional scaffold. Appendage of molecules such as Arg-Gly-Asp (RGD) and related fibronectin fragments and other growth factors make these materials resemble the natural ECM.16,17 Most of the commonly employed approaches for incorporation of bioactive molecules on nanofiber surface utilize methods like plasma treatment, wet chemical degradation methods and physical adsorption,18 techniques which can compromise fiber properties such as their mechanical durability. Over the past decade, since the advent of ‘click’ chemistry,19 utilization of polymeric nanofibers fabricated from copolymers containing reactive functional groups has gained wide interest.20 Click reactions have emerged as a powerful surface modification tool, given their high efficiency under mild reaction conditions, as well as excellent selectivity.21,22 One of the first examples of utilization of ‘click’ chemistry in nanofiber modification was performed by Jing and coworkers where they attached an azide-bearing protein onto alkyne-bearing polylactide nanofibers using the copper-catalyzed azide-alkyne cycloaddition (CuAAC).23 Lately, the usage of the toxic Cu catalyst in CuAAC has directed the interest towards metalfree click reactions.24 Recently, Becker and coworkers reported conjugation of an azide3



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containing peptide onto 4-dibenzocyclooctynol (DIBO)-terminated PCL nanofibers25 and azide-containing gold nanoparticles onto DIBO-terminated poly(γ-benzyl-L-glutamate) (DIBO-PBLG) nanofibers26 via the strain-promoted azide-alkyne cycloaddition (SPAAC). While the SPAAC reaction provides efficient conjugation, the multi-step synthesis of the cyclooctyne moiety limits its widespread adaptability. Other approaches have used functionalization of alkene group bearing nanofibers using the radical thiol-ene ‘click’ reaction,27 which requires utilization of photo-initiators and UV-irradiation. A survey of the tool box of ‘click’ reactions reveals that the Diels-Alder cycloaddition reaction between an electron rich diene and a deficient dienophile provides an attractive alternative for conjugation. The reaction has been investigated in creating different macromolecular architectures, functionalization of soluble polymers, and functionalization of surfaces.28-30 An attractive attribute of the reaction is that it proceeds in aqueous media, often at enhanced rates,31 under mild and ambient conditions. Furthermore, a suitable and readily available diene moiety, namely furan, can be easily introduced into various polymeric materials.32 To date, the furan-maleimide cycloaddition reaction has been widely used for functionalization of various types of polymeric interfaces such as thin films,33 polymer brushes,34 hydrogels,35 and nanoparticles.36 One reason for the increasing attention to the furan-maleimide pair is that the furan derivatives can be obtained from renewable resources which contributes to green chemistry.37 Recently, we reported the synthesis of polymeric nanofibers containing furan groups using non-degradable methacrylate based polymers.38 Although we demonstrated that these polymers could be functionalized efficiently using the Diels-Alder cycloaddition chemistry, the non-degradability of these materials limits their utilization for many applications such as using them as scaffolds for cell growth. Thus we envisioned that fabrication of their biodegradable counterparts will broaden their applications. Aliphatic polyesters synthesized via ring-opening polymerization (ROP) of lactides are widely used in biomedical applications such as drug delivery and tissue engineering because of their biodegradability.39,40 In recent years, synthesis of biodegradable polyesters using organo-catalysis has become prevalent since oftentimes they proceed under ambient conditions and do not require a metal catalyst.41,42 Although the polylactide-based materials have been used for various biomedical application, they are often devoid of functionalizable groups. A simple approach to obtain readily functionalizable polylactides involves their copolymerization with cyclic carbonate monomers containing various ‘clickable’ groups such as alkyne,43 azide,44 maleimide,45 furan46 and anthracene.47 While furan containing polylactide 4


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copolymer has been previously synthesized, they were utilized for fabrication of polycarbonate microstructures using nanoimprint lithography.46 Herein, we fabricated furan-containing hydrophilic biodegradable nanofibers that undergo facile functionalization with maleimide-containing molecules and ligands under benign conditions for protein or cell adhesion (Scheme 1). Polymeric precursors bearing a polylactide based copolymer containing furan groups as reactive handles and TEG groups for providing hydrophilicity and anti-biofouling characteristics were synthesized and electrospun to yield nanofibers. A series of copolymers with varying amount of TEG units on their side chains were evaluated for fiber formation and anti-biofouling characteristics. Efficient conjugation of maleimide containing fluorescent dye, and a biotin-based ligand onto nanofibers were demonstrated. The latter was used for ligand-directed immobilization of the protein, Streptavidin, in a specific manner. Furthermore, these nanofibers were conjugated with a cell-adhesive peptide, namely cRGDfK, to demonstrate that such thus modified nanofibers provide an attractive scaffold with enhanced cellular adhesion and proliferation.

Scheme 1. Schematic illustration of furan-containing biodegradable ‘clickable’ nanofibers for various applications.

RESULTS AND DISCUSSION Synthesis of side-chain furan-containing polylactides was performed using ring opening polymerization of L-lactide with furan (MTC-F) and TEG-bearing (MTC-TEG) cyclic carbonate monomers catalyzed by DBU/TU co-catalyst system using benzyl alcohol as an initiator (Figure 1). The furan containing cyclic carbonate monomer was obtained by reaction of furfuryl alcohol with a bis-MPA based anhydride (See ESI for details). Subsequent 5



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deprotection of the acetal group to the diol, followed by condensation with ethylchloroformate yielded the furan-containing carbonate monomer with high yield and purity. Likewise, the triethylene glycol (TEG) containing carbonate monomer was synthesized from triethylene glycol momnomethyl ether using a similar protocol. The hydrophilic TEG-based monomer was incorporated into the copolymers to provide the polymeric nanofibers with antibiofouling characteristics. To understand what is the minimal amount of the TEG-based monomer that will impart the nanofibers with desirable level of anti-biofouling ability, while preserving fiber formation ability, a series of copolymers containing varying amount of the MTC-TEG monomer were synthesized using ROP (Table 1). N N F3C O

O O O

+

O

S

O CF3

O

+

O

N H

N H O

O

O

OH O

O O

O

O

O O O X

O O

H O

O

O O

y

z

O

O

O O CH2Cl2, 25 °C, 20h

O

O

O

O

n

n

Figure 1. Synthesis of TEG and furan-containing hydrophilic biodegradable PLA copolymers.

Table 1. Details of furan- and TEG- containing PLA copolymers. Polymers

[TEG]/[Furan]/[LA]a

MTC-TEG5-F/PLA

10/40/150

MTC-TEG7.5-F/PLA

[M]/[I]b

Mnc (Da)

Mwc (Da)

PDIc

200

12200

16900

1.40

15/40/145

200

13000

18500

1.40

MTC-TEG10-F/PLA

20/40/140

200

7100

9800

1.37

MTC-F/PLA

0/40/160

200

13300

16000

1.20

a

Molar feed ratio; bI=benzyl alcohol; cDetermined using SEC using polystyrene standards (1150 kDa) in THF.

6


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With the polymeric precursors at hand, furan-containing nanofibers were prepared using the solution electrospinning method. After a brief survey of electrospinning conditions, bead-free nanofibers were successfully prepared from solutions of furan-containing copolymers using chloroform as a solvent with a concentration of 40 wt % and electrospun with an applied voltage of 15 kV, a tip-to-collector distance of 15 cm and a flow rate of 0.01 mL/min. The scanning electron microscopy images of nanofibers indicated that while beadfree uniform fibers were obtained using the copolymer devoid of TEG-groups and those containing 5 % and 7.5 % TEG groups, the 10 % TEG-monomer containing copolymers did not give stable nanofibers (Figure 2). The fibers obtained using the higher TEG content appeared to be in a melted state rather than stable nanofibers. Differential scanning calorimetric analysis of the copolymers revealed that increasing amount of TEG in the copolymers lowered the glass transition temperature (Tg) from 37.5 °C to 5.8 °C which explains the poor nanofiber formation from copolymers with high TEG content (Figure S12).

Figure 2. SEM images of A) 0 % B) 5 % C) 7.5 % D) 10 % TEG-containing MTCF/PLA nanofibers. 7



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The three nanofibers with stable fiber morphlogies containing TEG moeities ranging from 0-7.5 mol % were evaluated for their anti-biofouling characteristics. The fibers were treated with FITC-labelled Streptavidin (0.1 mg/mL in PBS) and after incubating for 30 min in the dark, were rinsed with copious amount of PBS (1×). While the nanofibers devoid of TEG groups adsorbed and retained high amount of protein, comparatively less amount of protein adsorbed onto fibers with 5 % TEG groups (Figure 3). Finally, it was only when the TEG content was increased to 7.5 mol %, nanofibers with significant antibiofouling character were obtained with an average diameter of 472 nm ± 160 nm. It is well established that hydrophilicity of the TEG units plays a role in providing anti-biofouling characteristics to the materials surface. Hence to correlate the observations here with surface hydrophilicity of nanofibers, water contact angle measurements were undertaken. An average of water contact angle for the furan-containing nanofibers (MTC-F/PLA) without TEG units was obtained as 128°, while the value for the 7.5 mol % TEG-containing nanofibers was obtained as 84°, similar to previously reported trend observed upon introduction of polymers with hydrophilic ethylene glycol units.48 Additionally, XPS analysis also supported presence of the TEG units on nanofiber surface through an increase in ether type C-O linkages for nanofibers containing the TEG comonomer (Figure 4).

Figure 3. Fluorescence microscopy images of FITC-labelled Streptavidin immobilization on non-biotinylated A) 0 % TEG-containing B) 5 % TEG-containing C) 7.5 % TEG-containing MTC-F/PLA nanofibers.

8


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Figure 4. XPS and water contant angle measurements of A) MTC-F/PLA B) MTC-TEG7.5F/PLA nanofibers.

The MTC-TEG7.5-F/PLA nanofibers were first treated with a maleimide-containing fluorescent dye in order to investigate the feasibility of the conjugation through the DielsAlder cycloaddition reaction (Figure 5). An aqueous solution of the fluorescent dye was dropped on the nanofibers in the dark at room temperature. After 4 h, the nanofibers were washed with PBS solution in order to remove unbound dye. The conjugation was confirmed with the bright green colour observed under fluorescence microscope. A control experiment was performed using an amine-containing fluorescent dye under the same conditions and negligible fluorescence was observed (Figure 5 inset). These results suggest that the conjugation was a result of Diels-Alder reaction between the furan-containing nanofibers and maleimide-containing fluorescent dye.

9



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Figure 5. Fluorescence microscopy images of MTC-TEG7.5-F/PLA nanofibers treated with maleimide-containing

fluorescent

dye

(inset

shows

fibers

after

treatment

with

fluoresceinamine).

After establishing that succesful functionalization of nanofibers occur through the Diels-Alder cycloaddition reaction, we focused on functionalization of these nanofibers with biomolecules. First, the feasability of a ligand directed bioconjugation was evaluated by conjugation of biotin onto these nanofibers. The MTC-TEG7.5-F/PLA nanofibers were treated with an aqueous solution of a maleimide-bearing biotin and thus biotinylated nanofibers were washed with ample amount of water and then treated with an aqueous solution of TRITCExtravidin. After incubation for 30 min, the nanofibers were washed with water to remove any unbound enzyme, followed by analysis using fluorescence microscopy. The bright red fluorescence indicated the successful immobilization of TRITC-Extravidin onto the nanofibers (Figure 6). As a control experiment, an aqueous solutions of TRITC-Extravidin was dropped on the non-biotinylated MTC-TEG7.5-F/PLA nanofibers under the same conditions. Negligible fluorescence observed for these nanofibers suggested that the immobilization was indeed specific and occured through ligand-mediated pathway (Figure 6 inset).

10


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Figure 6. Ligand-directed TRITC-Extravidin immobilization on MTC-TEG7.5-F/PLA nanofibers and fluorescence microscopy images (Control experiment on nonbiotinylated nanofibers is shown as inset).

The furan-containing polyester fibers with and without the hydrophilic TEG groups were evaluated for their biodegradability in PBS (1×) solution. It can be expected that the difference in their hydrophilicity will play a role in the extent of their degradation. After immersion in PBS for 20 days, the samples were analyzed with SEM (Figure 7). While the nanofibers devoid of any TEG units seemed to retain most of their structure, the nanofibers contaning TEG fragments appeared to lose their intact fibrous structure. This difference in stability suggests that attachment of hydrophilic TEG groups promotes their biodegradability, presumably due to increased swelling which increases the surface area and water penetration. Due to presence of biodegradable ester and carbonate linkages throughout their backbone, both of these nanofibers can be expected to undergo degradation with time, albeit at different rates.

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Figure 7. SEM micrographs indicating change in morphology of nanofibers with and without TEG units after immersion in PBS at 37 °C for 20 days.

Appropriately functionalized electrospun fibers can act as suitable substrate for cell adhesion and proliferation. To evaluate this aspect, L929 mouse fibroblasts were seeded on nonfunctionalized and RGD-functionalized MTC-TEG7.5-F/PLA nanofibers. The cell adhesion promoting peptide fragment cRGDfK was conjugated to the MTC-TEG7.5-F/PLA nanofibers via Diels-Alder reaction of cRGDfK(Mal). The fibers were incubated with an aqueous solution of the maleimide bearing peptide (1 mg/mL) for 4 h at room temperature. After rinsing with copious amount of water to remove any unbound peptide, the amount of peptide conjugated onto the nanofiber was quantified as 8.84 µg/mg using a bicinchoninic acid (BCA) assay kit, where a calibration curve was obtained from UV absorbance values of standard peptide solutions at 562 nm according to manufacturer’s protocol (Figure S13). Each nanofiber coated surface was used as substrate for cells culture for 3 days, and cell adhered surfaces were analyzed with confocal microscopy. The cells were fixed and stained with Alexa Fluor 488® phalloidin solution in PBS (1×) to investigate their morphology and the patterns of filamentous actin (F-actin) on the 1st and 3rd days. It was observed that the spherical shapes of the fibroblast cells tend to transform into spindle-like in all cases, indicating stronger binding on to the nanofiber coated platforms (Figure 8A). Nonfunctionalized nanofibers showed shorter F-actin bundles while the peptide-functionalized 12


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fibers showed cells with stretched morphology and longer F-actin fibers. It can be deduced that the functionalization of nanofibers with the cell adhesive peptide fragment enhanced the cell spreading and proliferation as expected. The cytotoxicity experiments indicated that both non-functionalized and RGD-functionalized nanofibers were not cytotoxic even at high polymer concentrations (Figure 8B). The cells seeded remained highly viable even at high concentration up to 1 mg/mL as indicated by almost unaffected cell viability levels. Furthermore, seeded fibroblast displayed spreading and proliferation on nanofiber surfaces over the course of 3 days which also suggested that the polymeric fibers were not only biocompatible but also provided a highly cyto-compatible environment.

Figure 8. Schematic illustration of peptide conjugation for cell growth and proliferation. A) Confocal microscopy images of scaffolds showing Alexa Fluor 488® phalloidin (green) and DAPI (blue) staining of L929 mouse fibroblasts after 1 and 3 days on MTC-TEG7.5-F/PLA 13



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nanofibers with and without RGD (scalebar 50 µm); B) Cytotoxicity of non-functionalized and RGD-functionalized MTC-TEG7.5-F/PLA nanofibers.

CONCLUSIONS Biodegradable nanofibers fabricated using copolymers containing pendant furan groups and anti-biofouling polyether groups undergo facile functionalization with maleimide-containing small molecules and ligands through the Diels-Alder cycloaddition reaction at ambient temperature in aqueous media without the need of any additional catalyst. Conjugation of ligands onto nanofiber surface enables immobilization of proteins in an efficient manner. The immobilization of protein occurs in a specific manner because of the anti-biofouling characteristics of these materials due to the presence of ethylene glycol based side chains. These non-toxic biodegradable nanofibers can be easily modified with appropriately functionalized cell adhesive peptides to promote cellular attachment and proliferation. Overall, the facile synthesis of these reactive yet anti-biofouling biodegradable nanofibers, coupled with their efficient functionalization in a benign manner to render them suitable for biomolecular immobilization, as well as cell proliferation, make them attractive substrates for a variety of biomedical applications.

EXPERIMENTAL Materials. L-Lactide (Aldrich) was recrystallized from toluene three times, dried under vacuo at room temperature for 6 h and stored in glovebox. 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU; Fluka) and benzyl alcohol (Merck) were dried over activated 4 Å molecular sieves and stored in a nitrogen filled glovebox. 1-(3,5-bis(trifluoromethyl)phenyl)-3-cyclohexylthiourea (TU) was synthesized according to the literature procedure.49 Polymerizations were performed in glovebox using anhydrous dichloromethane (DCM) from SciMatCo purification system. Chloroform was purchased from VWR Chemicals and used as received. N-(5Fluoresceinyl)maleimide and fluoresceinamine isomer I were obtained from Aldrich. BiotinPEG2-maleimide was purchased from ThermoScientific and FITC-labeled streptavidin was obtained from Pierce. TRITC-conjugated extravidin and DAPI were purchased from Sigma. Cyclo[Arg-Gly-Asp-D-Phe-Lys(3-Maleimide

propionic

acid)]

14


(cRGDfK(Mal))

was

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purchased from Peptides International. BCA assay kit was obtained from ThermoFisher Scientific, USA. Alexa Fluor 488® phalloidin was obtained from Invitrogen. Measurements. 1H NMR spectra of the polymers were recorded on a Bruker 400 MHz spectrometer using tetramethylsilane as an internal reference and deuterated chloroform as a solvent. The molecular weights of the copolymers were estimated by gel permeation chromatography (GPC) using a Shimadzu PSS-SDV (length/ID 8 × 300 mm, 10 µm particle size) mixed-C column calibrated with polystyrene standards (1–150 kDa) using a refractiveindex detector. Tetrahydrofuran (THF) was used as eluent at a flow rate of 1 mL.min−1 at 30 °C. The morphology of electrospun nanofibers was observed with Jeol Neoscope JCM-5000 scanning electron microscope (SEM). Fluorescence images of samples were recorded at room temperature on a Zeiss Observer Z1 fluorescence microscope. Static water contact angle were measured in air via the sessile-drop method using a goniometer (CAM 101 KSV instruments). Approximately 5 µL of deionized water was deposited on the surface and images were taken by an integrated digital camera. The software CAM2008 was used for image processing to determine the contact angle. Contact angle value for each sample was independently measured at three different locations and average values were measured. Differential scanning calorimeter (DSC) experiments were performed on a TA Instruments Q-2000 DSC apparatus at a heating rate of 10 °C/min. The X-ray source employed was a monochromatic Al Kα (1486.6 eV) at 72 W and 10−9 mbar. All XPS spectra were calibrated on the aliphatic carbon signal at 285.0 eV. Confocal microscopy images were taken by Leica TCS SP5 Confocal Microscope and processed using LAS X 3.02 software. Synthesis of furan-containing (MTC-F) and TEG-containing cyclic carbonate monomer (MTC-TEG). Full synthetic details regarding preparation and characterization of these monomers can be found in supporting information. Synthesis of furan and TEG-containing PLLA copolymers. In a typical experiment, MTC-F (157 mg, 0.65 mmol), MTC-TEG (75 mg, 0.25 mmol), L-lactide (348 mg, 2.42 mmol), TU (15.4 mg, 0.042 mmol) and benzyl alcohol (1.7 µL, 0.017 mmol) were added in a glass vial equipped with magnetic stirring bar and dissolved in anhydrous dichloromethane (3 mL) in a nitrogen-filled glovebox. Under rapid stirring, DBU (12.5 µL, 0.084 mmol) was added to the reaction mixture. The reaction was carried out at room temperature and quenched with benzoic acid (51.29 mg, 0.42 mmol) after 20 h. The resulting copolymer was purified by precipitation in 1:1 methanol/ether solution, followed by filtration and drying under vacuo to yield a white powder (360 mg, 65 %). 15



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Electrospinning of furan and TEG-containing PLLA copolymers. MTC-TEG-F/PLA copolymers were dissolved in CHCl3 at a concentration of 40 wt % and stirred for 6 h in order to obtain a homogenous solution. The solution was delivered at a constant flow rate to a needle connected to a high-voltage power supply. For a typical experiment, the tip to collector distance was 15 cm, the applied voltage was 15 kV and the flow rate was 0.01 mL/min. Fibers were collected on 22 x 22 mm glass slides attached on a grounded aluminum collector. Functionalization of Nanofibers. Conjugation of fluorescent dye to nanofibers. The MTC-TEG7.5-F/PLA nanofibers were treated with a water-soluble fluorescent dye, namely N-(5-fluoresceinyl)maleimide. The dye was dissolved in 1x PBS (1 mg/mL) and the solution (50 µL) was dropped on the nanofibers at room temperature. After 4 h in the dark, the surfaces were washed with ample amount of water, dried under nitrogen stream and examined under fluorescence microscope. As a control experiment, a solution of fluoresceinamine isomer I was prepared in 1× PBS solution (1 mg/mL) and this solution (50 µL) was dropped on the glass slide covered with furancontaining nanofibers. After 4 h in the dark, the nanofibers were gently rinsed with excess PBS solution. Subsequently, modified nanofibers were analyzed with fluorescence microscope. Ligand-directed immobilization of protein on nanofibers. An aqueous solution of biotinPEG2-maleimide (50 µL, 1 mg/mL) was dropped onto MTC-TEG7.5-F/PLA nanofibers. After incubating 4 h at room temperature in the dark, the coated-fiber surfaces were washed with copious amount of water. Aqueous solution of TRITC-Extravidin (0.1 mg/mL) was prepared and this solution (10 µL) was dropped onto the biotinylated nanofibers. After 30 min in the dark, the fibers were gently rinsed with ample amount of water to remove any unbound protein. The immobilization of protein was analyzed using fluorescence microscopy. Degradation studies. Electrospun nanofibrous samples were incubated in closed vials containing 2 mL of 1× PBS at 37 °C for different periods of time. The specimens were washed with ample water and dried under nitrogen stream at the end of each degradation period and examined using SEM. Bicinchoninic acid protein quantification. An aqueous solution of cRGDfK(Mal) (1 mg/mL, 30 µL) was dropped onto MTC-TEG7.5-F/PLA nanofibers at room temperature. After 4 h, nanofiber surfaces were washed with water and dried. The peptide (cRGDfK(Mal)) amount conjugated onto the furan-containing PLLA nanofibers was quantified using a bicinchoninic

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acid (BCA) kit (ThermoScientific, U.S.A.) according to manufacturer’s instructions. All measurements were done in triplicate. Cell culture on furan and TEG-containing PLLA nanofibers. L929 mouse fibroblasts were seeded onto prepared nanofiber coated slides with a density of 2 × 104 cells/cm2 using a stock solution 2 × 105 cells/mL. After dropping the cell suspensions onto coated slides, they were incubated at 37 °C in 5% CO2 containing atmosphere for 3 h. Afterwards, cell media (1 mL) was added into each well containing nanofiber coated slides. After incubation (24 or 72 h), cell media was removed, slides were washed with 1× PBS and cells were fixed with 3.7% formaldehyde solution at room temperature. For staining filamentous actins (F-actins), first, cells were incubated in 0.1 % Triton X-100 in PBS for 5 min, then after washing with 1× PBS, they were incubated in Alexa Fluor 488® phalloidin solution (5 units/mL concentration containing 1 % bovine serum albumin (BSA) in 1× PBS) for 20 min at room temperature. Subsequently, cell nuclei were stained with DAPI. Resulting images of cells attached onto nanofibers were taken and processed via LAS X software. Cytotoxicity of furan and TEG-containing PLLA nanofibers. In this study, L929 mouse fibroblast cells were used, which were grown in 5% CO2 containing atmosphere at 37 °C. Cells were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS) (PAN Biotech). For all experiments, cells were seeded at their exponential growth phase. For cytotoxicity assay, L929 cells were seeded into 96 well plates in quadruplicates. Nanofibers were pulverized and after addition of 1× PBS, they were sonicated for 20 min to obtain a cloudy solution with a final concentration of 10 mg/mL. Dilutions were made to obtain final polymer solutions with different concentrations. Cells were treated with polymer solutions at various concentrations and they were incubated continuously for 48 h in incubator. To determine cell viability CCK-8 assay was employed according to manufacturer’s recommendations and viability values were calculated via GraphPad Prism software.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Details of synthesis and characterizations of monomers, HNMR spectra of copolymers, nanofiber size histogram, DSC thermograms and BCA assay calibration curves. 17



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AUTHOR INFORMATION Corresponding Author *Email: [email protected], Tel: +902123597613 ORCID Rana Sanyal: 0000-0003-4803-5811 Amitav Sanyal: 0000-0001-5122-8329 Author Contributions The manuscript was written from contributions of all authors. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS The authors acknowledge financial assistance for this research by The Scientific and Technological Research Council of Turkey (TUBITAK Project No. 114Z139) and Ministry of Development of Turkey for Grant no. 2009K120520 and 2012K120480. O.I.K-A acknowledges TUBITAK BIDEB 2211-A for financial support.

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(2012)

Synthesis

and

functionalization

of

thiol-reactive

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polymers.

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