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Copper-Free Click Chemistry for Highly Luminescent Quantum Dot Conjugates: Application to in ViWo Metabolic Imaging Aude Bernardin,† Aure´lie Cazet,‡ Laurent Guyon,† Philippe Delannoy,‡ Franc¸oise Vinet,† David Bonnaffe´,§ and Isabelle Texier*,† CEA, LETI-MINATEC, De´partement des Technologies pour la Biologie et la Sante´, 17 rue des Martyrs, F-38054 Grenoble, France, Universite´ des Sciences et Technologies de Lille, UMR 8576, Unite´ de Glycobiologie Structurale et Fonctionnelle, 59655 Villeneuve d’Ascq, France, and Universite´ Paris-Sud 11, ICMMO UMR 8182, Laboratoire de Chimie Organique Multifonctionnelle, 91405 Orsay, France. Received December 18, 2009; Revised Manuscript Received February 9, 2010
Quantum dots (QD) are inorganic nanocrystals with outstanding optical properties, specially suited for biological imaging applications. Their attachment to biomolecules in mild aqueous conditions for the design of bioconjugates is therefore highly desirable. 1,3-dipolar [3 + 2] cycloaddition between azides and terminal alkynes (“click chemistry”) could represent an attractive QD functionalization method. Unfortunately, the use of the popular Cu(I)-catalyzed version of this reaction is not applicable for achieving this goal, since the presence of copper dramatically alters the luminescence properties of QD dispersions. We demonstrate here that copper-free click chemistry, between strained cyclooctyne functionalized QD and azido-biomolecules, leads to highly luminescent conjugates. In addition, we show that QD-cyclooctyne can be used at previously unreported low concentration (250 nM) for imaging the incorporation of azido-modified sialic acid in cell membrane glycoproteins.
Fluorescence imaging, from the cellular to the macroscopic range, is an invaluable tool to improve our knowledge of complex biological pathways, in particular, those not ruled by genomics, such as post-transcriptional modifications (e.g., phosphorylation, glycosylation). In the last ten years, inorganic semiconductor nanocrystals known as quantum dots (QD) have drawn attention as a potential alternative to classical organic fluorophores for imaging purposes (1-3). Their tunable, narrow, and symmetric emission band, long luminescence lifetime, and good photostability make them attractive objects for fluorescence in vitro diagnostics and in vivo imaging (4-9). Conjugation of QD with biomolecules such as antibodies, proteins, enzymes, DNA, and oligosaccharides is a prerequisite to their use as biological probes for fluorescence imaging. Therefore, the presence on QD surfaces of reactive moieties allowing further mild, selective, bioorthogonal, and water-compatible ligation with biomolecules, while keeping the outstanding optical properties of the nanoparticle, is of crucial importance. In this regard, Cu(I)-catalyzed 1,3-dipolar [3 + 2] cycloadditions between azides and terminal alkynes, considered by Sharpless “the cream of the crop” among “click” reactions that can be carried out in water (10-13), seems to be an attractive method for QD bioderivatization. Indeed, Cu(I) promoted click chemistry has been extensively used for the modification of numerous bio- or synthetic polymers (14-18) and capsules (18-20), as well as the functionalization of gold (21-23), silica (24), and iron oxide (18, 25-27) nanoparticles, as well as carbon nanotubes (18, 24). The selective and orthogonal reactivity of the azido and terminal alkyne groups has been used for the conjugation of a number of these materials with a wide range of synthetic molecules and biomolecules (peptides, proteins, enzymes, drugs, saccharides, dyes, organometallic compounds, and so forth) while keeping their functionalities (22, 23, 25, 27, 28). * E-mail:
[email protected]. Tel: +33 438 784 670, fax: +33 438 785 787. † CEA, LETI-MINATEC, DTBS. ‡ USTL, UMR 8576, UGSF. § UPS-11, UMR 8182, LCOM.
Moreover, the reaction leads to the formation of a very stable triazole linker, quantitatively, with a limited number of byproduct, and in mild conditions. Quite surprisingly, however, the use of Cu(I) promoted click chemistry for QD functionalization has only been sparingly reported (29). This may be accounted for by the fact that a strong inhibition of the QD luminescence has been observed for bare CdS nanocrystals in the presence of copper ions (29-31). It has been proposed that isolated Cu+ or ultrasmall CuxS (x ) 1-2) aggregates form on the QD surface, inhibiting their emission properties (30, 31). Despite their poly(ethylene glycol) (PEG) organic coating and their protective inorganic ZnS shell, commercial CdSe/ZnS/PEG2000-NH2 QD (Invitrogen ITK 545 aminoPEG, noted as QD-amine 1) used in this study also suffer a dramatic irreversible luminescence inhibition whenever incubated in the presence of CuSO4/sodium ascorbate (NaAsc), the Cu(I) catalyst classically described for Huisgen cycloadditions in water (Figure 1). Two main mechanisms can account for the QD luminescence inhibition in the presence of copper, either (i) luminescence quenching due to nondestructive energy transfer or photoinduced electron transfer between the semiconductor nanomaterial and the Cu cations, which could actually be chelated by the PEG chains coating the nanoparticles; or (ii) chemical reaction between copper and the CdSe/ZnS particles themselves, leading to a partial or complete destruction of the core-shell structure responsible for the luminescence properties (reduction of the number of luminescent dots and/or their intrinsic luminescence intensity). In addition to the formation of CuxS aggregates on the surface of CdS nanocrystals (30, 31), rapidly occurring cation exchange altering the inner core structure of CdS and CdSe quantum dots has been reported (32, 33). In this latter case, the modification of the crystalline structure and shape of the small particles, accompanied by the modification of their optical properties, has been observed (32, 33). More thorough studies on the copperinduced luminescence inhibition mechanism of QD-amine 1 are in progress and will be detailed elsewhere. However, evidence supporting hypothesis (ii) has already appeared: (1) Energy and
10.1021/bc900564w 2010 American Chemical Society Published on Web 03/11/2010
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Figure 1. Cu-induced luminescence inhibition of QD-amine 1 (17 nM) in 1× PBS. (A,B) Photographs of the suspensions under white (A) and UV (B) light before (left) and after (right) NaAsc/CuSO4 addition (25 µM). (C) Stern-Volmer plots depicting the luminescence inhibition of QD-amine 1. Φ (respectively τ) is QD luminescence quantum yield (respectively luminescence lifetime). 0-subscript stands for values in the absence of Cu (I). (D) Absorption spectra of QD-amine 1 (17 nM) in the presence of increasing concentrations of Cu(I). Inset: luminescence spectra of QD-amine 1 (17 nM) in the absence and in the presence of 5 µM Cu(I).
electron transfer has always been reported to affect both nanoparticle luminescence quantum yield (Φ) and lifetime (τ) (34-36). The Stern-Volmer plot Φ0/Φ depicting the ratio of the luminescence quantum yield of QD-amine 1 before (Φ0) and after (Φ) addition of Cu(I) solution (Figure 1) evidence strong luminescence inhibition (decrease by 50% for usual click conditions: 0.1 equiv Cu(I) per functional group). Moreover, even after five washes with PBS, which should have eliminated residual copper in solution or copper tied by weak interaction to the QD surface, Φ is not restored. The nature of the copperrelated QD luminescence inhibition is further investigated by performing luminescence lifetime measurement. As already accounted for in the literature, CdSe dispersions observed as a macroscopic sample display multiexponential luminescence decays (37, 38). However, biexponential fit of the data most often provides a satisfactory description in order to account for QD luminescence quenching by electron or energy transfer (34-36). The luminescence decays in 1× PBS buffer of the core-shell CdSe/ZnS QD-amine 1 used in this study can indeed be satisfactorily fitted by biexponential decays, with a major “long” component τ ≈ 20 ns and a minor short-lived component (2.7 ns), both insensitive to copper concentration (see details in Supporting Information). Since only modifications of Φ and not τ are observed, it suggests that it is the number of emitting nanocrystals that is affected by copper addition, rather than the involvement of QD exciton interactions. (2) Cu(I) or Cu(II) solutions do not display absorption bands in the 500-600 nm range that would overlap with the absorption and emission spectra of the particles and induce energy transfer, and the redox potentials of the species involved should not lead to electron transfer according to the study reported by Mattoussi et al. (36). (3) Only slight spectral modifications are observed upon copper addition in quantum dot dispersion: 2 nm blue shift for the plasmon absorption band, in addition to a global increase of the solution absorbance (Figure 1). These spectral modifications are similar to those observed by Isarov et al. (31), who accounted them for by the formation of small CuxS aggregates on the nanocrystal surface. (4) The possible exchange of shell Zn2+ by Cu2+ cations is highly thermodynamically favorable due to the lower solubility of CuS (Ks ) 8 × 10-37) in comparison to ZnS (Ks ) 2 × 10-25 (wurtzite) to 3 × 10-23 (zinc blende)) (39), whereas the number of copper ions added in the solution is in the same range as those of zinc cations constituting the QD shell (calculated using 4 nm as the CdSe/ZnS diameter according to the supplier, and 2.7 nm diameter CdSe core according to the emission wavelength (40)). Moreover, the ionic diameter of Cu2+ (0.72 Å) is identical to that of Zn2+ (0.72 Å),
and both ZnS and CuS can crystallize with a hexagonal structure, which should facilitate the exchange in the QD shell (33, 39, 41). The small size of the nanocrystals (4 nm total diameter, 2.7 nm core) means that only 5 to 6 atomic layers constitute the core-shell QD, which moreover display a high surface to volume ratio, which could account for an important solid reactivity (33). If the presence of copper induces the irreversible formation of nonemissive nanocrystals with a different composition and structure than the starting QD particles, Cu(I) click chemistry cannot be used for the functionalization of these objects while preserving their inner structure integrity. Copper-free click reactions are therefore highly desirable for QD derivatization in biological media. The possibility to use the Bertozzi’s modification of the 1,3-dipolar [3 + 2] cycloaddition between azides and alkynes, known as “copper-free click chemistry”, has been investigated to achieve this goal (42-47). The principle of copper-free click chemistry relies on the use of cycle’s strain, such as in the cyclooctyne moiety present in compounds 2 and 3 (Scheme 1), to replace the Cu(I) catalysis as the reaction’s driving force. Moreover, copper-free click chemistry avoids the presence of the metallic cation, potentially toxic for living cells, the presence of which can therefore be restrictive for in vivo reactions (43). In order to investigate the impact of the functionalizing conditions (classical and copper-free click chemistry) on QD luminescence, QD-amine 1 are derivatized with cycloot-1-yn-3-glycolic acid 2 by EDC coupling to afford QD-cyclooctyne 3 after size exclusion chromatography (Scheme 1). QD-cyclooctyne 3 are then reacted with a DMSO solution of azido-tagged mannosamine (ManNAz 4), a good model for further in vivo imaging studies, in the absence (leading to 5a) or in the presence (leading to 5b) of a “Cu-click buffer”. After purification by size-exclusion chromatography, the success of the coupling reactions is qualitatively assessed by agarose gel electrophoresis (Scheme 1), since the migration pattern of the nanoparticles are dependent on the QD size, charge, and coating (48). QD functionalization is further evidenced by modification of the particle hydrodynamic diameter and zeta potential measured using dynamic light scattering in PBS buffer (Table 1). QD-amine 1 has an inner negative polymer coating, which is partly counterbalanced by the terminal protonated amino groups at the end of the PEG chains. Overall, the zeta potential of QD-amine 1 is slightly negative (-3.5 ( 5.6 mV), and particles migrate toward the positive pole in the gel (Scheme 1). Alkyne derivatization induces a negative shift of the zeta potential of QD-cyclooctyne 3 (-25.6 ( 7.6 mV), as surface positive ammonium groups of QD-amine 1 are replaced by
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Bioconjugate Chem., Vol. 21, No. 4, 2010 585
Scheme 1. Functionalization of QD Surface by Copper-Free (a) and Cu(I)-Catalyzed (b) Click Chemistriesa
a
Modifications of the QD surface are qualitatively assessed by electrophoretic migration of the particles on agarose gel.
Table 1. Physical and Optical Properties of QD in Phosphate Buffer nanoparticles
hydrodynamic diameter (nm)a,b
zeta potential (mV)a,c
absorption maximum (nm)b
emission maximum (nm)b,d
luminescence quantum yielda,b,d
QD-amine 1 QD-cyclooctyne 3 5a 5b
14.7 ( 0.4 9.6 ( 0.7 16.3 ( 2.0 16.9 ( 1.2
-3.5 ( 5.6 -25.6 ( 7.6 -41.4 ( 2.1 -39.5 ( 3.7
530 531 530 530
552 552 551 550
100 137 ( 14 127 ( 13 47 ( 5
a Standard deviation for three different measures. b Measures are performed in 1× PBS buffer (10 mM phosphate, 137 mM NaCl, pH 7.3). Measures are carried out in 0.1× PBS buffer (1 mM phosphate, 13.7 mM NaCl, pH 7.3) in order to limit the buffer conductivity. Several peaks are observed in zeta potential analysis, even with commercial QD-amine 1. The values indicated refer to the main peak (>65%). d Excitation at 488 nm. c
Scheme 2. Cell Machinery Modification of Mannosamines Ac4ManNAz 6a and Ac4ManNAc 7a to Biosynthesize Sialic Acids 6b and 7b, Respectively
neutral alkyne groups. Interestingly, the hydrodynamic diameter of particles 3 (9.6 ( 0.7 nm) is reduced in comparison to that of QD-amine 1 (14.7 ( 0.4 nm), probably because the rather hydrophobic ending groups prefer to retract in the PEG chains of the QD-coating polymer than remain in aqueous solution. Moreover, QD-cyclooctyne 3 migrate in the agarose gel toward the negative pole. Such a migration pattern has already been reported for gold (49) and CdSeTe/ZnS (50) nanoparticles coated by poly(ethylene glycol) chains. It has been accounted for by the association of positively charged cations, such as sodium and potassium cations present in PBS or TBE buffer, with the ethyleneoxide motif (51). Therefore, PEG chains associated with positive charges would be the entities that appear on the particle surface for QD-cyclooctyne 3, accounting for their electrophoretic migration toward the negative pole. As previously suggested for other quantum dot conjugates, gel migration orientation would be mainly governed by the charges on the external surface of the particles, whereas zeta potential might
rather reflect the overall charges of the QDs (taking into account their inner negative charges) (50). ManNAz 4 grafting onto the QD 5a and 5b is evidenced by an important increase of the particle hydrodynamic diameter (∼16-17 nm) and a shift of the zeta potential (∼ -40 mV) (Table 1), along with an electrophoretic migration toward the positive pole. The presence of negative charges on the QD-ManNAz particles 5a and 5b might be due to the long-known reaction between carbohydrate diols and the borate ions of the electrophoresis buffer. Indeed, neutral carbohydrates are known to migrate toward the positive pole in such electrophoretic conditions (52). The two QDManNAz conjugates 5a and 5b display similar structural properties (size and zeta potential), regardless of the functionalization method used. In particular, the increase of the hydrodynamic diameter of the particles can certainly be attributed to the stretching of the highly hydrophilic PEGsaccharide chains in the aqueous phase. However, if alkyne derivatization does not alter the QD luminescence and integrity
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Figure 2. Fluorescence microscopy images of CHO cells treated with Ac4ManNAz 6a or Ac4ManNAc 7a (negative control) at 200 µM for 3 days, incubated with QD-cyclooctyne 3 (green) at 250 nM for 4 h at 4 °C, and fixed with paraformaldehyde 4%. Nuclei have been stained with DAPI (blue).
(QD-cyclooctyne 3 display even ∼30% improved Φ in comparison to QD-amine 1), the presence of copper for QD modified by classical click chemistry (conditions b) induces a loss of about half of the luminescence for the QD suspension (Table 1). Interestingly, copper-free click chemistry (conditions a) allows the modification of the QD surface with a high remaining luminescent quantum yield (Table 1). Strain-promoted Huisgens cycloaddition thus appears as a method of choice for the functionalization of QD using such a bioorthogonal and biocompatible “click” reaction. To outline the interest of QD modified by cyclooctynes for biological applications, the possibility to use QD-cyclooctyne 3 for the in vivo imaging of the metabolic incorporation of sialic acids on cell membrane glycoconjugates is investigated. The development of new fluorescent tools for the in vivo imaging of the biosynthesis of glycoconjugates is of tremendous interest. The modification of cell surface glycosylation is a common phenotypic change that occurs in a number of pathologies, including carcinogenesis (53-55). It mainly affects the outer part of the carbohydrate moiety of glycoproteins and glycolipids, leading to the expression of tumor associated carbohydrate antigens (TACA). Most of TACA are sialylated and the expression of TACA such as sialyl-Lewisx or sialyl-Tn is usually linked to tumor aggressiveness (56, 57). Acetylated azidomannosamine Ac4ManNAz 6a (or peracetylated mannosamine Ac4ManNAc 7a used as a negative control) is incubated with CHO (Chinese Hamster Ovary) cells for incorporation in membrane glycoconjugates via a metabolic savage pathway (58). Acetylated Ac4ManNAz 6a is used instead of ManNAz 4, since acetylation allows for a better cellular internalization and incorporation in glycoconjugates (59). Briefly, cells treated with Ac4ManNAz 6a (200 µM) or with Ac4ManNAc 7a (200 µM) for 3 days metabolize the mannosamine moiety into sialic acids 6b and 7b, which can subsequently be used by the cell machinery and be incorporated in the newly synthesized glycoproteins exposed on the cell membrane (Scheme 2) (60). The incorporation of the azido-modified metabolite 6b transferred to the cell’s membrane epitope is revealed using copperfree click chemistry with QD-cyclooctyne 3 as labeling agent (incubation at 250 nM for 4 h at 4 °C, to avoid particle endocytosis). Epi-fluorescence microscopy images are recorded and are displayed in Figure 2. Cell membranes are clearly labeled by the QD conjugate 3 when CHO cells have been incubated with Ac4ManNAz 6a, contrary to the negative control Ac4ManNAc 7a for which no background signal is detected, indicating the absence of QD agglomeration or precipitation at the cell surface. The low photobleaching rate of QD under microscopic illumination, as well as the multimeric presentation
of cyclooctyne moieties on their surface, could account for the improved detection sensitivity (250 nM of QD-cyclooctyne 3) in comparison to other organic dye based probes, used at >50 µM concentrations (43). The copper-free click reaction is therefore successfully performed in biological media and allows labeling with QD at submicromolar concentrations with a good signal/noise ratio. We have demonstrated that copper-free click chemistry offers a great opportunity for QD facile bioorthogonal derivatization, while bypassing the luminescence inhibition of the particles observed in the presence of Cu(I), the catalyst classically used for alcyne-azide conjugation. Because of their excellent optical properties in general, and their reduced photobleaching rate during microscopic observations in particular, QD modified by cyclooctyne groups prove to be an interesting alternative to organic fluorescent probes usually used for in vivo metabolic imaging (43). Numerous other applications, especially in the field of biology, can be envisioned using this new functionalization process of QD, taking benefit of both the outstanding optical properties of the nanocrystals as labeling agents and the versatile chemistry of copper-free azido/constraint alkyne cycloaddition that circumvent the impossibility of using the popular Cu(I)-catalyzed version of this reaction.
ACKNOWLEDGMENT We acknowledge Peter Reiss (CEA, INAC) for fruitful discussion of the results. This work is supported by the Commissariat a` l’Energie Atomique, France, through the “Technologies pour la sante´’” program (TIMOMA2). Supporting Information Available: Experimental procedures, QD absorption and emission spectra, luminescence lifetime measurement details, hydrodynamic diameter measurements. This material is available free of charge via the Internet at http://pubs.acs.org.
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