Nanocomputed Tomography Imaging of Bacterial Alkaline

Dec 7, 2016 - Alkaline phosphatase (ALP) is an important enzyme, but direct imaging of ALP activity with high spatiotemporal resolution remains challe...
0 downloads 0 Views 2MB Size
Download PDF
Letter pubs.acs.org/ac

Nanocomputed Tomography Imaging of Bacterial Alkaline Phosphatase Activity with an Iodinated Hydrogelator Zhen Zheng,†,¶ Anming Tang,†,‡,¶ Yong Guan,⊥ Liang Chen,⊥ Fuqiang Wang,∥ Peiyao Chen,† Weijuan Wang,†,‡ Yufeng Luo,† Yangchao Tian,⊥ and Gaolin Liang*,† †

CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, China ‡ Institute of Nuclear Physics and Chemistry, Chinese Academy of Engineering Physics, 64 Mianshan Road, Mianyang, Sichuan 621900, China ⊥ National Synchrotron Radiation Laboratory, University of Science and Technology of China, 42 Hezuohua South Road, Hefei, Anhui 230029, China ∥ Analysis Center, Nanjing Medical University, Nanjing, Jiangsu 210093, China S Supporting Information *

ABSTRACT: Alkaline phosphatase (ALP) is an important enzyme, but direct imaging of ALP activity with high spatiotemporal resolution remains challenging. In this work, we rationally designed an iodinated hydrogelator precursor Nap-Phe-Phe(I)-Tyr(H2PO3)-OH (1P) which self-assembles into nanofibers to form hydrogel under the catalysis of ALP. With this property of concentrating iodine atoms at the locations of ALP, 1P was successfully applied for direct nanocomputed tomography (nano-CT) imaging of ALP activity in bacteria for the first time. We envision that, on the basis of this pioneering work, new hydrogelators containing more iodine atoms (e.g., five iodine atoms in 1P) will be designed for better nano-CT imaging of ALP activity with higher CT contrast in the near future.

A

from their growth chamber and cryo-immobilized; thus, they are imaged much closer to their native state.20 Due to its efficiency in establishing three-dimensional images at nanometer resolution, soft X-ray CT (or called nano-CT) has become a useful tool for the direct imaging of the internal structures in bacteria, fungi, or mamallian cells.21 However, using nano-CT to image ALP activity in cells has not been explored. Inspired by the above-mentioned pioneering studies and considering the advantages of nano-CT, in this work, we rationally designed an iodinated hydrogelator precursor NapPhe-Phe(I)-Tyr(H2PO3)-OH (1P, Figure 1), which could be applied to directly image bacterial ALP activity with nano-CT. The rationales of our design are as follows: first, 1P was designed to contain an iodine atom which is the commonly used heavy atom to effectively absorb X-ray for CT contrast; second, in the absence of ALP, the water-soluble 1P is homogeneously distributed and will not induce CT contrast; last and importantly, upon the catalysis of ALP, the phosphate group on 1P will be removed to yield hydrogelator Nap-PhePhe(I)-Tyr-OH (1) which self-assembles into nanofibers and accumulates the iodine atoms at the ALP site to confer

lkaline phosphatase (ALP) is one of the phosphatases in the phosphatase/kinase pairs which is responsible for removing the phosphate groups from its substrate molecules.1,2 This process plays a key role in intracellular signal transmission and protein activity regulation, as well as cell growth and apoptosis.3,4 Dysregulation of ALP levels can induce significant development of inflammatory diseases or cancers.5 Therefore, direct imaging of ALP activity is of high importance for the early and quick diagnoses of ALP-related diseases.6 Traditional methods for imaging ALP activity are confined to radiation or fluorescence approaches.7−11 However, these approaches suffer from either a low spatial resolution or a poor stability of the probes.12−15 Recently, using ALP to trigger a supramolecular self-assembly of 19F nanofibers, Liang and co-workers developed a 19F nuclear magnetic resonance method for sensing ALP activity in vitro and in cells.16 Nevertheless, it remains challenging to directly image ALP activity with high spatiotemporal resolution. Soft X-ray computed tomography (CT) is one of the newly emerging, high resolution imaging modalities in which the image contrast is generated by the differential, quantitative absorption of X-ray photons.17,18 It is capable of quantitatively imaging the subcellular organelles in cells greater than 10 μmthick, contrary to the small sample thickness in cryo transmission electron microscopy (cryo-TEM) observations.19 Moreover, for soft X-ray CT iamging, cells are simply taken © XXXX American Chemical Society

Received: October 22, 2016 Accepted: December 7, 2016 Published: December 7, 2016 A

DOI: 10.1021/acs.analchem.6b04139 Anal. Chem. XXXX, XXX, XXX−XXX

Letter

Analytical Chemistry

mobility (Figure S9). Time-dependent conversion ratio curves for the dephosphorylation of 1P under different concentrations of ALP were plotted in Figure S10, and the calculated average dephosphorylation rates were summarized in Table S4. A stability study indicated that more than 95% of 1P remained undecomposed in the buffer at 37 °C for 24 h (Figure S11). To evaluate the viscoelastic properties of the as-formed hydrogel, we first used dynamic strain sweep to determine the proper condition for the dynamic frequency sweep of Gel I. As shown in Figure S12, the values of the storage modulus (G′) and the loss modulus (G″) of Gel I exhibit a weak dependence from 0.1% to 1.0% of strain (with G′ dominating G″), indicating that the sample is a hydrogel. After setting the strain amplitude at 1.0% (within the linear response regime of strain amplitude), we used dynamic frequency sweep to study Gel I. Figure 2A shows that G′ and G″ of Gel I slightly increase with the increase of frequency from 0.1 to 10 Hz. Moreover, the G′ values are significantly higher than those of G″, suggesting that Gel I is fairly tolerant to external force. To investigate the morphologies of the nanofibers in Gel I, we performed cryoTEM observations. The microscopic structure of Gel I under cryo-TEM exhibited sparse nanofibers with an average width of 3.46 ± 0.23 nm (Figure 2B). On the basis of cyro-TEM observations and the amphiphilic nature of hydrogelator 1, a possible molecular arrangement was proposed for the nanofibers in Gel I (Figure S13). In this model, the aromatic naphthalene moieties of 1 stack layer by layer with each neighboring Nap motif being positioned in the opposite orientation to form the hydrophobic inner layer. Meanwhile, the peptide segments of 1 stretch to the surrounding water to form the hydrophilic outer layer. From the molecular arrangement model,we proposed that the nanofiber in Gel I has a calculated width of 3.98 nm, which fits well with our cryoTEM observation (i.e., 3.46 nm). To verify the biocompatibility of the hydrogelator, we studied the cytotoxicity of 1P and 1 on HeLa cells (overexpressing ALP) using the 3-(4,5-dimethylthiazol-2yl)2,5 diphenyltetrazolium bromide (MTT) assay. As indicated by Figure S14, after being incubated with 1P (or 1) at 50, 100, 200, or 400 μM for 8 h, 94%, 91%, 85%, or 74% of the cells survived in 1P while 95%, 88%, 87%, or 81% of the cells survived in 1, respectively. These results suggested that neither 1P nor 1 is toxic to the cells in 8 h and 1P is safe for cell imaging. After the preliminary cytotoxicity test, we then verified whether the precursor 1P could be converted to the desired hydrogelator 1 by bacterial ALP as proposed. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE), we first confirmed the successful overexpression of ALP in E. coli (BL21) cells transfected with ALP plasmid (Figure S15). After incubation with 200 μM 1P at 37 °C for 4 h, the bacteria were harvested and lysed in dimethyl sulfoxide (DMSO) for HPLC analysis. The HPLC trace indicated that 81.6% of 1P was converted into 1 in the bacteria, suggesting a successful overexpression and high activity of ALP in the bacteria (Figure S16). When the E. coli cells were treated with an ALP inhibitor (phosphatase inhibitor complex II, Sangon, China), the dephosphorylation product of 1P (i.e., 1) decreased to 6.0% (Figure S16), which indicated that the above dephosphorylation of 1P was indeed triggered by the ALP in E. coli. After confirming that 1P could be effeciently converted to hydrogelator 1 in E. coli, we wondered whether the hydrogelator could in turn be used to image the enzyme activity with

Figure 1. Chemical structures of precursor 1P and corresponding hydrogelator 1 and schematic illustration of bacterial ALP-catalyzing 1P to yield 1 for the self-assembly of nanofibers inside and on E. coli.

enhanced CT contrast for ALP imaging (Figure 1). With this nano-CT method, one could visualize the internal distribution and activity of ALP at a single cell level and demonstrate the potential of 1P to serve as a new iodinated contrast agent with good ALP-targeting ability. The syntheses of 1P and 1 are simple and straightforward with solid phase peptide synthesis (SPPS), and the iodinated amino acid Phe(I) was used as the starting material for iodine modification. Their crude compounds were purified with reverse phase high performance liquid chromatography (RPHPLC). After 1P and 1 were fully characterized (Figures S1− S6), we examined their gelation ability. Plots of fluorescence emission intensity versus concentration revealed two regimes, indicating critical micelle concentration (CMC) of 13.4 μM for 1 (Figure S7). Thereafter, 2.0 mg of 1P was dissolved in 400 μL of phosphate buffer (PB, 0.2 M, pH 7.4) which resulted in a clear solution at 5.9 mM (inset of Figure 2A). After a 10 h

Figure 2. (A) Dynamic frequency sweep of the storage modulus (G′, black) and the loss modulus (G″, red) of Gel I. The rheological measurement was carried out at 37 °C and strain of 1.0%. Inset: 5.9 mM 1P solution in phosphate buffer (0.2 M, pH 7.4) (left vial) and corresponding Gel I obtained after a 10 h incubation of the solution with 200 U/mL ALP at 37 °C (right vial). (B) Cryo-TEM image of Gel I.

incubation with 200 unit/mL ALP at 37 °C, the solution changed to transparent Gel I (inset of Figure 2A). The efficient chemical transformation of 1P to 1 by ALP was validated by HPLC analyses (Figure S8). Under this condition, 73.7% of 1P was converted to 1 by ALP. A lower concentration of ALP (100, 50, or 25 U/mL) will also result in gelation but with longer gelation time, lower conversion ratio, and higher gel B

DOI: 10.1021/acs.analchem.6b04139 Anal. Chem. XXXX, XXX, XXX−XXX

Letter

Analytical Chemistry

81.6% of 1P was converted to 1 by bacterial ALP. However, when the cells were treated with an ALP-inhibitor, conversion of 1P to 1 decreased to 6.0%. These results indicated that our iodinated hydrogelator 1P could be subjected to efficient dephosphorylation by bacterial ALP. Nano-CT imaging showed that, while the blank cells had a CT contrast of 8.3%, the 1Ptreated E. coli cells had a high CT contrast of 18.3%. Moreover, dense objects (i.e., Gel I), which were not oberved in blank cells or inhibitor-treated cells, were clearly observed in and on 1P-treated E. coli cells. By this means, the bacterial ALP was directly imaged with our hydrogelator precursor 1P. Nevertheless, the E. coli cells used in this work were artificially overexpressed with ALP. To get a nano-CT image of ALP activity in natural bacteria with comparable contrast in this work, we need to design new hydrogelators containing more iodine atoms (e.g., five iodine atoms in 1P) and this work is underway.

a soft X-ray microscopy. As shown by the bottom panels in Figure 3, the soft X-ray image of the blank E. coli (without

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b04139. General methods; syntheses and characterizations of 1 and 1P; 1H and 13C NMR spectra and ESI-MS spectra of 1 and 1P; additional fluorescence spectra, HPLC traces, optical images of hydrogels, time-dependent conversion ratio curves, strain dependence plot, proposed molecular arrangement of 1, MTT assay on HeLa cells, SDS-PAGE distribution, and soft X-ray images; tables of HPLC conditions and conversion ratios for the dephosphorylation of 1P (PDF) Three-dimensional nano-CT images of ALP-overexpressing E. coli cells after being incubated with 200 μM 1P at 37 °C for 4 h; cells lyophilized prior to imaging (MPG) Three-dimensional nano-CT images of ALP-overexpressing E. coli cells after being incubated with ALP inhibitor (phosphatase inhibitor complex II, Sangon, China) and 200 μM 1P at 37 °C for 4 h; cells lyophilized prior to imaging (MPG) Three-dimensional nano-CT images of ALP-overexpressing E. coli cells; cells lyophilized prior to imaging (MPG)

Figure 3. Left column: representative soft X-ray images of E. coli cells treated with 1P (top), ALP-inhibitor together with 1P (middle), and blank E. coli cells without treatment (bottom). Middle column: Segmentation of the E. coli cells in the left column with different colors. Cyan: E. coli cells. Magenta: hydrogels. Segmentation detail was provided in the Supporting Information. Right column: Absolute soft X-ray absorptions for the images in the left column.

treatment of 1P) showed the cell to have a rhabditiform structure with a calculated X-ray absorption contrast value of 8.3%. From the soft X-ray image of the 1P-treated E. coli, clearly we observed dense objects in the cells and on the cell membrane (top left panel in Figure 3), which should be Gel I self-assembled from the hydrogelator 1.22 Additionally, the hydrogelator 1 should be yielded from the dephosphorylation of 1P by bacterial ALP. Compared with the blank cell, the 1Ptreated E. coli had a much higher calculated contrast value of 18.3% (top right panel in Figure 3). A soft X-ray image, showing a lot of 1P-treated E. coli cells in one field, was provided in Figure S17. For comparison, we also used one type of selenium quantum dot to treat the E. coli cells to provide positive control CT images of the cells. A comparable CT contrast value of 18.9% for the E. coli cell treated with selenium quantum dots suggested good contrast ability of our hydrogelator (Figure S18). To further validate that the above dense objects in and on 1P-treated E. coli were induced by bacterial ALP, together with 1P, we incubated the cells with the ALP inhibitor before imaging. The soft X-ray image of the cells showed the disappearance of the previously mentioned dense objects, and the contrast value of the cells decreased to 14.7% (middle row in Figure 3). The corresponding constructed three-dimensional nano-CT images of all these three groups of cells were provided as Videos S1, S2, and S3. All the above results indicated that the designed hydrogelator 1P could serve as an efficient contrast agent for direct nano-CT imaging of bacterial ALP activity. In summary, we rationally designed an iodinated hydrogelator precursor 1P, which could be applied for direct nanoCT imaging ALP activity in bacteria. Upon ALP catalysis in vitro, 1P was efficiently converted to hydrogelator 1 which selfassembled into the nanofibers in Gel I. Using ALP-overexpressing E. coli (BL21) cells incubated with 1P, we found that

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Gaolin Liang: 0000-0002-6159-9999 Author Contributions ¶

Z.Z. and A.T. contributed equally.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors are grateful to Prof. Jun Jiang and Maolin Xie for their assistance in the molecular arrangement model. This work was supported by Collaborative Innovation Center of Suzhou Nano Science and Technology, Hefei Science Center CAS (2016HSC-IU010), Ministry of Science and Technology of China (2016YFA0400904), and the National Natural Science Foundation of China (Grants U1532144 and 21675145). C

DOI: 10.1021/acs.analchem.6b04139 Anal. Chem. XXXX, XXX, XXX−XXX

Letter

Analytical Chemistry

■

REFERENCES

(1) Zhou, J.; Du, X.; Yamagata, N.; Xu, B. J. Am. Chem. Soc. 2016, 138, 3813. (2) Zheng, Z.; Chen, P.; Xie, M.; Wu, C.; Luo, Y.; Wang, W.; Jiang, J.; Liang, G. J. Am. Chem. Soc. 2016, 138, 11128. (3) Strater, N.; Lipscomb, W. N.; Klabunde, T.; Krebs, B. Angew. Chem., Int. Ed. Engl. 1996, 35, 2024. (4) Alonso, A.; Sasin, J.; Bottini, N.; Friedberg, I.; Friedberg, I.; Osterman, A.; Godzik, A.; Hunter, T.; Dixon, J.; Mustelin, T. Cell 2004, 117, 699. (5) de la Croix Ndong, J.; Makowski, A. J.; Uppuganti, S.; Vignaux, G.; Ono, K.; Perrien, D. S.; Joubert, S.; Baglio, S. R.; Granchi, D.; Stevenson, D. A.; Rios, J. J.; Nyman, J. S.; Elefteriou, F. Nat. Med. 2014, 20, 904. (6) Bialy, L.; Waldmann, H. Angew. Chem., Int. Ed. 2005, 44, 3814. (7) Kaneuchi, Y.; Hakozaki, M.; Yamada, H.; Hasegawa, O.; Tajino, T.; Konno, S. Hell. J. Nucl. Med. 2016, 19, 46. (8) Ren, C.; Wang, H.; Zhang, X.; Ding, D.; Wang, L.; Yang, Z. Chem. Commun. 2014, 50, 3473. (9) Dong, L.; Miao, Q.; Hai, Z.; Yuan, Y.; Liang, G. Anal. Chem. 2015, 87, 6475. (10) Gao, Y.; Shi, J.; Yuan, D.; Xu, B. Nat. Commun. 2012, 3, 1033. (11) Devaraj, N. K.; Hilderbrand, S.; Upadhyay, R.; Mazitschek, R.; Weissleder, R. Angew. Chem., Int. Ed. 2010, 49, 2869. (12) Li, G.; Fu, H.; Chen, X.; Gong, P.; Chen, G.; Xia, L.; Wang, H.; You, J.; Wu, Y. Anal. Chem. 2016, 88, 2720. (13) Han, A.; Wang, H.; Kwok, R.; Ji, S.; Li, J.; Kong, D.; Tang, B.; Liu, B.; Yang, Z.; Ding, D. Anal. Chem. 2016, 88, 3872. (14) Su, X.; Cheng, K.; Jeon, J.; Shen, B.; Venturin, G. T.; Hu, X.; Rao, J.; Chin, F. T.; Wu, H.; Cheng, Z. Mol. Pharmaceutics 2014, 11, 3947. (15) Fan, Q.; Cheng, K.; Hu, X.; Ma, X.; Zhang, R.; Yang, M.; Lu, X.; Xing, L.; Huang, W.; Gambhir, S. S.; Cheng, Z. J. Am. Chem. Soc. 2014, 136, 15185. (16) Zheng, Z.; Sun, H.; Hu, C.; Li, G.; Liu, X.; Chen, P.; Cui, Y.; Liu, J.; Wang, J.; Liang, G. Anal. Chem. 2016, 88, 3363. (17) Geisel, K.; Henzler, K.; Guttmann, P.; Richtering, W. Langmuir 2015, 31, 83. (18) Jensen, T. H.; Bech, M.; Bunk, O.; Thomsen, M.; Menzel, A.; Bouchet, A.; Le Duc, G.; Feidenhans’l, R.; Pfeiffer, F. Phys. Med. Biol. 2011, 56, 1717. (19) Parkinson, D. Y.; McDermott, G.; Etkin, L. D.; Le Gros, M. A.; Larabell, C. A. J. Struct. Biol. 2008, 162, 380. (20) Uchida, M.; McDermott, G.; Wetzler, M.; Le Gros, M. A.; Myllys, M.; Knoechel, C.; Barron, A. E.; Larabell, C. A. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 19375. (21) Schneider, G.; Guttmann, P.; Heim, S.; Rehbein, S.; Mueller, F.; Nagashima, K.; Heymann, J. B.; Muller, W. G.; McNally, J. G. Nat. Methods 2010, 7, 985. (22) Yang, Z.; Liang, G.; Guo, Z.; Guo, Z.; Xu, B. Angew. Chem., Int. Ed. 2007, 46, 8216.

D

DOI: 10.1021/acs.analchem.6b04139 Anal. Chem. XXXX, XXX, XXX−XXX