A New Family of Pyridine-Appended Multidentate Polymers As

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A New Family of Pyridine-Appended Multidentate Polymers As Hydrophilic Surface Ligands for Preparing Stable Biocompatible Quantum Dots Kimihiro Susumu,*,†,§ Eunkeu Oh,†,§ James B. Delehanty,‡ Fabien Pinaud,∥ Kelly Boeneman Gemmill,‡ Scott Walper,‡ Joyce Breger,‡,⊥ Maria J. Schroeder,# Michael H. Stewart,† Vaibhav Jain,† Craig M. Whitaker,# Alan L. Huston,† and Igor L. Medintz*,‡ †

Optical Sciences Division, Code 5611, and ‡Center for Bio/Molecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory, Washington, DC 20375, United States § Sotera Defense Solutions, Columbia, Maryland 21046, United States ∥ Department of Biological Sciences, Department of Chemistry, and Department of Physics and Astronomy, Dana and David Dornsife College of Letters, Arts and Sciences, University of Southern California, Los Angeles, California 90089, United States ⊥ American Society for Engineering Education, Washington, DC 20036, United States # Department of Chemistry, U.S. Naval Academy, Annapolis, Maryland 21402, United States S Supporting Information *

ABSTRACT: The growing utility of semiconductor quantum dots (QDs) in biochemical and cellular research necessitates, in turn, continuous development of surface functionalizing ligands to optimize their performance for ever more challenging and diverse biological applications. Here, we describe a new class of multifunctional polymeric ligands as a stable, compact and high affinity alternative to multidentate thiolated molecules. The polymeric ligands are designed with a poly(acrylic acid) backbone where pyridines are used as anchoring groups that are not sensitive to degradation by air and light, along with short poly(ethylene glycol) (PEG) pendant groups which are coincorporated for aqueous solubility, biocompatibility and colloidal stability. The percentages of each of the latter functional groups are controlled during initial synthesis along with incorporation of carboxyl groups which serve as chemical handles for subsequent covalent modification of the QD surface. A detailed physicochemical characterization indicates that the multiple pyridine groups are efficiently bound on the QD surface since they provide for relatively small overall hydrodynamic sizes along with good colloidal stability and strong fluorescence over a wide pH range, under high salt concentration and in extremely dilute conditions at room temperature under room light over extended timeframes. Covalent conjugation of dyes and metal-affinity coordination with functional enzymes to the QD surfaces were also demonstrated. Biocompatibility and long-term stability of the pyridine polymer coated QDs were then confirmed in a battery of relevant assays including cellular delivery by both microinjection and peptide facilitated uptake along with intracellular single QD tracking studies and cytotoxicity testing. Cumulatively, these results suggest this QD functionalization strategy is a viable alternative that provides some desirable properties of both compact, discrete ligands and large amphiphilic polymers.

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applications continues to be a primary research topic.3,9−11 Methods to modify the surface of as-prepared hydrophobic QDs have been mainly categorized by three different chemistry approaches: (i) ligand exchange, (ii) encapsulation, and (iii) silica coating.3,9−13 Silica coating has fallen out of favor probably due to the complexity of the chemistry and the large hydrodynamic size. While encapsulation by amphiphilic

INTRODUCTION The application of luminescent semiconductor quantum dots (QDs) within biological formats grows with each passing year and their roles have increased far beyond that of just a bright cellular/in vivo probe to include active participation in diverse biosensing and drug delivery formats along with growing potential as photodynamic therapy agents.1−8 Since biological systems exist primarily in aqueous environments, it is mandatory to render the as-prepared hydrophobic QDs water-soluble. Developing robust QD surface chemistries to ensure long-term colloidal stability for these challenging © 2014 American Chemical Society

Received: July 1, 2014 Revised: August 5, 2014 Published: September 11, 2014 5327

dx.doi.org/10.1021/cm502386f | Chem. Mater. 2014, 26, 5327−5344

Chemistry of Materials

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Scheme 1. Synthesis and Ligand Exchangea

a (A) Synthetic schemes for the polymer ligands (1−6) used in this study. Letters a−d represent the precursors of each functional group. The parameter (x:y:z) indicates the average percentages of the pyridine (x), PEG (y) precursors, and free carboxyl groups (z) on a single polymer chain. This is based on the molar amount of the precursors used for the coupling reaction compared to the molar amount of carboxyl groups available in the PAA used. (B) Schematic representation of the one-step and two-step ligand exchange processes using the polymer ligands. Note, not to scale.

biological sensing applications including fluorescence resonance energy transfer (FRET), where the sensitivity is directly related to the distance separating the energy donor and acceptor,8,16 and for efficient renal clearance in vivo.17,18 Cumulatively, this has led to ligand exchange with discrete molecules remaining the most popular approach to keep the hydrodynamic sizes of QDs small. For biological applications, QDs typically utilize ZnS as the outermost shell because of its role as a physical barrier to

polymers is still quite popular due to providing stable and bright materials by “insulating” the QD from the aqueous environment, this method also dramatically increases the hydrodynamic size of the resulting colloid.3,9−11 Larger-sized nanoparticles are often more difficult to deliver to cellular environments and show poor biodistribution. In the case of single particle tracking studies, larger materials are less mobile and may hamper the cellular processes to be monitored.14,15 A small overall QD hydrodynamic size is thus crucial for many 5328



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with pyridine itself, implying that the electronic properties of pyridine’s peripheral substituents may play an important role in QD surface binding ability. The binding of pyridine monomers to the QD surface, however, is not expected to be strong enough to maintain each ligand robustly over long time periods in aqueous media, and therefore, it is rational to utilize pyridines as ligand-anchoring groups in the context of multidentate structures. Here, we report the design and synthesis of a new family of pyridine-appended polymeric ligands which are intended to yield QDs with excellent colloidal stability. Our design rationale focuses on providing a viable alternative to utilizing thiols for multidentate coordination to QDs while exploiting some of the benefits of both small discrete ligands and polymers without incurring a large size penalty. The ligand design relies on multiple pyridine pendant groups attached to a polymer backbone to provide strong coordination onto QD surfaces; chemical structures of the polymer ligand series described here (1 − 6) are shown in Scheme 1A. QDs coated with these ligands were subjected to extensive physical, optical, and functional characterization in assays ranging from bioconjugation to intracellular single QD tracking.

prevent potentially toxic core materials such as CdSe from leaching out to the surrounding environments. In addition, the higher band gap of ZnS (∼3.7 eV) confines the excitons to the cores and prevents them from interacting with the surrounding environment, thereby maintaining high fluorescence quantum yields (QYs).3,9 Among the more common chemical groups able to coordinate Zn in the context of the QD surface, thiols form reasonably stable bonds leading to their ubiquitous use as the surface anchoring group on many discrete QD ligands. While thiol-based ligands still play a dominant role in providing QDs dispersibility in aqueous media, issues of long-term stability have also arisen.3,9 Thiolated ligands, especially the monothiolated variety, are prone to oxidation in air and under light with the presence of QDs potentially contributing to this as a photocatalyst.19 This can result in a dynamic off-rate and the formation of a disulfide bond leading to the liberation of the surface ligands from the QDs and eventual aggregation. To overcome this issue, a variety of multidentate thiolated ligands have been synthesized over the past decade by several groups, including our own, and these have cumulatively confirmed enhanced functional stability using this cooperative binding motif.20−27 This approach has been further extended to create polymeric ligands appended with multiple thiols as pendant groups; these yield robust hydrophilic QDs which are wrapped by the polymers.28−33 Despite these improvements, synthesizing, purifying, and handling discrete multidentate thiolated ligands and/or polymers is not a trivial undertaking. Moreover, the presence of so many thiols in such close proximity to each other makes them extremely susceptible to disulfide formation and cross-linking. This has spurred interest in replacing thiols with other QD anchoring groups that can be used in a similar multidentate fashion to overcome issues of lower affinity when presented in the context of a monomeric molecule that can only bind to the QD surface at a single point. A variety of other chemical groups have demonstrated QD surface binding or have been utilized as ligand anchors with QDs including carboxylic acids,34−36 amines,37−39 phosphines,40 imidazole,41,42 and pyridine.43 Among these, pyridine is well-known for use in replacing the original hydrophobic ligands on the QD surfaces and has been extensively utilized to fabricate QD-based thin film devices such as solar cells.44−46 Pyridine-coated QDs are favored in this role as their compact size minimizes the steric barrier on surfaces and helps enhance electronic communications in the solid state. To accomplish ligand exchange and replacement, the QDs are usually heated in a vast excess of pyridine at ≥60 °C, leading to efficient replacement of the original surface. QD films can be further annealed under an inert atmosphere to remove pyridines and prepare densely packed QD arrays for device fabrication. Indeed, Pearson’s Hard and Soft Acid and Base (HSAB) Principle categorizes Zn2+ and pyridine as borderline acid and base, respectively,47 confirming pyridine’s utility as a potential anchoring group with the Zn surface of QDs. Emrick successfully demonstrated the utility of pyridine groups to render QDs soluble in aqueous media almost a decade ago.43 They synthesized poly(ethylene glycol) (PEG) appended pyridine monomers in which the pyridine and PEG units work as surface anchoring and water solubilizing groups, respectively. These PEG-appended pyridines displaced the native surface ligands on CdSe QDs and rendered the QDs soluble in water. They also found that N,N-dimethylaminopyridine (DMAP) could completely displace the native ligands in a rapid manner which was, interestingly enough, not observed

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EXPERIMENTAL SECTION

Chemicals. 4-Bromopyridine hydrochloride, N,N′-dimethylethylenediamine, N-hydroxysuccinimide (NHS), methyl acrylate, and agarose (low EEO) were purchased from Acros Organics (Fisher Scientific, Pittsburgh, PA). Palladium (10 wt % on activated carbon), Poly(acrylic acid) (PAA; average MW ∼1800), N,N′-diisopropylcarbodiimide (DIC), 4-(aminomethyl)pyridine, and 2-(2-aminoethoxy)ethanol were purchased from Sigma-Aldrich (St. Louis, MO). N-EthylN′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS) were purchased from Pierce Biotechnology (Rockford, IL). MeO-PEG750-NH248 and N3-PEG400NH249 were prepared as described. All the other chemicals including solvents were purchased from Sigma-Aldrich or Acros Organics and used as received. Quantum Dots. 545 nm emitting CdSe/CdZnS/ZnS core−shell QDs were synthesized as previously described with some modifications.50,51 For this particular QD sample, a minimum amount of CdZnS (less than one layer) precursor was applied to overcoat the CdSe core, which allowed us to tune the emission maxima. 600 nm emitting CdSe/CdS/ZnS core−shell QDs were synthesized via modification of published procedures combining successive ion layer adsorption and reaction (SILAR) with thermal cycling techniques.52−54 Instrumentation. 1H NMR spectra were recorded on a Bruker SpectroSpin 400 MHz spectrometer. Chemical shifts for 1H NMR spectra are reported relative to tetramethylsilane (TMS) signal in deuterated solvent (TMS, δ = 0.00 ppm). All J values are reported in hertz. A Finnigan LCQ Classic electrospray ionization/ion trap mass spectrometer was used for mass spectral analysis. Each sample was dissolved in methanol and introduced by direct infusion using a syringe pump. Electronic absorption spectra were recorded using an HP 8453 diode array spectrophotometer (Agilent Technologies, Santa Clara, CA) or Shimadzu UV-1800 UV−vis spectrophotometer. Fluorescence spectra were collected using a Spex Fluorolog-3 spectrophotometer (Jobin Yvon Inc., Edison, NJ) equipped with a red-sensitive R2658 Hamamatsu PMT detector. Fluorescence quantum yields were measured at room temperature with Rhodamine 6G in methanol (Φf = 0.93)55 or Rhodamine 101 in ethanol (Φf = 1.0)56 as standards. The obtained fluorescence spectra were corrected using the spectral output of a calibrated light source supplied by the National Bureau of Standards. FT-IR spectra were measured using a Nicolet Nexus 870 FT-IR spectrometer (Thermo Fisher Scientific, Inc., Waltham, MA). Dynamic light scattering (DLS) measurements were performed on a CGS-3 goniometer system equipped with a He− 5329



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2 h. The palladium catalyst was filtered off through Celite, and the solvent was evaporated. Yield = 0.275 g (95% based on 0.301 g of N3PEG400-N(COOMe)2). 1H NMR (400 MHz, CDCl3): δ 3.57−3.73 (m, −CH2CH2O− and −OCH3), 3.48−3.55 (m, 4H, −CH2−), 2.86 (t, 2H, J = 7.2 Hz, −CH2−), 2.82 (t, 4H, J = 9.4 Hz, −CH2−), 2.67 (t, 2H, J = 8.4 Hz, −CH2−), 2.45 (t, 4H, J = 9.6 Hz, −CH2−). ESI MS m/z: 585.20 (n = 8), 629.13 (n = 9), 673.40 (n = 10) (M+H)+/ theoretical monoisotopic mass (M+H)+ was 585.360 (n = 8), 629.386 (n = 9), 673.412 (n = 10). Synthesis of Pyridine-Appended Polymers. Typical procedures for the synthesis of pyridine-appended polymers are described with polymer 4 as an example. Polymer 4: Poly(acrylic acid) (MW ∼1800: 0.150 g, ∼2.08 × 10−3 mol of carboxyl groups), NHS (0.108 g, 9.38 × 10−4 mol) and DMF (4.0 mL) were added to a 100-mL three-neck round-bottom flask. The reaction mixture was stirred at room temperature under N2 until the solids were completely dissolved. DIC (0.20 mL, 1.3 × 10−3 mol) in 3.0 mL of DMF was added dropwise through an addition funnel. The reaction mixture was stirred at room temperature for 3 h, and subsequently heated to 65 °C. N,N′-Dimethyl-N-(4-pyridyl)ethylenediamine (0.155 g, 9.38 × 10−4 mol) in 4.0 mL of DMF was added dropwise through the addition funnel, and the reaction mixture was stirred at 65 °C for 16.5 h. After checking the complete consumption of N,N′-dimethyl-N-(4-pyridyl)ethylenediamine by thin layer chromatography (TLC), DIC (0.20 mL, 1.3 × 10−3 mol) in 2.0 mL of DMF was added dropwise, and the reaction mixture was stirred at 70 °C for 2 h. MeO-PEG750-NH2 (0.689 g, ∼9.36 × 10−4 mol) in 4.0 mL of DMF was subsequently added dropwise, and the reaction mixture was stirred at 70 °C for 25.5 h. After cooling, the solvent was removed in vacuo. CH3CN was added to the residue, the precipitate was removed by filtration, and the solvent was evaporated (two times). Deionized (DI) water was added to the residue, and the precipitate was filtered off. The aqueous solution was dialyzed in DI water using a dialysis cassette (MWCO 2000; Thermo Scientific; Rockford, IL) for 2 days. During the dialysis, DI water in a reservoir was replaced several times. The product solution in the cassette was filtered, and the solvent was removed in vacuo to obtain an oily product. Yield 0.635 g. 1: 1H NMR (400 MHz, D2O): δ 8.4 (br s, Py), 7.3 (br s, Py), 4.4 (br s, −CH2−Py), 3.3−3.8 (m, PEG), 3.37 (s, −OCH3), 1.0−2.5 (m, PAA). IR (neat): 3514, 3388, 3281, 3069, 2877, 1722, 1673, 1626, 1562, 1547, 1466, 1416, 1359, 1346, 1327, 1281, 1243, 1146, 1111, 1062, 945, 844 cm−1. 2: 1H NMR (400 MHz, D2O): δ 8.1 (br s, Py), 6.9 (br s, Py), 3.2−4.0 (m, PEG and NCH2CH2N), 3.37 (s, −OCH3), 2.8−3.3 (m, N−CH3), 0.9−2.5 (m, PAA). IR (neat): 3516, 3387, 3275, 3076, 2882, 2741, 2696, 1703, 1644, 1630, 1548, 1467, 1413, 1360, 1345, 1326, 1281, 1242, 1199, 1147, 1113, 1061, 946, 843 cm−1. 3: 1H NMR (400 MHz, D2O): δ 8.3 (br s, Py), 7.2 (br s, Py), 4.3 (br s, −CH2− Py), 3.3−3.8 (m, PEG), 3.33 (s, −OCH3), 0.9−2.5 (m, PAA). IR (neat): 3494, 3284, 3066, 2882, 1722, 1669, 1629, 1604, 1562, 1545, 1466, 1417, 1360, 1345, 1281, 1243, 1200, 1147, 1113, 1062, 1042, 1001, 993, 944, 844 cm−1. 4: 1H NMR (400 MHz, D2O): δ 8.0 (br s, Py), 6.7 (br s, Py), 3.3−3.8 (m, PEG and NCH2CH2N), 3.33 (s, −OCH3), 2.8−3.3 (m, N−CH3), 0.9−2.5 (m, PAA). IR (neat): 3505, 3269, 3039, 2872, 1755, 1698, 1644, 1600, 1544, 1524, 1467, 1389, 1346, 1324, 1280, 1254, 1232, 1198, 1108, 993, 948, 844, 808 cm−1. 5: 1 H NMR (400 MHz, D2O): δ 8.4 (br s, Py), 7.2 (br s, Py), 4.3 (br s, −CH2−Py), 3.4−3.9 (m, PEG), 3.2−3.4 (m, −CH2−), 2.8−3.1 (m, −CH2−), 2.5−2.7 (m, −CH2−), 0.9−2.5 (m, PAA). IR (neat): 3290, 3054, 2919, 2872, 1948, 1723, 1672, 1603, 1563, 1547, 1452, 1416, 1377, 1349, 1302, 1251, 1221, 1172, 1106, 1036, 995, 954, 843 cm−1. 6: 1H NMR (400 MHz, D2O): δ 8.1 (br s, Py), 7.0 (br s, Py), 3.5−4.0 (m, PEG and NCH2CH2N), 3.3−3.5 (m, −CH2−), 3.0−3.3 (s, N− CH3), 2.9−3.0 (m, −CH2−), 2.5−2.8 (m, −CH2−), 0.9−2.5 (m, PAA). IR (neat): 3276, 3068, 2869, 1974, 1732, 1645, 1549, 1455, 1350, 1326, 1253, 1217, 1188, 1106, 953, 829 cm−1. Ligand Exchange of Pyridine-Appended Polymer Ligands onto Quantum Dots. Typical procedures for ligand exchange reaction are as follows.48−50 As-prepared 600 nm emitting QDs (5 nmol) were flocculated with a mixture of acetone and acetonitrile. The mixture was centrifuged at 3800 rpm for 5 min and the supernatant

Ne laser illumination at 633 nm and a single photon counting avalanche photodiode for signal detection (Malvern Instruments). The autocorrelation function was performed by an ALV-5000/EPP photon correlator and analyzed using Dispersion Technology Software (DTS, Malvern Instruments). All QD solutions were filtered through 0.02 or 0.1 μm syringe filters (Whatman), and sample temperature was maintained at 20 °C. The autocorrelation function was the average of three runs of 10 s each, and then repeated at different scattering angles ranging from 70° to 110°. The Laplace transform CONTIN analysis was applied to extract intensity versus hydrodynamic size profiles for the QD samples studied.57 Laser Doppler velocimetry measurements were performed using a ZetaSizer NanoSeries equipped with a He−Ne laser source (λ = 633 nm) and an avalanche photodiode for detection, controlled with DTS software. The QD solutions (∼100 nM) were loaded into disposable capillary cells, and data were collected at 25 °C. Three runs of the measurements were performed for each sample. Structural characterization of the QDs was carried out using a JEOL 2100-FE analytical high-resolution transmission electron microscope (HR-TEM) with a 200 kV accelerating voltage. Samples for TEM were prepared by spreading a drop of the QD dispersion onto the ultrathin carbon film on holey carbon support film on a fine mesh gold grid (300 mesh) (Ted Pella, Inc.) and letting it dry. The individual particle sizes were measured using Gatan Digital Micrograph (Gatan, Inc.) and ImageJ; average sizes and standard deviations were extracted from analysis of at least ∼100 nanoparticles. pKa values of pyridine derivatives were calculated by ACD/PhysChem Suite, version 12.01, Advanced Chemistry Development, Inc., Toronto, ON, Canada. Synthesis. A synthetic scheme of the polymer precursors is available in the Supporting Information (SI) (Figure S1). N,N′-Dimethyl-N-(4-pyridyl)ethylenediamine. 4-Bromopyridine hydrochloride (2.00 g, 1.03 × 10 −2 mol) and N,N′dimethylethylenediamine (4.63 g, 5.25 × 10−2 mol) were added to a 100 mL round-bottom flask and purged with N2, and the reaction mixture was refluxed for 5 h. After cooling, K2CO3 (3.60 g, 2.60 × 10−2 mol) was added. The mixture was filtered and the solid was extracted with toluene and isopropanol, and the filtrate was evaporated. NaOH solution (1 M; 100 mL) saturated with NaCl was added to the reaction mixture. The product was extracted with CHCl3 (∼5 times). The combined organic layers were dried over Na2SO4, inorganic salt was filtered off, and the solvent was evaporated. The residue was chromatographed on silica gel with CHCl3:MeOH (4:1) as an eluent. Yield = 1.381 g (81% based on 2.00 g of 4-bromopyridine hydrochloride). 1H NMR (400 MHz, CDCl3): δ 8.17 (dd, 2H, J = 7.2 and 2.0 Hz, pyridine-2,6-H), 6.65 (dd, 2H, J = 7.2 and 2.0 Hz, pyridine-3,5-H), 3.62 (t, 1H, J = 8.8 Hz, −CH2−), 3.08 (s, 3H, −CH3), 2.89 (t, 2H, J = 8.8 Hz, −CH2−), 2.51 (s, 3H, −CH3). The product was confirmed by electrospray ionization mass spectrometry (ESI MS) with a mass-to-charge ratio (m/z) of 166.13 (M+H)+/ theoretical monoisotopic mass (M+H)+ was 166.134. N3-PEG400-N(COOMe)2. N3-PEG400-NH249 (2.709 g, ∼6.18 × 10−3 mol) and methanol (30 mL) were added to a 250 mL roundbottom flask, and the mixture was cooled in an ice-bath under N2. Methyl acrylate (1.40 mL, 1.55 × 10−2 mol) dissolved in 10 mL of methanol was added dropwise over 1 h to the mixture through an addition funnel. The reaction mixture was gradually warmed up to room temperature and stirred overnight under N2. The solvent was then evaporated and residue was purified over silica gel column chromatography with CHCl3:MeOH (15:1) as the eluent. Yield = 3.130 g (83% based on 2.709 g of N3-PEG400-NH2). 1H NMR (400 MHz, CDCl3): δ 3.57−3.71 (m, −CH2CH2O− and −OCH3), 3.52 (t, 2H, J = 8.2 Hz, −CH2−), 3.39 (t, 2H, J = 6.8 Hz, −CH2−N3), 2.82 (t, 4H, J = 9.6 Hz, −CH2−), 2.67 (t, 2H, J = 8.2 Hz, −CH2−), 2.45 (t, 4H, J = 9.6 Hz, −CH2−). The product was confirmed by ESI MS with a mass-to-charge ratio (m/z) of 611.27 (n = 8), 655.13 (n = 9), 699.20 (n = 10) (M+H)+/theoretical monoisotopic mass (M+H)+ was 611.350 (n = 8), 655.377 (n = 9), 699.403 (n = 10). H2N-PEG400-N(COOMe)2. N3-PEG400-N(COOMe)2. (0.301 g, ∼4.93 × 10−4 mol) was dissolved in 8.0 mL of methanol. 10% Palladium on carbon (10.5 mg) was added, and the mixture was shaken under hydrogen atmosphere (40 psi) at room temperature for 5330



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was discarded. The QD pellet was mixed with CHCl3 (0.3 mL), ethanol (0.5 mL), and 2-(2-aminoethoxy)ethanol (0.15 mL). The reaction mixture was stirred at 60 °C for 1 h under N2. After cooling, the QDs were flocculated with a mixture of ethyl acetate and hexane, and centrifuged at 3800 rpm for 5 min, and the supernatant was discarded. The QD pellet was dissolved in 0.5 mL of ethanol, and pyridine-appended polymer 4 (15.0 mg) in 0.90 mL of ethanol was injected, and the reaction mixture was stirred at 60 °C for 3.5 h. The QDs were again flocculated with a mixture of ethyl acetate and hexane and the mixture was centrifuged at 3800 rpm for 5 min, and then, the supernatant was discarded. The QD pellet was dissolved in DI water and filtered through a Millex-LCR membrane filter (pore size 0.45 μm, Millipore) and transferred to a centrifugal filter unit (Amicon Ultra 50K, Millipore). The mixture was diluted with DI water and centrifuged at 3800 rpm for 5−10 min, and the clear filtrate was discarded. To remove excess unbound ligands and other byproducts, the QD dispersion was subject to a few additional rounds of centrifugation with DI water. Detailed descriptions of the characterization procedures including: pH and salt stability tests; protein expression, Hisn-based protein selfassembly and enzymatic assays; agarose gel electrophoresis; EDC coupling; FRET analysis; along with cellular experimental procedures including: cell cultures; microinjection; cellular uptake with cell penetrating peptide; cytotoxicity testing; and single QD tracking and analysis can all be found in the Supporting Information (SI).

ratios of the pyridine and PEG precursors in a controlled manner. In order to study the relationship between the number of pyridine units in the polymer chain and the colloidal stability of the polymer-coated QDs, two different pyridine grafting percentages of 30 and 45% were applied during polymer synthesis. The relative grafting percentages of the pyridine and PEG precursors for the coupling reactions were set as follows: (pyridine (%), PEG (%)) = (30, 50) for polymers 1 and 2; (45, 45) for polymers 3 and 4; (45, 50) for polymers 5 and 6, respectively. Reactions were monitored by thin layer chromatography (TLC) to ensure that the pyridine precursors were consumed before grafting PEG units; this is an important step for subsequently monitoring how the percentages of pyridine incorporated into the polymer affected QD colloidal stability. Completion of PEG grafting was checked by TLC again and the crude product was purified by membrane dialysis in DI water. The polymer ligands synthesized in this study were first characterized by 1H NMR spectra. Figure 1 presents 1H

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RESULTS AND DISCUSSION Design and Synthesis of the Pyridine-Appended Polymer Ligands. In addition to replacing the thiol QD anchoring group, other important design criteria for the pyridine-appended polymers were to ensure good QD colloidal stability in biologically relevant environments while making the overall QD size as small as possible. The polymers designed in this study incorporate poly(acrylic acid) (PAA) as a linear backbone, pyridine groups as anchors to the QD surface, and PEGs as hydrophilic units to ensure QD water solubility, see Scheme 1A. PEG groups have been extensively used within biocompatible hydrophilic polymers and to help QDs maintain colloidal stability while minimizing nonspecific adsorption in biological environments.3,9−11,58,59 PAA has repeating carboxylic acid groups within its linear chain, which can be simply modified with carbodiimide coupling chemistry to attach both the requisite QD anchoring groups and PEG. This coupling is effected using amine-terminated precursors to form stable amide bonds. The commercially available PAA used as the initial building block in this study has an average molecular weight of 1800 and has been previously used to prepare other hydrophilic polymeric ligands for QDs.29,60 We adopted this short polymer chain (∼25 average repeating units) to avoid potential cross-linking between the QDs during the ligand exchange process. One of the advantages in using pyridine units as the QD anchoring group is that the binding ability of the pyridine unit can be modified by the peripheral substituent. In this study, we used 4-(aminomethyl)pyridine and N,N′dimethyl-N-(4-pyridyl)ethylenediamine as pyridine precursors for the polymer synthesis: the p-positions of these pyridine units are modified with alkyl and amino groups, respectively. Compared with the alkyl substituent, the amino substituent is expected to enhance the electron donating property of the pyridine unit. The polymer ligands were synthesized by carbodiimide coupling chemistry using N,N′-diisopropylcarbodiimide (DIC) and N-hydroxysuccinimide (NHS) as coupling reagents in one pot (Scheme 1A), see also Experimental Section. This simple reaction scheme allowed us to modify the PAA with different

Figure 1. Representative 1H NMR spectra. Pyridine-appended polymers measured in D2O: (A) 3, (B) 4.

NMR spectra of polymers 3 and 4 as typical examples. The peaks ascribed to pyridine, PEG, and PAA units were relatively well separated though each peak was broad. The pyridine units show two broad peaks between 6.5 and 8.5 ppm. The repeating ethylene oxide units of PEG have an intense peak around 3.7 ppm, and the methoxy terminal group appears at 3.3 ppm as a singlet peak. The PAA units have broad peaks between 1.0 and 2.5 ppm while a residual NHS peak also appeared as a singlet at 2.6 ppm depending upon the batch. Comparison of the peak integration ratios for each unit helped provide an estimate of 5331



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a large excess needs to be used for efficient ligand exchange. Inefficient ligand exchange with the one-step method suggests that the surface binding ability of the pyridine ligands is not as strong as thiol-based ligands, which do not necessarily require the two-step ligand exchange methods to render QDs soluble in aqueous media. The QDs purified after the two-step ligand exchange were found to remain bright, well dispersed in water and colloidally stable over 6 months without any sign of aggregation even at room temperature under room light (see below). Such remarkable colloidal stability at ambient conditions contrasts with QDs coated with thiol-based ligands: the thiol-coated QD dispersions tend to precipitate after long exposure to light presumably due to photooxidation of the thiol ligands,19,64 and therefore, storage in a refrigerator in the dark is usually recommended. Characterization of the Pyridine Polymer Coated Quantum Dots. Spectroscopic Properties. We next examined the photophysical properties of the hydrophilic pyridine polymer-coated QDs to characterize the properties the ligands impart to the QDs. Absorption and photoluminescence (PL) spectra were recorded for the QDs capped with the native hydrophobic ligands in isooctane prior to ligand exchange and compared to those coated with the polymer ligands in water (Figure 2A and SI Figure S4A). The spectral shape and positions of the lowest absorption bands measured before and after ligand exchange were essentially the same and occasionally showed only a few nanometer shift of the lowest absorption maxima from batch to batch; PL spectra also showed similar trends. On the other hand, new absorption bands appeared around 255 and 270 nm for the QDs coated with the alkylpyridine and amino-pyridine polymers, respectively (Figure 2A and SI Figure S4A). These new bands originate from the pyridine units themselves, and the contributions from QD-2 and QD-4 is more intense than that noted for QD-1 and QD-3. Variability in the absorption intensities of the pyridineoriginated bands arises from the non-negligible differences in the extinction coefficients of the alkyl-pyridine (ε ∼2200 M−1 cm−1 at 256 nm) and amino-pyridine units (ε ∼12 000 M−1 cm−1 at 262 nm). Overall, analyzing these spectral characteristics indicate that the QD core−shell structure and surfaces were well preserved during the two-step ligand exchange in a manner analogous to that seen with other dihydrolipoic acid (DHLA) ligand types.21,23,59 We should also note that the average sizes of the QDs were unchanged before and after the ligand exchange (SI Figure S5), indicating that our ligand exchange method with the pyridine polymer ligands did not cause etching of the QD surfaces. FT-IR spectra were also measured for the QDs before and after ligand exchange to verify that the native synthetic ligands found on the QD surfaces were efficiently replaced by the pyridine-appended polymers (Figure 2B and SI Figure S4B). Following ligand exchange, the QD samples displayed spectral features quite similar to those of the polymer ligands used, indicating that the two-step ligand exchange process was efficient and the native QD surface ligands were mostly replaced by the pyridine-appended polymers. We note that the FT-IR features of the QD sample after the first-step ligand exchange with 2-(2-aminoethoxy)ethanol also have a strong resemblance to those of 2-(2-aminoethoxy)ethanol (SI Figure S6). The FT-IR results confirm that the intermediate amine ligands efficiently stripped off the native synthetic ligands and this, in turn, helped with the subsequent polymer wrapping chemistry. The discussion here was further supported by 1H

the grafting percentages. Table 1 shows the theoretical and experimental percentage of pyridine units attached to a single Table 1. Comparison of Theoretical and Experimental Pyridine Grafting Percentages 1 2 3 4

theoreticala (%)

experimentalb (%)

30 30 45 45

32 31 42 40

a

The percentage was obtained from the molar ratio of the pyridine precursors and the carboxyl groups of PAA. bThe percentage was obtained from the integration ratio of 1H NMR peaks of the pyridine and PAA units.

PAA backbone. Experimental values obtained from peak integration ratios between PAA and pyridine units are reasonably close to the theoretical values, which originate from the initial molar ratio between the pyridine precursors and carboxyl groups present on the PAA. The close matches of those percentages suggest that the numbers of pyridine units attached to the PAA backbone can be simply controlled by the amount of pyridine precursor used within the coupling reactions. Comparison of FT-IR spectra of the product and the precursors also confirmed the formation of the desired structures (SI Figure S2). Ligand Exchange Using the Polymeric Ligands. In order to efficiently replace the native hydrophobic ligands on the QD surface with the polymer ligands, we opted to use a two-step ligand exchange method, in which the native ligands were first replaced with intermediate and lower affinity ligands, followed by ligand exchange with the final polymer ligands, see Scheme 1B. In this study, we used 545 nm emitting CdSe/ CdZnS/ZnS and/or 600 nm emitting CdSe/CdS/ZnS as the QDs, and 2-(2-aminoethoxy)ethanol as the intermediate ligand. Amines are known to bind to the QD surface, yet the binding is relatively weak.61,62 Use of a large excess of this amine ligand in the initial step will strip off the native ligands by mass action, and the amine ligands bound on the QD surface can then be replaced with stronger binding ligands in a second step. Indeed, similar two-step ligand exchange methods have been successfully demonstrated with other intermediate ligands to prepare QDs coated with thiol-based ligands.61,63 The amine ligand used in this study is relatively inexpensive, and its hydrophilic nature renders QDs soluble in hydrophilic solvents such as methanol and ethanol making it easier to do subsequent manipulation: the original hydrophobic QDs are insoluble in such polar solvents. We also carried out direct one-step ligand exchange of the as-prepared hydrophobic QDs with the polymer ligands. However, the one-step methods often resulted in incomplete ligand exchange or larger hydrodynamic sizes compared with the two-step methods, in particular when the alkyl-pyridine polymers (1, 3, 5) were used (for example, see SI Figure S3). We should also note that the amount of the polymer ligands used for the ligand exchange was minimized in the second step to make full surface coverage with each single polymer chain more efficient. This can be important to help keep the overall hydrodynamic size of the QDs small by not allowing multiple QDs to colocalize into each final polymeric dispersion. Achieving a successful ligand exchange with a minimum amount of polymer ligand contrasts with the approach commonly used for small molecular ligands, where 5332



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Figure 2. Effects of ligand exchange. (A) Absorption spectra of 545 nm emitting CdSe/CdZnS/ZnS QDs with the original hydrophobic ligands in isooctane (black line) and with the polymer ligand in H2O (red line), and the corresponding pyridine precursor in H2O (blue line): (i) QD-3 and 4(aminomethyl)pyridine; (ii) QD-4 and N,N′-dimethyl-N-(4-pyridyl)ethylenediamine. Each inset shows the absorption spectra of the lowest absorption bands, and fluorescence spectra of the same QDs with the original hydrophobic ligands in isooctane (green line) and with the polymer ligand in H2O (purple line). (B) FT-IR spectra of the as-prepared 545 nm emitting QDs with native ligands (black line), the same QDs coated with the pyridine-appended polymer ligands (red line), and the polymer ligands only (blue line): (i) QD-3; (ii) QD-4. (C) Fluorescence QYs of (i) 545 nm emitting CdSe/CdZnS/ZnS QDs and (ii) 600 nm emitting CdSe/CdS/ZnS QDs functionalized with 1−4 in H2O along with the original “asprepared” sample in isooctane. (D) Hydrodynamic size distributions of (i) 545 nm emitting CdSe/CdZnS/ZnS QDs and (ii) 600 nm emitting CdSe/CdS/ZnS QDs coated with different ligands measured by dynamic light scattering: QD-1 (black line), QD-2 (green line), QD-3 (red line), QD-4 (blue line), and QD-DHLA-PEG750-OMe (orange line). The chemical structure of DHLA-PEG750-OMe is shown at the top of part D(i).

NMR spectra of the QDs before and after ligand exchange (SI Figure S7). The 1H NMR spectrum of the as-prepared QDs is

represented by a sharp intense peak at 1.26 ppm and a triplet peak at 0.88 ppm, which are ascribed to long alkyl chain and its 5333



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Figure 3. pH stability testing. (A) Luminescence images for a set of 0.5 μM QDs coated with the indicated polymer ligands in different buffers at pH 2−13, taken 24 h and 3 weeks following sample preparation. 600 nm emitting CdSe/CdS/ZnS QDs were used and were excited with a UV lamp at 365 nm to take the images. (B) pH-dependent fluorescence intensities of 0.25 μM 600 nm emitting QDs coated with the pyridine-appended polymers measured at room temperature in 24 h after sample preparation: (i) 1 (navy bar) and 2 (pink bar); (ii) 3 (navy bar) and 4 (pink bar). The intensities were normalized with that of the same QD sample in DI water incubated for 24 h in the same manner.

terminal methyl group, respectively. On the other hand, the 1H NMR spectrum of the pyridine polymer coated QDs simply represents that of the polymer used (Figure 1). Importantly, the peaks ascribed to the long alkyl chain disappeared in the 1H NMR spectrum of the pyridine polymer coated QDs, suggesting that the original hydrophobic ligands were efficiently stripped off during the ligand exchange. Since the proton signals of the long alkyl chain and the PAA unit appear in the same range, it is still difficult to assess the efficiency of ligand exchange in a quantitative manner. Nevertheless, based on the present FT-IR and 1H NMR data, it is reasonable to conclude that the present two-step ligand exchange method leads to nearly quantitative ligand exchange with the pyridine polymer ligands used in this study. It is quite common to see a decrease in the fluorescence QY of QD samples following ligand exchange and phase-transfer from organic to aqueous media; this arises primarily from incomplete surface passivation.65,66 Here, the relative QYs of the two types of QDs (545 nm emitting CdSe/CdZnS/ZnS and 600 nm emitting CdSe/CdS/ZnS) after ligand exchange showed different trends depending on the QD core−shell structures (Figure 2C and SI Table S1). The QYs of the 545 nm emitting CdSe/CdZnS/ZnS after ligand exchange with the pyridine polymer dropped by half in water (∼20−30%) to that measured for the as-prepared hydrophobic QDs in isooctane (48%). The QY decrease after ligand exchange appeared to be far more attenuated for the CdSe/CdS/ZnS materials. The QYs of these QDs were 69% (in isooctane) and ∼40−70% (in water), respectively. The different trends observed for CdSe/ CdZnS/ZnS and CdSe/CdS/ZnS QDs can most likely be attributed to lattice mismatch in the core/shell structures. CdSe and ZnS have a large lattice mismatch (∼12%), which makes it difficult to eliminate the interfacial strain and defects. In order

to alleviate the surface strain and improve the QYs, insertion of a CdS layer in between CdSe core and ZnS layer has been successfully applied since CdSe and CdS have much smaller lattice mismatch (∼3.9%).67 Moreover, the CdSe/CdZnS/ZnS QDs utilized here have only a half layer of CdZnS intermediate shell applied during the overcoating process, and presumably such a thin intermediate layer was still not effective enough to alleviate the interfacial strain and defects. In contrast, the CdSe/CdS/ZnS QDs have four layers of CdS along with a further overcoating of two layers of ZnS, which should help minimize the interfacial strain and provides better overall passivation of the CdSe core. A well-protected emissive core makes the fluorescence less sensitive to surface modification, and therefore, reduction of QY following ligand exchange is minimized. This result implies that the quality of the QD itself, and especially the integrity of the shell layers, largely influences the final QYs after ligand exchange as expected. Supporting this notion, a similar trend has been previously observed for QDs coated with DHLA-based ligands as well.68 It should also be noted that the amino pyridine polymer series gave slightly higher QYs for the same QDs compared with the alkylpyridine analogues, perhaps reflecting the enhanced electron donating character of the amino pyridine anchoring groups. Hydrodynamic Size. We next determined the hydrodynamic diameters (HD) of the pyridine polymer-coated QD series with dynamic light scattering (DLS) analysis. As shown in Figure 2D and SI Table S2, the HD of the 545 nm emitting CdSe/CdZnS/ ZnS QDs coated with 1−4 ranged from 9.7 to 11.5 nm. HD of the same QDs functionalized with discrete DHLA-PEG750OMe23,59 ligands (10.0 nm) falls into the same range: both the pyridine-appended polymers and DHLA-PEG750-OMe incorporate the same PEG precursors in their structures. The hard diameter of the 545 nm QD sample used was directly measured 5334



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by transmission electron microscopy (TEM) to be 4.8 ± 0.39 nm. The difference in sizes between the HD and the hard core− shell size measured in all samples arises from the hydrodynamic interactions of the ligand layer.23,57 Pyridine-appended polymer contributions to the overall hydrodynamic radius are in the ∼2.5−3.4 nm range based on the TEM size of the QD. Values measured for the 600 nm emitting CdSe/CdS/ZnS QDs (TEM size: 8.2 ± 0.46 nm) followed a similar trend with HD increasing over the hard size by ∼2.2−2.8 nm yielding a 12.5 to 13.8 nm range. These values are also comparable to that measured for the same QDs coated with DHLA-PEG750-OMe (13.0 nm). Although the pyridine-appended polymers are larger in size/mass than DHLA-PEG750-OMe, they still provide for QD dispersions with a similar HD suggesting that the polymers are well wrapped around the individual QDs. More importantly, these HD values are smaller than that of QDs functionalized by either longer PEGylated- or larger amphiphilic polymer ligands (usually ≥20 nm).3,28,57,69,70 Long-Term pH and Salt Stability. We examined the longterm colloidal stability of the pyridine polymer-coated QDs across a wide pH range. Representative images in Figure 3A show 0.5 μM 600 nm emitting QDs coated with 1−4, respectively, dispersed in buffer solutions spanning a pH 2−13 range at 24 h and 3 weeks following sample preparation. Images of the intervening time points can be found in SI Figure S8. All QD samples showed good colloidal stability from neutral to basic pHs (pH ≥ 7) for long time periods. Lower pHs gradually led to a partial or complete fluorescence quenching and sedimentation of the QDs; similar results were observed for QDs coated with multidentate thiol ligands at acidic pHs.23,59 We believe that the strongly acidic environment results in “acidic” etching of the QD surface or destabilizes the ligand-QD coordination due to protonation of the pyridine units. The remaining QD dispersions were colloidally stable in weakly acidic to strongly basic pH conditions without apparent quenching for at least up to 3 weeks. Two factors appeared to play important contributing roles toward pH-dependent colloidal stability in acidic media: (1) the type of pyridine anchoring group and (2) their grafting percentages on the polymer chain. First, QD-1 and QD-3 were more resistant than QD-2 and QD-4 in acidic media: the alkylpyridine anchoring groups provide better colloidal stability than the amino analogues. This difference is most likely correlated with the pKa of the pyridine groups. The pKa of pamino substituted pyridine (pKa = 8.8) is higher than that of palkyl substituted pyridine (pKa = 5.6), implying that p-amino substituted pyridine is more susceptible to protonation, especially in acidic media. Protonation of the pyridine group leads to loss of the coordination ability of the ligands for the QD surface, which would result in liberation of the polymer ligands from the QD surface and subsequent aggregation of the QDs. Since the p-alkyl substituted pyridine has a lower pKa, it is less susceptible to protonation and helps maintain the colloidal stability in weakly acidic media for a longer period of time. Second, we observed that within the polymers appended with the same pyridine groups, the higher the grafting percentage of pyridine, the better the ensuing colloidal stability. This trend was expected given the importance of multidentate chemistry for enhancing colloidal stability by higher affinity with the QD surface.22,28,29 However, for amino-pyridine polymers 2 and 4, the benefits of multidentate structures may be overwhelmed by the susceptibility to protonation suggesting that increasing the number of anchoring points bound to the QD by itself is only

sufficient up to a point and environment can be a more determinative factor. pH-dependent PL intensities were also monitored up to 24 h since any changes from pH could directly affect the sensitivity of subsequent sensing and imaging formats using these materials. QDs were dispersed in buffers spanning pH 3−11, and the intensities were measured at

A New Family of Pyridine-Appended Multidentate Polymers As

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