Research ArticleAPPLIED SCIENCES AND ENGINEERING

Directly observing intracellular nanoparticle formation with nanocomputed tomography

See allHide authors and affiliations

Science Advances  23 Oct 2020:
Vol. 6, no. 43, eaba3190
DOI: 10.1126/sciadv.aba3190

Abstract

Directly observing intracellular nanostructure formation remains challenging. In this work, using a rationally designed small-molecule 4-nitrobenzyl carbamate–Cys(SEt)-Asp-Asp-Phe(iodine)–2-cyano-benzothiazole (NBC-Iod-CBT), we directly observed intracellular nanoparticle formation with nanocomputed tomography (nano-CT). In vitro, upon glutathione reduction and nitroreductase (NTR) cleavage, NBC-Iod-CBT undergoes a CBT-Cys click condensation reaction to self-assemble nanoparticles Iod-CBT-NPs with an average linear absorption coefficient (LAC) value of 0.182 ± 0.078 μm−1 to x-ray. Nano-CT imaging of the NBC-Iod-CBT–treated, NTR-overexpressing HeLa cells showed the existence of Iod-CBT-NPs in their cytoplasm with an average LAC value of 0.172 ± 0.032 μm−1. We anticipate that our strategy could help people to deeply understand the formation mechanism of intracellular nanostructures in the near future.

INTRODUCTION

Using small molecular precursors to assemble nanostructures inside cells is an intelligent strategy, which has shown great advantages in molecular imaging and drug delivery (1). As we know, small molecules are easily taken up by cells but clear fast. Different from small molecule, a tens of thousands of therapeutic (or imaging) agent-containing nanostructure has longer retention time in cells, thus affording much more potent (or higher) effect (25). However, it is much more difficult for a nanostructure to be taken up by cells than a small molecule. Thus, normally, scientists endow a nanostructure with active targeting ability by modifying its surface with targeting warheads (68). However, this after-modification of a nanostructure notably lowers down the reproducibility of the nanocomplex. Recently, an emerging “smart” strategy of intracellular nanoparticle formation (i.e., incubating cells with a small molecular precursor but ending up with a nanostructure inside cells) well adopts above complementary advantages of small molecule and nanostructure and has shown exciting application potentials in molecular imaging and drug delivery (911). Because there are numerous nanostructures inside a cell, up to date, how to differentiate above artificially formed nanostructures from the intrinsic cellular ones remains challenging.

Normally, by conjugating the small molecular precursor with a fluorescence tag and using a confocal fluorescence microscope, scientists are able to characterize the intracellularly formed nanostructures (e.g., nanoparticle or nanofiber) with concentrated or aligned fluorescence signals (1214). For example, by conjugating the hydrogelator precursor with environmentally sensitive fluorophore 4-nitro-2,1,3-benzoxadiazole, Xu et al. (12, 13) used a confocal microscope to characterize the intracellularly lined up fluorescence signals as the enzyme-instructed self-assembled nanofibers. However, because of its resolution limitation (>200 nm), an optical microscope cannot display the real morphology of a nanostructure in a diameter of several nanometers or several tens of nanometers. Super-resolution imaging technology breaks through the resolution limit of optical microscopes and has a spatial resolution of tens of nanometers. For examples, it was applied to unambiguously image intracellularly self-assembled nanofibers (15) or nanoparticles (16). Recently, super-resolution imaging of fluorescent nanoparticles has been applied to resolve subcellular structures and track intracellular protein dynamics. However, this approach is limited in imaging the distribution of a few fluorescence-labeled targets and cannot reveal the full ultrastructural features or real morphology of subcellular structures in cells. Because the target proteins require high-density fluorescent labeling, label-induced perturbation by this method is inevitable (1718). Electron microscopy (EM) is a suitable and powerful tool to characterize the real morphology of nanostructure inside a cell. However, limited by its penetration thickness, EM can only image the nanostructures within an ultrathin cell slice (~80 nm) in two dimensions (2D). Cryo-EM is suitable for resolving the complicated 3D structure of a biomacromolecule, but it is unable to penetrate a sample with thicknesses more than 1 μm (19, 20). Therefore, although EM techniques can spatially display the real morphology of a nanostructure inside a cell slice, it cannot temporally image the nanostructure in a whole cell in real time. Nanocomputed tomography (nano-CT) is an ideal technique for observing the 3D nanostructures in a whole cell (2125). Water window technology of nano-CT allows an unstained, ~10-μm-thick, frozen-hydrated cell to be 3D imaged in its near native state with unique high contrast and resolution (22, 23). Because the cryopreserved cell does not need to be sliced, spatial distribution of the cellular organelles, as well as the artificially formed nanostructures (size, >40 nm in this work), can be directly observed (24). However, to the best of our knowledge, using nano-CT to directly observe intracellularly formed nanoparticles has not been reported.

Inspired by the above studies, here, we intended to design an iodine (Iod)–containing small molecular precursor, which subjects to intracellular enzyme–instructed self-assembly to form nanoparticles, and use nano-CT to directly observe the intracellularly formed nanoparticles with high contrast. To achieve this, as shown in Fig. 1, an iodinated probe 4-nitrobenzyl carbamate–Cys(SEt)-Asp-Asp-Phe(iodine)–2-cyano-benzothiazole (NBC-Iod-CBT) was rationally designed to contain four parts: (i) a 4-nitrobenzyl carbamate (NBC) substrate for nitroreductase (NTR) cleavage, (ii) a latent cysteine (Cys) motif and 2-cyano-benzothiazole (CBT) structure for CBT-Cys click condensation reaction, (iii) an iodinated phenylalanine (Phe) structure for CT contrast enhancement, and (iv) two hydrophilic aspartic acid (Asp) motifs bearing carboxyl groups to endow the probe with good water solubility under physiological conditions (2628). As illustrated in Fig. 1, after NBC-Iod-CBT entering NTR-overexpressing hypoxic cancer cells (2930), its disulfide bond is reduced by intracellular glutathione (GSH), and its NBC substrate is cleaved by NTR whereafter to yield the reactive intermediate Cys-Iod-CBT. Instantly, the CBT-Cys click condensation reaction occurs between two Cys-Iod-CBTs to yield the amphiphilic cyclic dimer Iod-CBT-Dimer, which self-assembles into its nanoparticles Iod-CBT-NPs. Thus, the iodine-rich, intracellularly formed nanoparticles can be directly imaged by nano-CT with high contrast.

Fig. 1 Schematic illustration of NTR-triggered self-assembly of NBC-Iod-CBT into Iod-CBT-NPs inside a cell.

Under glutathione (GSH) reduction and nitroreductase (NTR) cleavage, small-molecule 4-nitrobenzyl carbamate–Cys(SEt)-Asp-Asp-Phe(iodine)–2-cyano-benzothiazole (NBC-Iod-CBT) undergoes an intracellular CBT-Cys click condensation reaction and self-assembles into iodinated nanoparticles (i.e., Iod-CBT-NPs).

RESULTS

NTR-triggered Iod-CBT-NP formation in vitro

We began the study with the validation of NTR-triggered nanoparticle formation in vitro. After synthesis and characterization of NBC-Iod-CBT (schemes S1 and S2 and figs. S1 to S6), we incubated 500 μM NBC-Iod-CBT with 2 mM tris(2-carboxyethyl)phosphine (TCEP) in phosphate-buffered saline (PBS; 10 mM, pH 7.4) at 37°C for 1 hour, followed by further incubation with 5 mM nicotinamide adenine dinucleotide (NADH) and NTR (5 U/ml) for 2 hours. After incubation, visible absorbance at 500 to 700 nm of the mixture notably increased (fig. S7), indicating the formation of nanostructures in it. When dicoumarin (an NTR inhibitor) was added to the solutions simultaneously, visible absorbances at 500 to 700 nm of the mixtures decreased again with dicoumarin concentration (fig. S8). These results confirmed that formation of nanostructures in the above mixture was induced by NTR. Transmission EM (TEM) image showed the appearance of nanopaticles (i.e., Iod-CBT-NPs) with an average diameter of 470.6 ± 10.6 nm in the mixture (Fig. 2A). High-performance liquid chromatography (HPLC) traces showed that, after incubation, with the disappearance of NBC-Iod-CBT peak at a retention time of 24.5 min, a new peak at 18.9 min appeared (Fig. 2B). High-resolution matrix-assisted laser desorption (MALDI)/ionization mass spectrometry (MS) analysis indicated that the new peak was Iod-CBT-Dimer for Iod-CBT-NP formation (fig. S9). After that, we obtained the 3D nano-CT images of the mixture with a soft x-ray microscopy nano-CT. First, a series of 2D projection images of the above mixture were acquired, and Iod-CBT-NPs could be seen in a representative 2D projection (Fig. 2C). Tomographic reconstruction of the 2D projections resulted in 3D nano-CT images (fig. S10). Then, the reconstruction volumes in the 3D images were segmented on the basis of their gray values and morphological appearance, resulting in a 3D rendering image of the Iod-CBT-NPs (Fig. 2D and movie S1). As we know, different substances have different x-ray absorption capacities, characterized by their different linear absorption coefficient (LAC) values (31). From their LAC histogram (fig. S11), the average LAC value of the Iod-CBT-NPs in the above 3D rendering image was calculated to be 0.182 ± 0.078 μm−1 by Gaussian fitting (32, 33). In contrast, the average LAC value of the background of above 3D rendering image (i.e., buffer salts) was much lower (0.016 ± 2.043 × 10−4 μm−1) (fig. S12).

Fig. 2 In vitro characterizations of Iod-CBT-NPs.

(A) TEM image of Iod-CBT-NPs. (B) HPLC traces of 500 μM NBC-Iod-CBT (black), 500 μM NBC-Iod-CBT incubated with TCEP (2 mM) for 1 hour, and a further incubation with NADH (5 mM) and NTR (5 U/ml) for 2 hours in 10 mM PBS at 37°C (red). Wavelength for detection: 320 nm. (C) 2D projection image of Iod-CBT-NPs. (D) 3D rendering image of Iod-CBT-NPs (yellow). LAC, linear absorption coefficient.

Intracellular formation of Iod-CBT-NPs

Before applying NBC-Iod-CBT for NTR-triggered self-assembly of Iod-CBT-NPs inside cells, we tested its selectivity toward NTR among the intracellular biothiols, reductants, oxidants, and amino acids. The results indicated that NBC-Iod-CBT has good selectivity toward NTR among these possible intracellular interferences (fig. S13). Human cervical HeLa cancer cells are known to overexpress NTR under hypoxic conditions. Our results indicated that NTR expression reached the highest level at 8 hours in HeLa cells cultured at 37°C under hypoxic conditions (1% O2) (table S1). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay results showed that, at a compound concentration of up to 500 μM, NBC-Iod-CBT did not exert observable cytotoxicity on HeLa cells for 8 hours (fig. S14). After incubating hypoxic HeLa cells with 250 μM NBC-Iod-CBT at 37°C for 4 hours and the cells were lysed, we also found Iod-CBT-Dimer as the main product in the cell lysate, suggesting that Iod-CBT-NPs could form in hypoxic HeLa cells (figs. S15 and S16). NTR is known existing in the cytoplasm of a mammalian cell (34, 35). EM image of the NBC-Iod-CBT–treated hypoxic HeLa cells showed the existence of Iod-CBT-NPs with an average diameter of 53.3 ± 2.8 nm in the cytoplasm of the cells (Fig. 3 and fig. S17). However, Iod-CBT-NPs were not found in the four control groups of cells (fig. S18).

Fig. 3 TEM image of NBC-Iod-CBT–treated hypoxia HeLa cell.

(A) Low-magnification TEM image of hypoxia HeLa cell incubated with 250 μM NBC-Iod-CBT for 4 hours. (B) High-magnification TEM image of the red square area in (A).

Directly observed Iod-CBT-NPs with soft x-ray microscopy nano-CT imaging

To directly observe Iod-CBT-NP formation inside cells, we froze the hypoxia, NBC-Iod-CBT–treated HeLa cells and imaged them with a soft x-ray microscopy nano-CT. Hypoxia HeLa cells pretreated with dicoumarin, or normoxia (i.e., 20% O2) HeLa cells, both incubated with the compound, were designated as two positive controls. The untreated hypoxia or normoxia HeLa cells were assigned as two negative controls. Figure 4A showed the representative 2D projection images of three groups of NBC-Iod-CBT–treated HeLa cells, each of which shows the morphology of a single HeLa cell. Quantitative analysis indicated that, while the cytoplasm of the untreated HeLa cells has CT contrasts around 30% (fig. S19, A and B), NBC-Iod-CBT–treated hypoxia HeLa cells have the highest cytoplasm CT contrast of 64.2% among all five groups (Fig. 4B), suggesting that Iod-CBT-NPs formed in the cytoplasm of hypoxia HeLa cells. Upon NTR inhibitor treatment, cytoplasm CT contrast of the hypoxia cells decreased to 52.1%, indicating that the above CT contrast enhancement was induced by NTR (Fig. 4B). Normoxia HeLa cells, whose NTR expression was validated low (table S2), consistently show the lowest cytoplasm CT contrast of 33.8% among the three compound-treated groups (Fig. 4B). We then conducted tomographic reconstruction of the 2D projections to obtain the 3D nano-CT images (fig. S20). As we know, different cellular structures have their own morphologies with different gray values. On this basis, the reconstruction volumes in the 3D images of five groups of HeLa cells were segmented to yield their respective 3D rendering images (Fig. 4C and movies S2 to S4, fig. S19D, and movies S5 and S6, respectively). We observed the existence of nanoparticles in the experimental group cells but not in other four control groups (Fig. 4, C and D, and fig. S19D). By Gaussian fitting of the LAC histogram of the segmented nanoparticles in the whole experimental group cell (i.e., all the yellow structures in the left image of Fig. 4C), their average LAC value was calculated to be 0.172 ± 0.032 μm−1, which is very close to that of Iod-CBT-NPs formed in vitro (Fig. 2D), suggesting that the nanoparticles in hypoxia HeLa cell cytoplasm are Iod-CBT-NPs. In addition, calculated average LAC values of the cytoplasm of experimental cells and the cytoplasm of four control groups of cells, which are obviously smaller than 0.172 ± 0.032 μm−1 (figs. S21 and S22), echoed the above conclusion. To validate the feasibility of our method, we also used one type of polymer iodine nanoparticles poly(methacrylated iopamidol) (PMAI) provided by the Yang’s group (36). TEM image showed PMAI with an average diameter of 129.4 ± 9.9 nm (fig. S23, A and B). We first obtained the 3D nano-CT image of PMAI in vitro (fig. S23, C and D, and movie S7). The average LAC value of PMAI in vitro was calculated to be 0.210 ± 0.036 μm−1 (fig. S24). After that, PMAI-treated HeLa cell was imaged with soft x-ray microscopy nano-CT. Quantitative analysis indicated that the representative 2D projection image of PMAI-treated HeLa cell has a CT contrast of 68.1%, preliminary indicating the existence of PMAI in HeLa cell (fig. S25, A and B). By tomographic reconstruction and segmentation, we also obtained 3D rendering image of PMAI-treated HeLa cell (fig. S25, C and D, and movie S8). As shown in fig. S25E, the average LAC value of PMAI nanoparticles in cell was calculated to be 0.213 ± 0.065 μm−1, which is very close to that of the nanoparticle in vitro (fig. S24), and these two LAC values are significantly different from those of our Iod-CBT-NPs in vitro and in cell, respectively, suggesting that our method of using LAC to confirm intracellular nanoparticle formation is feasible. Consistently, the average LAC value of 0.051 ± 3.814 × 10−4 μm−1 of the cytoplasm of PMAI-treated cell (fig. S26) was much lower than that of the nanoparticle.

Fig. 4 Directly observed Iod-CBT-NPs with soft x-ray microscopy nano-CT imaging.

(A) The 2D projection images of hypoxic HeLa cells treated with 250 μM NBC-Iod-CBT for 4 hours (left), hypoxic HeLa cells treated with 500 μM dicoumarin (an NTR inhibitor) and then treated with 250 μM NBC-Iod-CBT for 4 hours (middle), and normal HeLa cells treated with 250 μM NBC-Iod-CBT for 4 hours (right). (B) Corresponding absolute soft x-ray absorptions for the red lines in (A) part. (C) Corresponding 3D segmented HeLa cells in (A). In the segmented regions, the yellow structures are Iod-CBT-NPs, the green structures are cytoplasm, and the blue structures are nucleus. (D) Magnified view of the red rectangle area in (C) part. (E) LAC histogram of whole intracellular Iod-CBT-NPs [the yellow structures in the left image of (C)] and its corresponding Gaussian fitting curve (black).

DISCUSSION

In summary, by rational design of an iodinated small-molecule NBC-Iod-CBT, we are able to directly observe nanoparticle formation inside cells with nano-CT. Upon GSH reduction and NTR cleavage in vitro, NBC-Iod-CBT subjects to a CBT-Cys click condensation reaction to self-assemble nanoparticles Iod-CBT-NPs with an average LAC value of 0.182 ± 0.078 μm−1 to x-ray. Under hypoxic conditions, NTR was found highly expressed in the cytoplasm of HeLa cells. HPLC analysis and TEM imaging showed Iod-CBT-NP formation in NBC-Iod-CBT–treated hypoxia HeLa cells. Nano-CT imaging of the cells showed the existence of Iod-CBT-NPs in the cytoplasm with an average LAC value of 0.172 ± 0.032 μm−1. Using one type of polymer iodine nanoparticles PMAI as a control, we verified that our method of using LAC to confirm intracellular nanoparticle formation is feasible. We anticipate that our strategy could help people to deeply understand the formation mechanism of intracellular nanostructures, either artificially assembled or naturally formed, in the near future.

MATERIALS AND METHODS

Experimental materials and instruments

All the starting materials were purchased from Aladdin or Sangon Biotech. Commercial reagents were directly used, unless otherwise noted. All chemicals are in reagent grade or better. NTR was purchased from Sigma-Aldrich [1 U is defined as the enzyme activity that cleaves 1 μmol of the standard substrate per minute in the presence of menadione and NADH at 37°C]. A 300-MHz Bruker AV 300 spectrometer was used to obtain the 1H nuclear magnetic resonance (NMR) and 13C NMR spectra. Electrospray ionization–MS spectra were obtained on an LCQ Advantage MAX ion trap mass spectrometer (Thermo Fisher Scientific). MALDI ionization–time-of-flight (TOF)/TOF mass spectra were obtained on a TOF Ultraflex II mass spectrometer (Bruker Daltonics). An Agilent 1200 HPLC system, which was equipped with a G1322A pump, an in-line diode array ultraviolet detector, and an Agilent Zorbax 300SB-C18 RP column, was used for HPLC analyses. TEM observations were conducted on a JEM-2100F electron microscope with a working acceleration voltage of 100 kV. Normal HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; HyClone) containing 10% fetal bovine serum at 37°C, 5% CO2, and humid atmosphere. Hypoxic HeLa cells were cultured in DMEM (HyClone) with 10% fetal bovine serum at 37°C, 1% O2, and 5% CO2.

Selectivity studies

NBC-Iod-CBT (500 μM) was incubated with 2 mM TCEP for 1 hour and further incubated with 5 mM NADH in the presence of NTR (5 U/ml), 1 mM biothiols [Cys, homocysteine, and dithiothreitol], 1 mM arginine (Arg), 1 mM glutamic acid (Glu), 1 mM serine (Ser), 1 mM vitamin C, 1 mM vitamin E, 100 μM reactive oxygen species (H2O2), or 1 mM glucose, respectively. Compared with absorbance value induced by other selected species, NTR (5 U/ml) notably showed absorbance enhancement after the addition to the mixture (about 10-fold of those induced by the other tested species).

NTR activity assay

HeLa cells growing in log phase were incubated under normoxic conditions overnight at 37°C and then incubated under hypoxic or normoxic conditions for 4, 8, or 12 hours, respectively. After washing three times with PBS buffer, the cells were disrupted in lysis buffer [50 mM tris and 150 mM NaCl (pH 8.0)] on ice. The NTR activity of HeLa cells was calculated by measuring increased optical density values at 550 nm within 1 min using a commercially available kit.

MTT assay

The cytotoxicity of NBC-Iod-CBT on HeLa cells was measured by MTT assay. HeLa cells growing in log phase were seeded into 96-well cell culture plate at 3 × 103 per well and then incubated at 37°C for 12 hours. Then, the cells were incubated with NBC-Iod-CBT at concentrations of 62.5, 125, 250, or 500 μM, respectively, for 2, 4, or 8 hours, respectively. MTT dissolved in PBS buffer (pH 7.4) was added to each well of the 96-well plate (5 mg/ml, 10 μl). After 4-hour incubation, 100 μl of dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan. An enzyme-linked immunosorbent assay reader (Varioskan Flash) was used to detect the absorption at 490 nm of each well. Cell viability was calculated using following formula: viability (%) = A/A0 × 100 (A, mean of absorbance value of treatment group; A0, mean of absorbance value of control).

HPLC analysis of NBC-Iod-CBT–treated hypoxic HeLa cells

HeLa cells growing in log phase were incubated under hypoxic conditions (1% O2) for 8 hours. After incubation with 250 μM NBC-Iod-CBT at 37°C for 4 hours, the cells were washed three times with PBS buffer and then lysed in DMSO for HPLC analysis. HPLC trace showed a main peak of Iod-CBT-Dimer at a retention time of 19.0 min.

HeLa cell preparation for nano-CT imaging

HeLa cells growing in log phase were seeded into 12-well cell culture plate, with each well being preplaced with a 100-mesh nickel grid. The cells were incubated under normoxic conditions overnight at 37°C and then incubated under hypoxic or normoxic conditions for 8 hours, respectively. After treating hypoxic HeLa cells with 250 μM NBC-Iod-CBT for 4 hours (left), hypoxic HeLa cells with 500 μM dicoumarin (NTR inhibitor) and then with 250 μM NBC-Iod-CBT for 4 hours, and normal HeLa cells with 250 μM NBC-Iod-CBT for 4 hours, all the cells were washed three times with PBS buffer and fixed with 4% paraformaldehyde. Grids with cells were mounted in the homemade freezer plunge and rapidly frozen in liquid nitrogen in the movable cryopreserving container. The rapid plunge procedure was adopted to avoid ice crystallization contamination and protect the cells from radiation damage. Then, the grids with cells were transferred into the vacuum cryogenic chamber by the side-entry holder for soft x-ray imaging.

Nano-CT imaging

The 3D imaging experiments were performed using a transmission soft x-ray microscope at the beamline BL07W of the National Synchrotron Radiation Laboratory, Hefei, China. The x-ray beam is focused on the sample by an elliptical capillary condenser. Then, the objective zone plate generates a magnified image of the sample on a 16-bit 1024 × 1024 charge-coupled device camera with a 16-μm field of view and a spatial resolution of 40 nm. Initially, a tilt series consisted of 121 images taken at 1° intervals were collected at 520-eV x-ray energy. Each projection was collected with an exposure time of 2 s. The 3D reconstruction volumes were obtained by the simultaneous algebraic reconstruction technique–the total variation (SART-TV) method and then were segmented by Amira software (FEI Visualization Science Group, France).

SUPPLEMENTARY MATERIALS

Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/43/eaba3190/DC1

https://creativecommons.org/licenses/by-nc/4.0/

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

REFERENCES AND NOTES

Acknowledgments: We thank X. Yang from the Tianjing Medical University for providing polymer iodine nanoparticles PMAI. Funding: This work was supported by the National Natural Science Foundation of China (grants 21725505, 81821001, and 21675145) and the Ministry of Science and Technology of China (2016YFA0400904). Author contributions: M.Z. conducted the synthesis and characterization of NBC-Iod-CBT and cell experiments and helped with nano-CT imaging. Y.G. conducted nano-CT imaging and data processing. Z.D. helped with nano-CT image reconstruction and segmentation. P.Z. assisted in cell sample preparation. Z.Z. helped with project design. L.C. operated the nano-CT instrument. W.K. and C.W. helped with TEM imaging. G.L. supervised the whole project and wrote the paper. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

Stay Connected to Science Advances

Navigate This Article