Letter Cite This: ACS Catal. 2018, 8, 5941−5946
pubs.acs.org/acscatalysis
Nanocopper-Doped Cross-Linked Lipoic Acid Nanoparticles for Morphology-Dependent Intracellular Catalysis Jingsheng Huang,† Liang Wang,†,‡ Pengxiang Zhao,§ Fuqing Xiang,† Jie Liu,∥ and Shiyong Zhang*,†,‡ †
National Engineering Research Center for Biomaterials, Sichuan University, 29 Wangjiang Road, Chengdu, 610064, China College of Chemistry, Sichuan University, 29 Wangjiang Road, Chengdu 610064, China § Institute of Materials, China Academy of Engineering Physics, No. 9, Huafengxincun, Jiangyou 621908, China ∥ State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu 610041, China ‡
S Supporting Information *
ABSTRACT: The metal catalysts encapsulated in nanomaterials have recently been applied successfully in bioorthogonal chemistry for intracellular generation of bioactive compounds. However, the nanomaterial-involved intracellular catalysis is intrinsically different from that in solution or in extracellular fluid. Except for the reactivity of metal catalyst itself, the supporting material’s morphology and biocompatibility are essential factors for building optimal nanocatalysts. Herein, we present a new nanocopper-doped cross-linked lipoic acid nanoparticle (Cu@cLANP) that meets the stringent requirements for the intracellular nanocatalyst. It comes from endogenous lipoic acid and can easily achieve the morphology change by different reduction methods. The optimal rugbylike Cu@cLANPs I did show much better catalytic efficiency for intracellular azide−alkyne cycloaddition than that of the other two spherical nanocatalysts Cu@cLANPs II and III, hinting at the importance of nanoparticle morphology for the intracellular bioorthogonal transformation. KEYWORDS: nanocatalyst, intracellular catalysis, lipoic acid, morphology, biocompatibility
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achieving the optimal catalyst formula. Yet, as far as we know, there is no report regarding the effect of nanoparticle morphology on intracellular catalysis. Another important consideration is the biocompatibility of supporting materials. Although the reported nanomaterials are claimed to be biologically benign under their work conditions, they are after all exogenous matters, and would no doubt have hazards to some extent. Taking the popular supporting material polystyrene as an example, it was listed as a Class 3 carcinogen by the World Health Organization on Oct. 27, 2017.15 Herein, we present a new nanocopper-doped cross-linked lipoic acid nanoparticle (Cu@cLANP; see Scheme 1), which meets the stringent requirements mentioned above for the nanocatalyst of intracellular catalysis. On one hand, the supporting material consists of endogenous lipoic acid, a coenzyme existing in the mitochondria;16 as a result, it has extremely good biocompatibility. On the other hand, the copper-doped cross-linked lipoic acid nanoparticles, by virtue of a large number of interlaced disulfide groups, could reassemble under different reduction conditions and nanocatalysts with different morphologies could be constructed. The experimental
enefitting from high catalytic efficiency, good water solubility, reduced toxicity, and structural similarity with metalloenzymes, the metal catalysts encapsulated in various nanomaterials, which have been widely applied over decades in synthetic chemistry,1−4 has recently been successfully expanded in bioorthogonal chemistry in intracellular generation of bioactive compounds for therapeutic and labeling applications. For example, Bradley and co-workers developed a series of nanopalladium catalysts supported by polystyrenes for intracellular drug synthesis;5−7 Rotello and colleagues reported the supramolecular regulation of bioorthogonal catalysis in cells with gold nanoparticle-embedded ruthenium and palladium catalysts for imaging and therapeutic applications;8 Zimmerman and co-workers synthesized a copper-containing metal-polyolefin nanoparticle for catalyzing cellular azide−alkyne cycloaddition at parts-per-million (ppm) catalyst levels;9 and Weissleder et al. developed poly(lactic-co-glycolic acid)-bpolyethylene glycol supported palladium catalysts for intracellular activation of prodrugs.10 However, the nanomaterial-involved intracellular catalysis is intrinsically different from that in solution or in extracellular fluid.11−14 Except for the reactivity of catalyst itself, the endocytosis of nanoparticles would be taken into account for efficient transformations. This requires that the nanocatalyst would be better prepared in morphology controlled for © XXXX American Chemical Society
Received: April 5, 2018 Revised: May 21, 2018 Published: May 24, 2018 5941
DOI: 10.1021/acscatal.8b01337 ACS Catal. 2018, 8, 5941−5946
Letter
ACS Catalysis Scheme 1. Schematic Illustration of the Preparation of Nanocatalysts Cu@cLANPs I−III
results showed that the resulting three morphological nanocatalysts Cu@cLANPs (I−III) demonstrated similar ppm catalytic efficiency in solution for azide−alkyne cycloaddition, but gave distinct catalytic activity in creating the same compounds in living cells, hinting at the importance of nanoparticle morphology for the intracellular bioorthogonal transformation. The supporting material, namely cross-linked lipoic acid nanoparticles (cLANPs), was prepared simply by dissolving lipoic acid in water above the critical aggregation concentration (CAC) of 17.3 μg/mL (Figure S1 in the Supporting Information), followed by reduction with 10 mol % dithiothreitol (DTT) and dialysis against deionized water (see the Supporting Information for details).17 The successful synthesis of the cLANPs was directly proven by the broadened proton signals in 1H NMR spectra (Figure S2 in the Supporting Information), the great increase in absolute weight-average molecular weight determined by static light scattering (SLS) measurement [molecular weight of Mw = (3.79 ± 0.13) × 104 g mol−1 (Figure S3 in the Supporting Information)], and the enhanced stability both in water and in 10% fetal bovine serum (Figure S4 in the Supporting Information). The transmission electron microscopy (TEM) image disclosed the formation of spherical nanoparticles with an average size of ∼93 nm by dynamic light scattering (DLS) measurement (Figure 1a). The resulting aqueous solution of cLANPs was treated with 1.0 equiv NaBH4 and 20 mol % CuCl2·2H2O (relative to LA) to generate the nanocopper-doped cross-linked lipoic acid nanoparticle I (Cu@cLANP I), which gave the similar hydrophobic size with cLANPs, but greatly changed the morphology from a spherical structure to a rugbylike structure with an aspect ratio of ∼2.8:1, as determined by TEM measurement (Figure 1b). The high-resolution TEM (HRTEM) confirmed the formation of nanocopper(0) with the clear lattice stripe of the particles (Figure S5 in the Supporting Information). We rationalize that the rugbylike Cu@cLANP I would be attributed to partial cleaved disulfide bonds by NaBH4. The resulting thiol intermediates, which were detected by using Ellmann’s reagent (Figure S6 in the Supporting Information),18 recombined and/ or anchored to the newly formed nanocopper(0) to achieve the establishment of the observed rugbylike structure (Scheme 1). Notably, despite the absence of metal particles, the cLANPs could still be partially reduced by NaBH4, the morphology of the obtained nanoparticles was basically the same as that of the original cLANPs, indicating the synergistic effect of metal
Figure 1. TEM and DLS characterization of the supporting material cLANPs and nanocatalysts Cu@cLANPs I−III: (a) cLANPs, (b) Cu@ cLANP I, (c) Cu@cLANP II, and (d) Cu@cLANP III.
nanoparticles in the morphological change of cLANPs. More intriguingly, the morphology of Cu@cLANP I can be further changed by different reducing agent doses or reducing methods. As exhibited in Scheme 1, under the condition of 10 equiv NaBH4, the spherical Cu@cLANP II, with a size of only ∼11 nm, was obtained (Figure 1c); when exposed under a hand-held ultraviolet (UV) lamp (365 nm, 8 W), the rugbylike Cu@cLANP I changed to spherical Cu@cLANP III with a size of ∼47 nm (Figure 1d). The nanocatalysts Cu@cLANPs I−III were further characterized via energy dispersive spectroscopy (EDS) analyses, which indicated that the copper was successfully encapsulated (Figure 2a). Moreover, X-ray photoelectron spectroscopy (XPS) analysis at the Cu 2p region showed two distinct peaks at 932.6 eV (Cu 2p3/2) and 952.6 eV (Cu 2p1/2), implying the successfully fabrication of copper(0) nanoparticles (Figure 2b).19 Note that the Cu content of all above three nanocatalyts were determined via inductively coupled plasma− atomic emission spectroscopy (ICP-AES), and the leaching 5942
DOI: 10.1021/acscatal.8b01337 ACS Catal. 2018, 8, 5941−5946
Letter
ACS Catalysis
S1, entries 7−9). The ppm catalyst level is comparable to the lowest catalyst dosage reported so far for “click” reaction in pure water.9,20,21 To further compare the catalytic efficiency of the three morphological nanocatalysts, the substrate 3-azido-7coumarin was employed to “click” with phenyl to generate the well-known fluorogenic coumarin derivative 1 with high fluorescence (see Figure 3a, as well as Figure S8 in the Supporting Information).22 By monitoring the real-time fluorescence change of the system, the reaction process can be easily tracked. Figure 3b shows the emission intensity at 460 nm (I460) of the reaction system, as a function of reaction time, which disclosed that the three nanocatalysts Cu@cLANPs I− III have almost uniform conversion efficiency at each given time point, suggesting that the morphology has little effect on the solution reaction rate. This result is reasonable in consideration of the homology of the copper nanoparticles buried in the three nanocatalysts. Moreover, the three nanocatalysts Cu@cLANPs I−III showed similar reaction activity with a wide scope of substrates (6 h; see Table S2 in the Supporting Information) and gave the corresponding 1,2,3triazoles with final yields ranging from 83% to 97% (24 h, Table S2). After confirmation of the solution reactivity of the three nanocatalysts, their potential for intracellular catalysis was tested in living cells using confocal microscopy. Briefly, human cervical cancer (HeLa) cells were preincubated with Cu@ cLANPs I−III ([Cu] = 15 ppm in Dulbecco’s modified Eagle medium, DMEM) for 2 h. The DMEM was removed and the cells were washed three times to eliminate extracellular Cu@
Figure 2. Physicochemical properties of the nanocatalysts: (a) EDS spectra of Cu@cLANPs I−III and (b) XPS spectrum of Cu@cLANP I.
levels were also estimated and shown to be
