“Clickable” Nanogels via Thermally Driven Self-Assembly of Polymers

Article pubs.acs.org/Biomac

“Clickable” Nanogels via Thermally Driven Self-Assembly of Polymers: Facile Access to Targeted Imaging Platforms using Thiol− Maleimide Conjugation Bugra Aktan,† Laura Chambre,† Rana Sanyal,†,‡ and Amitav Sanyal*,†,‡ †

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

‡

S Supporting Information *

ABSTRACT: Multifunctionalizable nanogels are fabricated using thermally driven self-assembly and cross-linking of reactive thermoresponsive copolymers. Nanogels thus fabricated can be easily conjugated with various appropriately functionalized small molecules and/or ligands to tailor them for various applications in delivery and imaging. In this study, a poly(ethylene glycol)-methacrylate-based maleimide-bearing copolymer was cross-linked with a dithiol-based cross-linker to synthesize nanogels. Because of lower critical solution temperature (LCST) around 55 °C in aqueous media, these copolymers assemble into nanosized aggregates when heated to this temperature, and they are cross-linked using the thiol−maleimide conjugation. Nanogels thus fabricated contain both thiol and maleimide groups in the same cross-linked nanogels. Postgelation functionalization of the residual maleimide and thiol groups is demonstrated through conjugation of a thiol-bearing hydrophobic dye (BODIPY-SH) and N-(fluoresceinyl) maleimide, respectively. In addition, to demonstrate the utility of multifunctionality of these nanogels, a thiol-bearing cyclic-peptide-based targeting group, cRGDfC, and N-(fluoresceinyl)-maleimide-based fluorescent tag was conjugated to nanogels in aqueous media. Upon treatment with breast cancer cell lines, MDA-MB-231, it was deduced from cellular internalization studies using fluorescence microscopy and flow cytometry that the peptide carrying constructs were preferentially internalized. Overall, a facile synthesis of multifunctionalizable nanogels that can be tailored using effective conjugation chemistry under mild conditions can serve as promising candidates for various applications.

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INTRODUCTION Over the past decade, nanosized hydrophilic gels, often referred to as nanogels, have attracted growing interest due to their potential applications in biomedical areas such as drug delivery, tissue engineering, protein encapsulation and release, and bioimaging platforms.1−4 Reasons for employing nanogels are primarily due to their capacity to carry molecules of interest such as drugs, biomolecules, and imaging agents with a high degree of stability and dispersibility in biological medium. Their nanometric size also enables passive targeting of tumors based on the enhanced permeability and retention (EPR) effect.5−8 Based on the chemical handles present on the nanogels, they can be appended with specific biologically relevant motifs for active targeting, as well as engineered to be responsive toward specific biological stimuli, thus allowing “on demand” delivery. Generally, preparation of nanogels are achieved either by performing an in situ simultaneous polymerization and crosslinking of hydrophilic monomers, or through cross-linking of hydrophilic copolymer using cross-linkers in a controlled fashion. However, without the addition of surfactants, size control is a challenge when obtaining nanogels from monomers.9,10 Synthesis of water-dispersible nanogels have also been achieved by using polymerization in an inverse © 2017 American Chemical Society

microemulsion with the use of a two-phase system and surfactants.11 Another widely used approach to form nanogels employs cross-linking of polymers in emulsions, instead of monomers, thus circumventing any concerns of toxicity from leftover monomers.12 Despite the advantages of using surfactants to achieve well-controlled narrow size distributions, complete removal of surfactants can be challenging. Needless to say, efficient cross-linking chemistry is important for fabrication of nanogels in a controlled manner. To date, “click” transformations such as the azide−alkyne and thiol−ene reactions have been employed for cross-linking of polymers to obtain nanogels.13−15 As an alternative, self-assembly of amphiphilic random copolymers provides a facile approach to form stable aggregates in aqueous solutions that can be crosslinked. As seminal contributions, Thayumanavan and coworkers reported fabrication of nanogels by harnessing the lower critical solution temperature (LCST) driven selfassembly of thermoresponsive copolymers. Nanogel synthesis was achieved by heating amphiphilic polymers above their Received: October 25, 2016 Revised: December 31, 2016 Published: January 4, 2017 490

DOI: 10.1021/acs.biomac.6b01576 Biomacromolecules 2017, 18, 490−497

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nanosize aggregates via self-assembly. The cross-linked nanogels were either modified with thiol-bearing dye or modified with both a maleimide-bearing dye and a thiol-containing peptide-based ligand to demonstrate multifunctionalization. In particular, to achieve imaging via active targeting, the nanogels were modified with fluorescein-maleimide (a water-soluble fluorescent dye) and cRGDfC peptide (a peptide targeting group with affinity for integrin receptors overexpressed in many cancer cells).

LCST temperature to form stable aggregates, followed by their cross-linking.16 In particular, pyridyl disulfide bearing poly(ethylene glycol)methacrylate (PEGMEMA) containing copolymers undergo self-assembly to form nanosized aggregates upon heating in aqueous solution, which are cross-linked via disulfide linkages mediated by thiol groups generated in situ by addition of dithiothreitol. These carriers are capable of encapsulating hydrophobic cargo and transport them into cells.17,18 Not only synthetic polymers but also several biopolymers have been utilized to fabricate nanogels because they are biodegradable and nontoxic.19 Oh and co-workers recently used a disulfide-based cross-linking via disulfide-thiol exchange reaction to obtain nanogels from PEGMEMA-grafted cellulose polymers.20−22 Facile multifunctionalization of nanogels is important to render these constructs functional. In particular, covalent attachment of biologically active ligands helps augment their selectivity and binding to tumor tissue.23,24 Small peptide groups are widely used as targeting groups for tumor tissues.25,26 For the bioconjugation of peptide-based ligands to the nanogels, generally thiol and amine groups present on the amino acid building blocks are employed as handles for attachment. Amine-based conjugation may exhibit challenges due to abundance of amine groups on biomolecules, which may lead to complications like undesired cross-linking. However, the thiol groups of cysteine residues that are in lower abundance or can be specifically installed can provide more specificity during bioconjugations.27 Conjugation of maleimide to a thiol group is a powerful tool that is widely employed in the field of bioconjugation such as protein labeling and biomolecular immobilization. This reaction is known to proceed under mild conditions in aqueous media with high efficiency.28−32 Nevertheless, in order to efficiently incorporate maleimide groups in polymeric materials, their protection before polymerization is necessary due to their susceptibility to reaction with radicals. A Diels−Alder/retro Diels−Alder reaction sequencebased strategy that allows protection of the maleimide groups with furan has been used to protect the maleimide groups during the polymerization.33−38 On the basis of this approach, a methacrylate based water-soluble thiol-reactive copolymer containing maleimide groups as side chains was recently reported.39 In this work, we present the design and synthesis of poly(ethylene glycol)-based nanogels amenable toward facile multifunctionalization through thiol−maleimide chemistry to engineer a construct for cellular imaging via active targeting Scheme 1. For the formation of the nanogels, polymers containing reactive maleimide groups as side chains were crosslinked using a dithiol based cross-linker. Prior to the crosslinking, LCST behavior of PEG groups are utilized to form

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EXPERIMENTAL SECTION

Materials. Chemicals. Poly(ethylene glycol) methyl ether methacrylate (Mn = 300 g mol−1) (PEGMEMA, 99%, Sigma-Aldrich) is passed through a basic alumina column to remove the inhibitor. Copper(I)bromide (CuBr, 99%, Sigma-Aldrich), 2,2′-bipyridine (99%, Sigma-Aldrich), maleic anhydride (>99%, Merck), furan (>99%, Sigma-Aldrich), 3-amino-1-propanol (>99%, Sigma-Aldrich), 2,2′(ethylenedioxy)diethanethiol, (95%, Sigma-Aldrich), trimethylamine (>99%, Merck), N-ethylmaleimide (98%,Alfa Aesar), 2-mercaptoethanol (>99%, Merck), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) (>98%, Sigma-Aldrich), Tris-HCl (>99%, Sigma-Aldrich), N-(5fluoresceinyl)maleimide (>90%, Sigma-Aldrich), 2,2-dimethyl-3-ethylpyrrole (95%, Sigma-Aldrich), oxalyl chloride (98%, Alfa Aesar), 11bromoundecanoic acid, (99% Aldrich), boron trifluoride-diethyl ether complex (Merck), and potassium thioacetate (98%, Alfa Aesar) were purchased and used as received. The dialysis bags (Spectra/Por Biotech Regenerated Cellulose Dialysis Membranes, MWCO 25 kDa) were purchased from Spectrum Laboratories. BODIPY-Br and the peptide cRGDfC were synthesized according to literature.40,41 Peptide cRGDfV was obtained from Peptides International (U.S.A.). Furanprotected maleimide containing methacrylate (FuMaMA) monomer and 2-(2-(2-methoxyethoxy)ethoxy)ethyl 2-bromo-2-methylproponoate were synthesized according to previously reported protocols.34,42 Column chromatography was performed by using Silicagel-60 (43−60 nm), and thin-layer chromatography was performed by using silica gel plates (Kiesel gel 60 F254, 0.2 mm, Merck). Pierce BCA (bicinchoninic acid) Protein Assay Kit was used for peptide assay (Thermo Fischer Scientific). Cell Studies. Human breast adenocarcinoma MDA-MB-231 cell line is obtained from ATCC (Virginia, US). The cells are maintained in RPMI-1640 culture medium (Roswell Park Memorial Institute) (Gibco, Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Lonza), 100 U/mL penicillin, 100 g/mL streptomycin, 0.25 g/ mL, 5% CO2, and 95% relative humidity. DAPI and Cell Counting Kit8 (CCK-8) were obtained from Sigma-Aldrich. Instrumentation. NMR spectra were recorded by using a 400 MHz Bruker spectrometer at 25 °C. Measurements were performed in deuterated chloroform (CDCl3). The molecular weight of copolymer was determined by size exclusion chromatography by using a Shimadzu PSS-SDV (length/ID 8 × 300 mm, 10 μm particle size) linear Mixed C column calibrated with 1−400 kDa polystyrene standards using a refractive−index detector. THF was used as eluent at a flow rate of 1 mL/min at 30 °C. Fourier transform infrared (FTIR) spectra were obtained using a Thermo Scientific Nicolet 380 FT-IR spectrometer. UV−visible spectra were collected on a Varian Cary 100 Scan UV−vis spectrophotometer. Fluorescence spectra were collected on a Varian Cary Eclipse spectrophotometer. Zeta potential and hydrodynamic radii of nanogels and polymers were determined from 1 mg/mL samples filtrated via 0.2 μm cellulose acetate membrane using an off-line dynamic light scattering analysis (DLS, Malvern, Zetasizer Nano ZS). Hydrodynamic size of nanogels were analyzed using an online gel permeation chromatography (GPC) system with 0.22 μm filtered 1× PBS as eluent at a flow rate of 0.5 mL/min on an HPLC instrument (LC-20AD Pump, Shimadzu, Japan) equipped with an analytical polymer-based size exclusion column (Shodex Protein KW803, Waters Corporation, MA). The refractive index (RI) measurements were obtained using a refractive index detector (Optilab rEX, Wyatt Technology Corporation, U.S.A.). A multiangle light scattering

Scheme 1. Schematic Illustration of Fabrication and Functionalization of Multi-Functionalizable Nanogels

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Biomacromolecules detector was employed for determination of size in aqueous media (DAWN HELEOS II equipped with QUELS, Wyatt Technology Corporation, U.S.A.), and the UV extinction was monitored at 254 and 280 nm (SPD-20A Photodiode Array Detector, Shimadzu). Images of nanogels were obtained using a LVEM5 electron microscope system (Delong America) on transmission electron microscopy (TEM) mode. The MALDI-TOF MS spectrum acquisitions were conducted on a Shimadzu AXIMA Performance Instrument (Shimadzu Biotech, Manchester, U.K.) equipped with a 337 nm N2-laser, calibrated using TOF-mix from Laser BioLabs (Sophia-Antipolis Cedex, France). All spectra were acquired in the positive ion reflectron mode. DHB (20 mg/mL) solution in acetonitrile with 0.1% TFA was used as matrix. The obtained spectra were analyzed using MALDI-MS software (Shimadzu Biotech, Manchester, UK). Cell viability results of cytotoxicity experiments were determined via measuring the absorbance of 96-well plates at 450 nm by using Multiscan FC Microplate Photometer from Thermo Scientific equipped with a quartz halogen light source of a precision CV ≤ 0.2% (0.3−3 Absorbance), excitation wavelength range of 340−850 nm, with excitation filters installed at 405, 450, 620 nm. Cell internalization experiments were performed with Zeiss Observer Z1 fluorescence microscope connected to Axiocam MRc5 using a Zeiss filter set 38 (excitation BP 470/40, emission BP 525/50) for imaging the cells, and Zeiss filter set 49 (Excitation G365, emission BP 455/50) was used for imaging DAPI stained nuclei. Images were obtained to visualize cell nuclei and morphology using Zeiss AxioVision software. Flow cytometry analysis was done using a Guava easyCyte (Merck Millipore) instrument. Synthesis of Furan-Protected Maleimide-Containing Copolymer. To synthesize the copolymer (P1), the furan-protected monomer FuMaMA (0.2 g, 0.69 mmol) and PEGMEMA (1.00 mL, 3.47 mmol) were dissolved in degassed MeOH (3 mL). The monomer solution was added into the flask containing CuBr (5.1 mg, 0.041 mmol) and 2,2′ bipyridine (12.7 mg, 0.082 mmol) in degassed H2O (3 mL). TEGME-Br (12.6 mg, 0.041 mmol) initiator was introduced into the stirring solution, and the reaction was stirred at 25 °C for 15 min. The reaction was quenched by opening the flask to the atmosphere. MeOH was removed under vacuum, whereas lyophilizer was used for the removal of H2O. The polymer was dissolved in minimum amount of THF and precipitated in cold diethyl ether. After precipitation, the residual copper complex was removed via passing the polymer solution through an aluminum oxide column. After evaporation of solvent, the polymer was obtained as a colorless viscous material. ([M0]/ [I0] = 100, [FuMaMA0]: [PEGMEMA0] = 1:5, [I0]: [CuBr]: [PMDETA] = 1:1:2, conversion 75%, Mn,theo = 30135 g mol−1, Mn,GPC = 20000 g mol−1, Mw/Mn = 1.54 relative to PS. In the obtained polymer, the ratio of monomers was [FuMaMA]:[PEGMEMA] = 1:4.6, as determined from 1H NMR spectroscopy. 1H NMR, (CDCl3, δ ppm) 6.53 (s, 2H, CHCH), 5.25 (s, 2H, CH bridgehead protons), 4.07 (s, 2H, OCH2 ester protons of PEGMEMA), 3.91 (br s, 2H, OCH2), 3.65−3.54 (m, 4H, OCH2 of PEGMEMA an NCH2), 3.37 (s, 3H, OCH3 of PEGMEMA), 2.88 (s, 2H, CH−CH, bridge protons), 1.88−0.85 (m, 7H, NCH2CH2CH2O, CH2 and CH3 protons along polymer backbone) (see Figure S4 in Supporting Information (SI)). Activation of Maleimide Groups of Copolymer via the Retro Diels−Alder Reaction. The copolymer P1 (400 mg) was dissolved in anhydrous toluene and heated for 24 h at 110 °C to obtain the thiol-reactive maleimide containing copolymer P2 (380 mg, 95% yield). Complete removal of the oxabicyclic group was confirmed by 1 H NMR analysis. ([FuMaMA]:[PEGMEMA] = 1:5) 1H NMR, (CDCl3, δ ppm) 6.77 (s, 2H, CHCH), 4.07 (s, 2H, OCH2 ester protons of PEGMEMA), 3.94 (br s, 2H, OCH2), 3.65−3.54 (m, 4H, OCH2 of PEGMEMA an NCH2), 3.37 (s, 3H, OCH3 of PEGMEMA), 1.91−0.86 (m, 7H, NCH2CH2CH2O, CH2 and CH3 protons along polymer backbone) (see Figure S5 in SI). Synthesis of Nanogels. Nanogels with different copolymer to cross-linker ratio were synthesized. A typical procedure for nanogel (NG2) synthesized using a maleimide/thiol mol ratio of 2:1 is as follows: In a round-bottom flask, copolymer (5 mg) was dissolved in water (1.3 mL) and heated at 60 °C for 10 min. After 10 min, 0.1 mL

of 16.6 mM 2,2′-(ethylenedioxy)diethanethiol aqueous solution was added into the polymer solution. After addition of cross-linker, 0.1 mL of 4.2 mM Et3N aqueous solution was added into the copolymer solution and the reaction was stirred at 60 °C for 1 h. The nanogels were centrifuged at 7000 rpm for 25 min to remove the non-crosslinked copolymer and catalyst to obtain nanogels (2.2 mg, 44% yield). Likewise, nanogels NG1 and NG3 were synthesized using a maleimide/thiol ratio of 4:1 and 1:1 respectively. Synthesis of BODIPY-SH. Synthesis is schematically outlined in Figure S1, SI. BODIPY-Br (100 mg, 0.204 mmol) was added into potassium thioacetate (28 mg, 0.236 mmol) in acetone (5 mL) and stirred under reflux for 2 h. Thereafter, acetone was evaporated, and the crude mixture was dissolved in CH2Cl2 (150 mL) and washed with water. It was further purified by silica column chromatography using hexane:ethyl acetate (90:10 v/v) as the eluent to obtain BODIPYSC(O)CH3 as a reddish orange solid (86 mg, 0.176 mmol, 86% yield). The BODIPY-SC(O)CH3 (50 mg, 0.104 mmol) was added to potassium carbonate (43 mg, 0.312 mmol) and stirred in ethanol for 6 h at 40 °C under N2 atmosphere in the dark. It was then washed with saturated NH4Cl solution and extracted with CH2Cl2. It was further purified using silica gel column chromatography using hexane:ethyl acetate (90:10 v/v) as the eluent to furnish an orange solid (50 mg, 91% yield). 1H NMR (400 MHz, CDCl3) δ 3.04−2.91 (m, 2H), 2.60− 2.46 (m, 8H), 2.40 (q, J = 7.5 Hz, 4H), 2.33 (s, 6H), 1.71−1.20 (m, 22H), 1.05 (t, J = 7.5 Hz, 6H) (Figure S2, SI). 13C NMR (101 MHz, CDCl3) δ 151.9 (s), 144.9 (s), 135.6 (s), 132.5 (s), 130.9 (s), 34.0 (s), 31.8 (s), 30.3 (s), 29.4 (d, J = 7.0 Hz), 29.0 (s), 28.5 (s), 28.3 (s), 24.6 (s), 17.2 (s), 14.8 (s), 13.3 (s), 12.4 (s) (Figure S3, SI). Molecular weight of the dye was determined via MALDI-TOF mass spectrometry. MALDI-TOF-MS m/z: calculated 476.3; found 477.2 [M + H]+. Conjugation of BODIPY-SH to Nanogels. For the conjugation of the BODIPY−SH, nanogels were collected via centrifugation and dispersed in THF (1 mg/mL). BODIPY-SH (0.1 mg, 0.22 μmol) and Et3N (0.22 μmol) were added into nanogel solution (1 mg/mL), and the solution was stirred at room temperature for 24 h. After the reaction, the nanogels were centrifuged at 7000 rpm for 25 min and were dispersed into water. Conjugation of N-Ethyl Maleimide to Nanogels. Nanogels (1 mg/mL) were dispersed in THF; subsequently, ethyl maleimide (125 μg, 1 μmol) was introduced into the nanogel solution, and the reaction was stirred for 24 h at room temperature. For removal of excess Nethyl maleimide, nanogels were centrifuged at 7000 rpm for 25 min and dispersed in water. Conjugation of 2-Mercaptoethanol to Nanogels. The nanogels conjugated with N-ethylmaleimide (1 mg/mL) were dispersed in H2O, and 2-mercaptoethanol (23 μg, 0.3 μmol) was added to the solution and stirred for 24 h at room temperature. Excess ethylmaleimide was removed by dispersing the nanogels in water followed by centrifugation at 7000 rpm. The process was repeated three times to ensure complete removal of unbound N-ethylmaleimide. Conjugation of cRGDfC to Nanogels. To the 1 mL nanogel solution (2 mg/mL) in water, cRGDfC (250 μg, 0.430 μmol) was added, and the reaction was stirred for 24 h at room temperature. For the removal of the excess peptide, both centrifugation and dialysis were used. After purification of the nanogels, 100 μL of the sample was mixed with 2 mL of BCA working reagent. The blue mixture was incubated for 30 min at 60 °C. After 30 min, the blue mixture turned into purple, and its absorbance was measured at 562 nm via UV spectroscopy. Conjugation of N-(Fluorescein) Maleimide to Nanogels. In a typical procedure, N-(fluoresceinyl) maleimide (25 μg, 0.058 μmol) was added to nanogel NG7 (1 mg/mL), and the reaction was stirred for 24 h at room temperature. After the reactions, residual N(fluoresceinyl) maleimide was removed via centrifugation. In Vitro Experiments. Cytotoxicity Experiments. Cytotoxicity of the nanogels were investigated via CCK-8 viability assay on MDA-MB231 adenocarcinoma cells. Cells (5000 cells/well) were seeded on a 96-well plate as triplicates in 100 μL of culture medium and incubated 492

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Biomacromolecules at 37 °C for 24 h for cells to grow and adhere completely. Nanogel dispersions with different concentrations (1.0−0.001 mg/mL) were prepared in PBS (pH 7.4) and added to the cell media, and the cells were incubated at 37 °C for 48 h. After 48 h, solutions were removed, and the cells were washed with PBS (2 × 100 μL). CCK-8 reagent (6 μL) was used to form the 60 μL of cell media, and the cells were incubated with CCK-8 reagent carrying media for 3 h. After 3 h of incubation, the absorbance values at 450 nm were measured via microplate reader. Results were obtained by GraphPad prism software using in nonlinear regression mode. Internalization Experiments. For the cellular internalization experiment, MDA-MB-231 adenocarcinoma cells (50 000 cells/well) were seeded in 12-well plate as triplicate in 1 mL of culture media. The cells were incubated at 37 °C for 24 h. One of the plates were treated with nanogel (0.1 mg/mL) conjugated to only N-fluoresceinyl) maleimide whereas the other one is treated with the nanogel (0.1 mg/ mL) conjugated to both N-(fluoresceinyl) maleimide and cRGDfC. After addition of nanogels cellular media were removed at several time points (3, 6, and 24 h) during the incubation. After removal, cells were washed with PBS (500 μL) and then DAPI (5 mg/mL) containing PBS were added to the plates. Cells were incubated at 37 °C for 20 min for nuclei staining. Cell images were obtained by using Zeiss Observer Z1 fluorescence microscope. For flow cytometry experiment MDA-MB-231 adenocarcinoma cells (50 000 cells/well) were seeded into 12-well plate as duplicates in 1 mL of culture media. The cells were incubated at 37 °C for 24 h. One of the plates was treated with 0.1 mg/mL NG2F, whereas the other one was treated with the 0.1 mg/mL NG8. After 3, 6, and 24 h, cell media were removed, and the cells were trypsinized with 0.05% trypsin solution. After trypsin neutralization, cells were centrifuged at 300 rpm for 5 min. Then the cells were resuspended in 1× PBS buffer and analyzed via flow cytometry.

Figure 1. (a) Synthesis of copolymer P2. (b) Aggregation of copolymer in aqueous solution above its LCST temperature. (c) Change in hydrodynamic size as determined by DLS at 25 and 60 °C.

embedded in these nanogels provides handle for their postgelation functionalization. Prior to gel formation, the polymer was heated in aqueous solution to 60 °C to obtain self-assembly. The size of this aggregate was investigated by using dynamic light scattering (DLS). It is measured as 192 nm at 60 °C and 7 nm at room temperature (Figure 1c). To determine the LCST temperature, an aqueous solution of copolymer P2 (2 mg/mL) was analyzed using UV−vis spectroscopy. Measurements were made at 600 nm between 5 °C intervals from 20 to 70 °C. The LCST of copolymer P2 was determined to be around 55 °C based on the change in transmittance with increase in temperature (Figure 2).

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RESULTS AND DISCUSSION Synthesis of Thiol-Reactive Maleimide Group Containing Copolymers. The synthesis of maleimide containing hydrophilic copolymer was accomplished using PEG-based methacrylate monomer (PEGMEMA) and a furan-protected maleimide containing methacrylate (FuMaMA) monomer, according to a previously reported protocol.39 Briefly, furanprotected maleimide containing monomer was polymerized with commercially available PEGMEMA (Mn = 300 g mol−1) at room temperature using atom transfer radical polymerization (ATRP) (Figure 1a). The PEGMEMA monomer was selected in order to obtain hydrophilic polymers with antibiofouling and thermoresponsive characteristics. A poly(PEGMEMA-co-FuMaMA) copolymer was synthesized using a 5:1 feed ratio of PEGMEMA to FuMaMA to furnish copolymer P1 will similar monomer ratio (Mn = 21 000 Da, PDI = 1.5). The copolymer was heated to 110 °C to remove the furan protecting group to yield a thiol-reactive maleimide containing copolymer P2. The degree of incorporation of the monomers and complete unmasking of the maleimide monomer through removal of the furan moiety via the retro Diels−Alder reaction were verified by using 1H NMR spectroscopy (Figure S4 and S5, SI). Synthesis of Multiclickable Nanogels. The next step involves the assembly of these thiol-reactive copolymers into nanogels through cross-linking by using dithiol-containing cross-linkers. It is well-established that PEGMEMA-based hydrophilic copolymers form stable nanosize aggregates above their LCST (Figure 1b).43 A two-stage protocol was followed to obtain the cross-linked nanogels; first, the copolymers were assembled into nanoaggregates, followed by their cross-linking via the thiol−maleimide “click” reaction.44−46 Furthermore, the residual unreacted maleimide and thiol functional groups

Figure 2. Change in % transmittance of copolymer (P2) solution at 600 nm with temperature.

To synthesize the nanogels (NG1−3), the copolymer was dissolved in H2O and heated to 60 °C, whereby the clear solution became turbid due to aggregation of copolymers. After formation of the aggregates, the water-soluble dithiol 2,2′(ethylenedioxy)diethanethiol was added to this suspension. The molar ratios of the maleimide to thiol functional groups 493

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(3.3 mg/mL) was conducted at 60, 65, and 70 °C. No significant size increase of nanogels was observed with change in temperature (Figure S10, SI). However, aggregates around 500−600 nm (less than 10%) were observed for nanogels synthesized at 65 and 70 °C. The yields were 52% and 90% for the nanogels synthesized at 65 and 70 °C respectively. As expected, increase of the temperature led to faster gelation; however, it disrupted the size control and caused larger aggregates. Functionalization of Nanogels. One can expect that these nanogels possess two reactive functional groups as handles for further modifications: the unreacted maleimide groups from the copolymer and residual thiol groups from the cross-linker. Because nanogels were fabricated with varying maleimide:thiol ratios, it can be expected that more residual thiols will be present in nanogels formulated with lower maleimide-to-thiol ratios. Indeed, upon addition of N-(5fluoresceinyl)maleimide to these nanogels, an increase in the amount of conjugated dye was observed for gels fabricated using higher amount of cross-linker, as deduced from UV−vis analysis (Figure S11). Also, to investigate the stability of free thiol groups in these nanogels, N-(fluoresceinyl) maleimide was conjugated to aliquots from the same batch of nanogel with 24 h interval. After conjugation and removal of residual unbound dye, the fluorescence of these nanogels was analyzed and was found to be similar, thus indicating that thiol content of the nanogels did not change through this duration (Figure S12, SI). To determine the maleimide content of the nanogels, 2mercaptoethanol was added to N-ethylmaleimide treated nanogels. Thereafter, the decrease in the amount of 2mercaptoethanol in nanogel solution was analyzed using Ellmans analysis. By using the decrease in the amount of thiol in the solution, it was concluded that maleimide content in the nanogel NG2 was 0.144 μmol/mg. On the basis of the amount of free maleimide groups present in the nanogels, it was deduced that the thiol−maleimide conjugation proceeded with an efficiency of 76% during the nanogel formation. Conjugation of BODIPY−SH to Nanogels. To demonstrate the presence of maleimide functional groups within these nanogels, a thiol-bearing hydrophobic dye, namely, BODIPYSH, was used for conjugation. A thiol-containing BODIPY dye was synthesized for the conjugation to the maleimide groups of the nanogels (Figure S1, SI). Conjugation of the dye to the nanogels was carried out in THF because the dye is insoluble in water (Figure 5). Moreover, to prove that the dye was not

were varied as 4:1, 2:1, and 1:1 to obtain nanogels NG1, NG2, and NG3, respectively. Variation in molar ratio of functional groups did not induce a notable change in the size of obtained nanogels, as deduced by DLS analysis. Normally, while the aqueous polymer solution was clear at room temperature, it became turbid upon heating to 60 °C. This self-assembly and formation of turbidity was reversible for polymer; however, the solution maintained its turbidity at room temperature after the gelation reaction. Nanogels were characterized by using DLS and TEM. Hydrodynamic diameter of the nanogel NG2 in water was found close to 90 nm via DLS and 85 nm via aqueous SEC using a sephadex column and light-scattering detectors (Figure S6−S8). The zeta potential of the nanogels were measured as −0.2 mV. A nearly similar size (76.3 ± 9.2 nm) was obtained for the nanogels by analysis of the solventcasted film using TEM (Figure 3). FTIR analysis revealed the presence of intact maleimide groups in the nanogels (Figure S9).

Figure 3. Schematic presentation of gelation reaction of copolymer P2, hydrodynamic size of nanogel at 25 °C using DLS and TEM image.

To investigate the effect of concentration on the size and yield of nanogels, polymer solutions with two different concentrations were used. Both 5 and 2.5 mg/mL polymer concentrations were used to synthesize the nanogels, and 111 and 76 nm size were observed, respectively, using DLS (NG4, NG5, Figure 4). A further decrease in the concentration of

Figure 4. Sizes of the nanogels prepared with different copolymer P2 concentrations.

Figure 5. Conjugation of BODIPY-SH to nanogel NG2.

copolymer P2 (e.g., 1.6 mg/mL) did not yield any cross-linked nanogels. It can be concluded that an increase in the concentration of the polymer solution results in an increase in the size of nanogels. To investigate the effect of temperature on the size and yield of nanogels, the gelation of a copolymer containing solution

incorporated in the nanogel through encapsulation but was covalently bonded through thiol−maleimide coupling, a BODIPY dye (BODIPY-Br) devoid of the thiol group was used for conjugation with a second batch of the nanogel. After the reaction of the nanogel with either BODIPY−SH or BODIPY−Br, nanogels were dispersed in water. Upon UV 494

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Biomacromolecules irradiation, while there was no fluorescence observed for the nanogel treated with BODIPY−Br, the nanogels treated with BODIPY−SH (NG6) showed strong fluorescence (Inset, Figure 6). This experiment while demonstrating dye con-

Figure 6. Fluorescence spectra and UV images of nanogels after treatment with BODIPY-Br (left) and BODIPY-SH (right).

jugation showed that there was no observable physical encapsulation of the dye. Using fluorescence spectroscopy (Figure 6), amount of conjugated BODIPY-SH was calculated as 1.63 nmol/mg of nanogel. The strong fluorescence observed through solubilization of the hydrophobic dye in aqueous solutions upon conjugation to these nanogels suggests their potential application as macromolecular imaging agents. Conjugation of cRGDfC and N-(Fluoresceinyl) Maleimide to Nanogels. To show the multifunctionalizability of these nanogels, the maleimide groups were conjugated with a cyclic peptide bearing a free thiol, cRGDfC (NG7, Figure 7). Due to the hydrophilic nature of the cRGDfC, the reaction was run in aqueous media at room temperature. The residual peptide was removed via dialysis. After the modification, no appreciable change in the size of nanogels was noticed via DLS (78 nm). In order to calculate the total amount of conjugated cRGDfC, BCA assay was used. The nanogel samples were incubated in the BCA working reagent for 30 min at 60 °C. After incubation, absorbance of the solution was measured using UV spectroscopy at 562 nm. Calibration curve of BSA standard was used to calculate the amount of cRGDfC in nanogels (0.13 μmol/mg NG). In addition, BCA assay was performed on the fractions during the removal of cRGDfC via dialysis to confirm that all unbound cRGDfC was removed. After the conjugation of the peptide to the nanogels NG2, the fluorescent dye N(fluoresceinyl) maleimide was conjugated to obtain doubly functionalized nanogels (NG8, Figure 7). No significant change in the size of nanogels was observed (87 nm) via DLS (Figure S13, SI). Zeta potential of nanogel constructs bearing the peptide and fluorescent dye was determined as −15.8 mV. Concentration of the residual nonconjugated dye in the solution was deduced using a UV calibration curve at 480 nm to suggest conjugation of 0.026 μmol/mg dye onto these nanogels. To ascertain the tunability of amount of free thiols in the nanogels, a maleimide-bearing hydrophilic dye, namely, N(fluoresceinyl) maleimide, was conjugated to the nanogels through Michael addition. As expected, it was observed that the amount of residual thiol groups on the nanogel depended on

Figure 7. Conjugation of cyclic peptide cRGDfC and N(fluoresceinyl) maleimide to nanogels.

the molar ratio of maleimide group in the copolymer to the thiol group in the cross-linker. When the ratio of cross-linker over P2 was increased, nanogels could be expected to possess more residual thiol groups. To investigate this phenomenon, we synthesized three different nanogels NG1, NG2, and NG3 with the maleimide to thiol group molar ratio of 1:0.25, 1:0.5, and 1:1, respectively. These nanogels were treated with equal amounts of the fluorescent dye. It was observed that the UV absorbance at 480 nm increased for nanogels containing higher amounts of the cross-linker (Figure S11, SI). As a control experiment, nanogel NG2 was first treated with N-ethyl maleimide to yield a thiol-depleted nanogel. This nanogel was treated with N-(fluoresceinyl) maleimide to yield nanogel NG9F. No absorbance at 480 nm was observed in the UV−vis spectrum for this nanogel, thus suggesting that the dye was indeed conjugated via the thiol−maleimide conjugation. In Vitro Experiments. In Vitro Cytotoxicity. In order for the nanogels to be utilized as imaging agents, they should be nontoxic toward cells. To investigate their cellular compatibility, MDA-MB-231, human breast adenocarcinoma cells, were treated with nanogels. Cells were incubated in 96-well plates and treated with nanogels for 48 h at 37 °C. CCK-8, a colorimetric assay, was performed to distinguish cell viability. Absorbance values at 450 nm were processed by GraphPad prism software using nonlinear regression curve with a sigmoidal dose response equation. The nanogels did not show any appreciable cytotoxicity even at highest concentration (Figure 8). This high degree of biocompatibility could be expected due to the PEG-based matrix of these nanogels. In Vitro Internalization Assays. To investigate the cellular uptake of the fluorescein conjugated nanogels, adenocarcinoma 495

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and cRGDfC. They were then analyzed via flow cytometry, which indicated increased internalization for the peptide containing construct, results consistent with fluorescence microscopy analysis (Figure 10; Figures S14−15). Additionally,

Figure 10. Flow cytometry analysis of cellular uptake for nanogels: (A) control, (B) nanogels conjugated with only N-(fluoresceinyl) maleimide, and (C) nanogels conjugated with both cRGDfC and N(fluoresceinyl) maleimide.

Figure 8. Cytotoxicity assay of NG2 on MDA-MB-231 cells as determined via CCK-8 assay.

MDA-MB-231 human breast cells were treated with the nanogels. Green fluorescence ability of the fluorescein enabled the visualization of the cells. To stain and visualize the nuclei of the cells DAPI, 4′,6-diamino-2-phenylindole, was used due to its blue fluorescence. Cultured cells were treated with nanogel samples (0.1 mg/mL) and incubated at 37 °C. Two constructs were designed for the experiment; one is conjugated to only fluorescein, and the other is conjugated to both fluorescein and cRGDfC. The overexpressed integrin proteins on the surface of MDA-MB-231 cells make them a good target for the constructs carrying cRGDfC on their surface. Cellular uptake of the nanogels was screened at designated time points (3, 6, and 24 h) via fluorescence microscopy (Figure 9). From the

internalization of peptide ligand and dye-conjugated nanogel NG8 in the presence of a competitive ligand for the integrin binding site, namely, cRGDfV, showed internalizations similar to the nanogels devoid of the targeting peptide, thus suggesting that the observed preferential internalization is due to the peptide ligand (Figure S16).

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CONCLUSIONS In conclusion, novel, multifunctionalizable nanogels containing maleimide and thiol functional groups were synthesized through cross-linking of thermoresponsive copolymers composed of pendant PEG and thiol-reactive maleimide functional groups. Because of the presence of PEG based side chains, these polymers possessed a LCST temperature above which they formed nanosized aggregates in aqueous media. These self-assembled aggregates could be cross-linked using the thiol− maleimide conjugation to yield nanogels. Cytotoxicity experiments showed that these nanogels were nontoxic up to 1 mg/ mL, and hence, they could be used for various biological applications. Because the nanogels carried residual maleimide and thiol functional groups, they could be functionalized with molecules/ligands containing either thiol or maleimide groups. Thus, sequential multifunctionalization of these nanogels could be used to obtain multifunctional constructs. In this study, a thiol-bearing targeting group (cRGDfC peptide) and a maleimide-bearing fluorescein dye as imaging group were conjugated to these nanogels to design a targeted imaging agent. Cellular uptake experiments showed that the presence of targeting group improved the internalization of these nanogels. Overall, facile synthesis of thiol and maleimide group containing reactive nanogels provided easy access to a class of biocompatible nanomaterials that could find potential applications in imaging and delivery.

Figure 9. Merged fluorescence images of MDA-MB-231 cells are treated as follows: (A) control, (B) nanogels with only N(fluoresceinyl) maleimide, and (C) nanogels first conjugated with cRGDfC then N-(fluoresceinyl) maleimide. Cells were incubated at 37 °C for different time points (3, 6, and 24 h). The scale bar is 50 μm.

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micrographs, it could be clearly observed that the cells treated with nanogels containing the cyclic RGD group show higher fluorescence intensity than the cells treated with nanogels lacking the targeting peptide group. The results suggested that the presence of the cyclic-peptide-based targeting group, cRGDfC, on these nanogels boosted their cellular internalization. For further confirmation of the cellular internalization, MDA-MB-231 cells were treated with nanogels conjugated to only fluorescein and nanogels conjugated to both fluorescein

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.6b01576. Synthetic details and spectra of BODIPY derivatives, spectra of copolymers, hydrodynamic radius determi496

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nation DLS plots, flow cytometry plots and internalization micrographs (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +902123597613. ORCID

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

A.S. planned and supervised the project. R.S. supervised the biological experiments. B.A. and L.C. carried out the experiments. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank The Scientific and Technological Research Council of Turkey (TUBITAK Project No. 114Z307) for financial support of this research and The Ministry of Development Turkey (Grant No. 2009K120520) for infrastructure support.

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