Quantitative Analysis of Caspase-1 Activity in Living Cells Through

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Quantitative Analysis of Caspase-1 Activity in Living Cells Through Dynamic Equilibrium of Chlorophyllbased Nano-Assemblies Modulated Photoacoustic Signals Li-Li Li, Qian Zeng, Wei-Jiao Liu, Xue-Feng Hu, Yongsheng Li, Jie Pan, Dong Wan, and Hao Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b05795 • Publication Date (Web): 24 Jun 2016 Downloaded from http://pubs.acs.org on June 28, 2016

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ACS Applied Materials & Interfaces

Quantitative Analysis of Caspase-1 Activity in Living Cells Through Dynamic Equilibrium of Chlorophyll-based Nano-Assemblies Modulated Photoacoustic Signals Li-Li Li ‡,†, Qian Zeng ‡,†, Wei-Jiao Liu †,§, Xue-Feng Hu †,£, Yongsheng Li £, Jie Pan §, Dong Wan , and Hao Wang †,*

§

†

CAS Center for Excellence in Nanoscience, CAS Key Laboratory for Biological Effects of

Nanomaterials and Nanosafety , National Center for Nanoscience and Technology (NCNST) Department Institution, No. 11 Beiyitiao, Zhongguancun, Beijing, China. E-mail: [email protected] §

State Key Laboratory of Hollow Fiber Membrane Materials and Processes, School of

Environmental and Chemical Engineering, Tianjin Polytechnic University £

Lab of Low-Dimensional Materials Chemistry, Key Laboratory for Ultraﬁne Materials of

Ministry of Education School of Materials Science and Engineering, East China University of Science and Technology

ACS Paragon Plus Environment

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KEYWORDS. Chlorophyll, Self-Assembly, Dynamic Equilibrium, Caspase-1, Bacterial Infection ABSTRACT. In situ construction of self-assemblies with unique property in living systems is a promising direction in the biomedical field. The noninvasive methods for significant enzyme activity in living cells or living subjects are imperative and meantime challenge tasks. The dynamic process of self-assembly of chlorophyll-based molecules in complex biological systems can be monitored by photoacoustic signals, which supports a noninvasive way to understand and quantitatively measure the activity of caspase-1. Furthermore, the activity of caspase-1 enables to be reflecting the bacterial infection in the early stage. Here, we present a biocompatible selfassembly from chlorophyll-peptide derivatives and first correlate the dynamic equilibrium with ratiometric photoacoustic signals. The intracellular equilibrium was managed by a bacterial infection precaution protein, i.e., caspase-1. This system offers a trial of non-invasive method to quantitative detection and real-time monitoring of bacterial infection in the early-stage. 1. INTRODUCTION Construction of supramolecular complex in vivo mimicking the well-ordered natural systems for sensitive bio-imaging or detection is one of the most promising fields in supramolecular chemistry.

1-4

In these cases, the small molecules have been designed with enzymatic-response

and self-assembled inside cells or living bodies for determining enzyme activity. The pilot studies based on this strategy proved to be effective in biology and medicine4-6. However, investigations on understanding of dynamic equilibrium of assemblies and design guideline of building blocks for controlled super-structures with desired properties in physiological environment remains a bottleneck for in-depth application.7


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As a non-invasive laser-induced technique,8-10 photoacoustic tomography (PAT, also called optoacoustic tomography) have been widely used for imaging blood vessels,9 tumors11-12 or organs,13-14 which can generate real-time,15-16 high resolution

scans10,

17

and 3D

reconstructions.13, 18 Thus, besides of spectrum signals,19-21 PA provides us a possibility for in situ monitoring the aggregation process of small molecules. Previously, highly ordered supramolecular structures19, 21-23 and photosynthesis24-26 have been reported, but there are few examples of their applications in living subjects owing to lack of well-controlled structure in complex condition. Chlorophyll derivatives, with self-organization property in nature, show distinct PA signals during the dynamic aggregation process. Reasonably, the modified seminatural chlorophyll derivatives with good biocompatibility could offer a molecular-level ordered self-assembly in physiological conditions. 27 For early-stage bacterial infection detection,28 the direct method is highly-sensitive bacterial cells detection;

29-34

however, the uncontrollable physiological environment makes it application

in vivo uncomfortably. Nevertheless, the innate immune pathway35-36 exhibited significance for monitoring the immune reaction and alerted bacterial infection as well. Thus, caspase-1 in this pathway could be an ideal infectious-imaging target, because their activation necessarily commits the infection by pathogen-associated molecular patterns (PAMPs). In our previous works, we have successfully applied chlorophyll-peptide systems for living body tumor and infection imaging through an in vivo supramolecular structure construction16, 37. However, how the sophisticated dynamic superstructures affect the ultimate signals is unclear so far. We therefore attempt to reveal the relationship of assembly and PA signals and develop an emerging quantitative and real-time imaging method for bacterial infection detection.


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Here, we designed a substrate molecule of P18-YVHDC-TAT, which can be cleaved by active caspase-1 and initialize the assembly process from monomeric to aggregated states (Scheme 1). The dynamic equilibrium of these binary states was monitored and analyzed by ratiometric photoacoustic (PA) signal and the correlation between aggregation tendency and ratiometric PA signal was revealed for the first time. Depending on the ratiometric PA signals, the activity of caspase-1 can be successfully imaged quantitatively without lysis of cells. This de novo noninvasive approach paves the way for quantitatively monitoring the activity of caspase-1 inside cells and further precaution of bacterial infection in the early stage. 2 EXPERIMENTAL SECTION 2.1 Synthesis of peptides. Peptides were synthesized by solid-phase methods using standard Fmoc-chemistry. The 0.35 mM scale protocol was used with C-terminal amide protection. Deprotection of the N-terminal Fmoc group was carried out using piperidine (20%, v/v) in anhydrous DMF for 30 min. Qualitative Fmoc deprotection was confirmed by a ninhydrin test (ninhydrin, phenol, VC 1:1:1, v/v). Amino acid activation was achieved by NMM (0.4 M) and HBTU (used at the same molar concentration with the amino acid) in anhydrous DMF. The amino acid link was reacted at RT for 2 h. Purpurin-18 (P18) was treated similar with amino acid in the protocol described above. Cleavage from the resin and deprotection of the amino acid side chains was performed by reaction with a mixture of TFA (95%, v/v), H2O (2.5%, v/v) and TIPS (2.5%, v/v) for 30 min in an ice bath and then at room temperature for 3 h or more. After separation from the resin, the above mixture was vacuum rotary evaporated to remove the TFA. The P18 peptides were then precipitated in cold anhydrous diethyl ether, collected by centrifuge, dried under vacuum and purified by column chromatography on silica gel.


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2.2 The calculation of heat conversion efficiency ( ) for monomers and aggregates. The time constant of monomers or aggregates was calculated in a linear fitting of vs. with the equation = − ln (

).

The heat conversion efficiency of aggregates and monomers was calculated with the equation (750nm) =

( ) !"# $# %(&&'()(*+,-.) )

where /(750 01) = 2(750 01)34 (Beer-Lambert law), 3 is the pathlength (cm), 4 is the concentration (M), 2(750 01) is molar extinction coefficient at 700 nm. The results showed that at 750 nm with a concentration of 2.5 mg mL-1, the heat conversion efficiencies of aggregates (i.e. P18-YVHD in aqueous solution) and monomers (i.e. P18-YVHD in DMSO solution) were 23.7% and 4.6%, respectively. 2.3 Calculation of aggregated degree of P18-YVHD. The degree of aggregation (5677 ), which is fraction of molecules present in the aggregates, was calculated by the Equation38 below: 5677 ≈ 1 − (

: − :;?@ is the emission intensity measured in pure DMSO. 2.4 Preparation of RAW 264.7 lysates. The protocol of preparation of RAW 264.7 lysates was following the report of literature.39 RAW 264.7 cells were pre-chilled for 15 min on ice in media with 1% FBS before adding bacteria (S. aureus). S. aureus clusters were disrupted by repeated passage through a 30-gauge needle. Then, RAW 264.7 cells with S. aureus were incubated with a number ratio of 1: 100 at 37 ºC for 15 min to activate caspase-1. Next the cells were washed by PBS twice and lysed in ice-bath for lysates collection.


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2.5 UV-vis and fluorescence spectra. Absorption and fluorescence spectra were recorded at 25°C

using

an

F-280

fluorescence

spectrophotometer

and

a

Cary100Bio

UV-vis

spectrophotometer with a 10 mm optical path length. P18-YVHD was dissolved with a final concentration of 10-5 M in different DMSO/water mixtures (1%-100% DMSO) by vortexing, and then left for 10 min before spectrophotometric analysis. Absorption and fluorescence spectra of P18-YVHD, P18-YVHDC-TAT and P18-YVHDC-TAT with RAW 264.7 lysates (2 × 107 cells mL-1) were recorded in aqueous solution (1% DMSO, 0.1% BSA, pH 6.0) with a concentration of 10-5 M. 2.6 CD spectra. CD spectra were obtained using a circular dichroism spectrometer (JASCO J 1500, Japan) with a cell path length of 1 mm at room temperature. The measurements were performed at a scanning speed of 500 nm min-1 and a resolution of 0.5 nm. The spectra were corrected by subtracting the solution background. For each sample, two spectra were obtained and averaged. Results for CD spectra are expressed in terms of molar ellipticity. The sample preparation of P18-YVHD, P18-YVHDC-TAT and P18-YVHDC-TAT with RAW 264.7 lysates was the same above with a concentration of 10-4 M. 2.7 PA signal detection. PA signal were recorded at 25°C using a MSOT 128 Multi-Spectral Optoacoustic Tomography. P18-YVHD was dissolved with a final concentration of 10-5 M in different DMSO/water mixtures (1%-100% DMSO) by vortexing, and then left for 10 min before analysis. The PA imaging of P18-YVHD, P18-YVHDC-TAT and P18-YVHDC-TAT treated with RAW 264.7 lysates (2 × 107 cells mL-1) with a final concentration of 10-4 M were respectively obtained by detection PA signal in the windows of 750 nm and 685 nm. The PA (I750 nm/I685 nm) for aggregated or monomeric state of P18-YVHD, P18-YVHDC-TAT and


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P18-YVHDC-TAT treated with RAW 264.7 lysates were calculated by the related PA intensity per area in 750 nm to 685 nm. 2.8 The ultrathin sections of cells for TEM. P18-YVHDC-TAT treated RAW 264.7 cells and control cells (without P18-YVHDC-TAT treatment) were harvested by centrifugation and fixed with 2% formaldehyde and 2.5% glutaraldehyde in PBS buffer overnight. After washing with PB buffer for 10 min/ time triple times, the cells were fixed with 1% osmium containing PB buffer for 2 h at room temperature. Subsequently, samples were washed several times with PB buffer similar with the protocol above. Samples were then dehydrated with a graded series of ethanol (50, 70, 80, 90, 95, 100%) on ice for 30 min for each step. Samples were infiltrated with a graded of series of mixtures (ethanol/EPON 812 resin: 2/1, 1/1, 1/2) at RT for 1h for each step. Pure resin was added the next day on ice, changed and left for overnight. Finally, samples were placed into gelatine capsules and filled with pure EPON 812 resin at 37ºC and 45ºC for 24 h, respectively. EPON 812 resin was polymerized for 48 h at 60°C. Ultrathin sections were cut with a diamond knife and sections picked up with formvar-coated copper grids (300 mesh). Counterstaining of the sections was performed with 4% aqueous uranyl acetate for 15 min. After airdrying samples were examined in HITACHI HT7700 transmission electron microscope at an acceleration voltage of 80 kV. The energy dispersive analysis was carried out by Tecnai G2 F20 U-TWIN electron microscope. 2.9 Cell culture and S. aureus infection. Typically RAW 264.7 cells were pre-chilled for 15 min on ice in media with 1% FBS before adding bacteria. Bacterial clusters were disrupted by repeated passage through a 30-gauge needle. Cells with bacteria were kept on ice for 30-40 min allowing the synchronization of phagocytosis. RAW 264.7 cells were then incubated at 37 ºC for the indicated times (0 min, 5 min, 15 min, 30 min, 60 min and 120 min).


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2.10 Caspase-1 activity detection by fluorescence substrate kit. The infectious RAW 264.7 cells (2×106 cells) were collected, washed by PBS and lysed with 100 µL lysis buffer. Then, the lysate was centrifuged (16000-20000 g) at 4 ºC for 15 min to remove the precipitation. The supernatant with a protein concentration of 1-3 mg mL-1 was incubated with fluorescence substrate (Ac-YVAD-pNA, 2 mM) at 37 ºC for 2 h for chromogenic reaction. Next, the mixtures were recorded at 405 nm for absorbance. The standard curve was obtained by detecting absorbance at 405 nm with different concentrations of pNA. 2.11 In situ caspase-1 activity monitoring by PAT. The infectious RAW 264.7 cells (8×106 cells) were collected, washed by PBS and incubated with PA substrate (P18-YVHDC-TAT, 0.2 mM) at 37 ºC for 1 h for diffusion. After wash the P18-YVHDC-TAT extracellular, the final intracellular concentration was approximated to 6.5×10-5 M. Next, RAW 264.7 cells were further collected for PA signal detection (with detection windows in 750 nm and 685 nm). 3 RESULTS AND DISCUSSION 3.1 Characterization of aggregation properties of chlorophyll-peptide derivatives and the relationship between the aggregation process and ratiometric PA signals. The molecule of P18-YVHDC-TAT is composed of three modules: P18 as a signal molecule, peptide YVHDC as a responsive peptide linker and TAT as a cell-penetrating peptide (Scheme 1). The building blocks (P18-YVHDC-TAT and P18-YVHD) were synthesized, purified and characterized comprehensively (see Supporting Information Scheme S1 and S2 for synthetic details). The responsiveness of P18-YVHDC-TAT (P18-YVHDCGYGRKKRRQRRR) to caspase-1 was investigated in Figures S1, the cleavage site was between Asp (D) and Cys (C). 40-41 In the innate immune pathway (Figure S2), PAMPs such as bacterial mRNA etc., can activate inflammasome, which is an intracellular protein complex consisting of NLRP3, recruitment


8

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domain (ASC) and pro-caspase-1. Then, the inflammasome sequential activated caspase-1. The molecules P18-YVHDC-TAT, which can penetrate into cells as monomer, specific cleaved by activated caspase-1 and formed the P18-YVHD residue. The enhanced hydrophobicity and reduced hindrance of P18-YVHD resulted in self-assembly of the building blocks. The in situ formed aggregates exhibited an obvious PA signal change compared to monomers. The ratios of PA signals between aggregates and monomers were well-correlated to the aggregation states of molecules.

Finally, we validated that our system can successfully realize quantitative

determination of caspase-1 activity in living infectious cells. The UV-vis absorption spectra for the aggregation process were carried out in mixing solutions (Figure 1a). With the increase of the ratio of H2O in DMSO, the Qy bands red-shifted (from 700 nm to 715 nm) and broadened, this is consistent with self-aggregation of the molecules.9, 42 Simultaneously, along with the transformation of P18-YVHD from monomeric to aggregated state, the fluorescence quenching (zoom in Figure 1a) also associated with such an aggregation process, implying the hydrophobic and/or π-π interactions drives the aggregate formation. Well-ordered aggregates of P18-YVHD significantly enhanced the heat conversion efficiency ( ), which increased from 4.6% for monomers to 23.7% for aggregates (Figure 1b, the calculation details see in Supporting Information and Figure S3). The equation 43 (1) explains why the PA signal intensity A(λ) is enhanced in aggregates compared to that of monomers : A(λ) ∝ C D6 E

(1)

where C is the thermodynamic property (dimensionless) called the Grüneisen parameter; is the heat conversion efficiency;44 D6 is the optical absorption coefficient (cm-1); D6 = 382I2(J) , which is proportional to the molar concentration ( I ) and molar extinction


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coefficient (2(J)) at a certain wavelength; and F is the local optical fluence (J/cm2). Combining these equations, the photoacoustic signal under certain optical conditions can be expressed as: A(λ) ∝ I2(J)

(2)

This suggests that under the same measurement condition and molecular concentration, the PA intensity was proportional to the heat conversion efficiency and molar extinction coefficient at certain wavelength. That is why the enhanced heat conversion efficiency for aggregates attributed to the increased PA intensity. Otherwise, the detection window for molar extinction coefficient of monomer and aggregate also should be considered (Figure S4). The suitable detection window for monomer and aggregate of P18-YVHD was at 685 nm and 750 nm respectively, according to the lowest and highest PA intensity ratio. Then, the transformation process of P18-YVHD from the monomeric to the aggregated state was monitored by PA intensity at 685 nm and 750 nm (Figure S5). Comparing to the standard analysis of aggregation property by fluorescence spectra, 45 the PA intensity ratio of 750 nm to 685 nm can also be used to monitor the dynamic equilibrium of aggregation process. The curve of aggregation degree, which means the fraction of molecules presented in aggregated state, 38 is well corresponding to the aggregation tendency monitored by ratiometric PA signal (Figure 2a). The aggregation process can be arbitrarily divided into three steps: aggregated state (1-40% DMSO/H2O, I750/I685 ˃ 2, αagg>0.75), transited state (40%-63% DMSO/H2O, 1˂ I750/I685 ˂2, 0.25

Quantitative Analysis of Caspase-1 Activity in Living Cells Through

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