Real-Time Visualization of Cell Membrane Damage Using Gadolinium

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Biological and Medical Applications of Materials and Interfaces

Real-Time Visualization of Cell Membrane Damage Using Gadolinium-Schiff Base Complex-Doped Quantum Dots Amitava Moulick, Zbynek Heger, Vedran Milosavljevic, Lukas Richtera, Joaquín Barroso-Flores, Miguel Angel Merlos Rodrigo, Hana Buchtelova, Robert Podgajny, David Hynek, Pavel Kopel, and Vojtech Adam ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b15868 • Publication Date (Web): 28 Sep 2018 Downloaded from http://pubs.acs.org on September 28, 2018

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ACS Applied Materials & Interfaces

Real-Time Visualization of Cell Membrane Damage Using Gadolinium-Schiff Base Complex-Doped Quantum Dots Amitava Moulick1,2, Zbynek Heger1,2, Vedran Milosavljevic1, Lukas Richtera1,2, Joaquin Barroso–Flores3, Miguel Angel Merlos Rodrigo1,2, Hana Buchtelova1, Robert Podgajny4, David Hynek1,2, Pavel Kopel1,2 and Vojtech Adam1,2* 1

Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, CZ-613

00 Brno, Czech Republic 2

Central European Institute of Technology, Brno University of Technology, Purkynova 123, CZ-

612 00 Brno, Czech Republic 3

Centro Conjunto de Investigación en Química Sustentable UAEM-UNAM, Carretera Toluca–

Atlacomulco Km 14.5, Unidad San Cayetano, CP-50200 Toluca, Estado de México 4

Jagiellonian University, Faculty of Chemistry, Ingardena 3, PL-30060 Krakow, Poland

KEYWORDS: Cell-penetrating peptides; Core-shell; Quantum dots; Fluorescence labelling; Nanotechnologies

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ABSTRACT Despite the importance of cell membranes for maintenance of integrity of cellular structures, there is still a lack of methods that allow simple real-time visualization of their damage. Herein, we describe gadolinium-Schiff base-doped quantum dots (QDs)-based probes (GdQDs) for a fast facile spatial labeling of membrane injuries. We found that GdQDs preferentially interact through electron-rich and hydrophobic residues with a specific sequence motif of NHE-RF2 scaffold protein, exposed upon membrane damage. Such interaction results in a fast formation of intensively fluorescent droplets with a higher resolution and in a much shorter time compared to immunofluorescence using organic dye. GdQDs have high stability, brightness and considerable cytocompatibility, which enable their use in long-term experiments in living cultures. To the best of our knowledge, this is the first report, demonstrating a method allowing real-time monitoring of membrane damage and recovery without any special requirements for instrumentation. Because of intensive brightness and simple signal pattern, GdQDs allow easy examination of interactions between cellular membranes and cell-penetrating peptides or cytostatic drugs. We anticipate that the simple and flexible method will also facilitate the studies dealing with hostpathogen interactions.


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Introduction Biological membranes play a pivotal role in various cellular processes, particularly in maintenance of structural integrity of cells and organelles. Membranes also participate in intraand intercellular communication and control the transfer of information between organs in the form of action potentials. Although it has been known for several decades that wounded eukaryotic cells tend to repair their membranes within a few seconds,1 many pathological states result from membrane injury. For instance, bacterial toxins (e.g., listeriolysin O produced by Listeria monocytogenes and others2) or non-enveloped viruses (e.g. Adenoviridae and Picornaviridae) directly penetrate the host membranes.3 A few reports have also provided evidence of a link between the conformational changes in amyloid-β that are accompanied by oligomerization and neuronal membrane penetration and the development of Alzheimer's disease.4-6 Notably, membrane penetration and pore formation is a specific effect of a large group of cell-penetrating peptides (CPPs) with a broad spectrum of applications, including delivery of nucleic acids, proteins or contrast agents into the cell.7 Moreover, CPPs are considered as a potent alternative to conventional antimicrobial and antiviral agents.8 This short overview indicates the importance of the investigation of membrane injuries. However, to the best of our knowledge, the methods applicable for such purposes are lacking, and the few existing methods have specific drawbacks. For instance, Martinez and colleagues reported a high-resolution approach to the spatial visualization of adenovirus entry to cells. However their method relies on the stable expression of galectin-3 fused to a fluorophore, and thus lacks versatility.9 Membrane penetration can be further studied using electron and probe microscopy techniques,10 but without the possibility of the real-time imaging of living cells.


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Hence, probes for spatial visualization of membrane damage with broad applicability are currently of a high interest. Semiconductor nanocrystals called quantum dots (QDs) are among the most promising advanced nanomaterials. Currently, a wide range of forming materials, functional coatings, and bioconjugate techniques are available for QDs to demonstrate their applicability.11-12 The synthesis of core-shell QDs is highly desirable because the core is stabilized with various surface ligands or caps to promote solubility in aqueous media and specific functionalities.13 Therefore, surface modification is vital for QDs in biological applications, and surface-modified QDs have been used for a myriad of applications, including in vitro diagnostics, energy transfer-based sensing, drug delivery, theranostics and in vivo imaging.14-16 However, to the best of our knowledge, QDs have not been applied to living cells imaging of membrane injuries. In our study, for the first time, we synthesized QDs formed from CdTe surface which was doped with a novel gadolinium-Schiff base complex (Gd-SB). The resulting nanostructure (hereinafter abbreviated as GdQDs) demonstrated clear red emission and exceptional colloidal properties. The primary aim of the present study was to develop an easy and fast method to visualize cell membrane damage. The stability of GdQDs for live cell imaging and their interaction with cell membrane should be studied well. To fulfil this purpose, we performed fluorescence microscopy experiments in various unfavourable conditions, including osmolysis or the administration of the Hecate17 or platinum cytostatics (carboplatin, cisplatin) on human cells. The results revealed that the GdQDs are highly suitable for a facile spatial visualization of cellular membrane damage. To shed light into the mechanism behind the specificity of GdQDs-based visualization process, we carried out a series of experiments that indicated that GdQDs preferentially interact with the Na+/H+ exchange regulatory cofactor NHE-RF2, a scaffold protein that supports the actin


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cytoskeleton.18 To our knowledge, this is the very first method for real-time monitoring of membrane damage and recovery without any special requirements for instrumentation. We anticipate that the discovered method with novel GdQDs will be used in a broad range of applications, such as evaluation of CPP activity, determination of cell membrane status and recovery in various undesirable conditions, and observation of host-pathogen interactions. Experimental Section Chemicals 2-Pyridinecarboxaldehyde, diethylenetriamine, gadolinium nitrate, cadmium acetate, MSA, sodium tellurite, sodium borohydride and all other chemicals were obtained from Sigma Aldrich (St. Louis, MO, USA) with ACS purity unless noted otherwise. Preparation of GdQDs The

Schiff

base,

[(2-[(E)-2-pyridylmethyleneamino]-N-[2-[(E)-2-pyridylmethylene-

amino]ethyl]ethanamine)] was prepared according to19 with few modifications. Briefly, 1900 µL of 2-pyridinecarboxaldehyde and 1080 µL of diethylenetriamine were mixed and heated under reflux in methanol (35 mL) for 6 h. After cooling, MeOH was added again to bring the volume to 50 mL to prepare the desired Schiff base solution. In a separate beaker, 10 mL of methanol was mixed with 5 mL of an aqueous solution of gadolinium nitrate (90 mg/mL), which was subsequently added to 5 mL of the Schiff base solution. The solutions were mixed for 2 h at 40°C, and the volume was brought to 100 mL with deionized water (Sigma-Aldrich). The prepared Gd-SB solution was stored at 25°C until use. Microwave preparation of the CdTe QDs was carried out according to our previous study20 with necessary modifications. Briefly, 53.2 mg of cadmium acetate was mixed with 86 mL of ACSgrade water on a magnetic stirrer, followed by the addition of 60 mg of MSA. Next, 1.8 mL of an


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ammonia solution (1 M) was then added to attain neutral pH (tested by a pH meter, inoLab pH 720, WTW, Weilheim, Germany). Then, 1.5 mL of a sodium tellurite solution (221 mg/mL) was added, and the solution was mixed well. Subsequently, 50 mg of sodium borohydride was added to the solution, which was stirred for approximately 2 h until bubble formation ceased, and the volume of the solution was brought to 100 mL with deionized water. Two millilitres of this solution was removed; placed in a small glass vessel and heated at 60°C, 300 W, for 10 min (ramping time, 10 min) under microwave irradiation (Multiwave 3000, Anton-Paar GmbH, Graz, Austria) to prepare the CdTe QDs. Next, 50 µL of the Gd-SB solution was added to 2 mL of the prepared CdTe QD solution, followed by heating at 100°C, 300 W, for 12 min (ramping time, 10 min) under microwave irradiation to prepare the GdQDs. For the control, deionized water was used in place of Gd-SB. The sample and control particles were filtered through 0.22 µm membranes and subsequently dialysed against deionized water several times to remove the unreacted initiators. Then, the particles were dispersed in deionized water for further characterization and use. Characterization of GdQDs The prepared GdQDs and CdTe QDs were visualized under a UV transilluminator (Transilluminator Multiband TFX-35.MC, Torcy, France; λex: 312 nm). The absorbance and fluorescence spectra of the GdQDs and control solutions were obtained using a microtitration plate reader (Tecan infinite M200 PRO, Männedorf, Switzerland). A total of 100 µL of each of the solutions was analysed in a microtitration plate. The absorbance was measured from 230 to 850 nm, and the fluorescence was measured using λex of 410 nm and λem of 450–850 nm. The photoluminescence quantum yield of the prepared GdQDs was determined using Rhodamine 6G as the reference according to a reported protocol.21 The TEM observations were made on an HR-


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TEM instrument (FEI Tecnai Osiris, FEI, Hillsboro, OR, USA) equipped with an X-FEG Schottky field emitter (200 kV) and Super-X EDX windowless detector system with a 4-sector silicon drift detector. The prepared samples were loaded into a beryllium double-tilt lowbackground holder to reduce the production of spurious X-rays and were then transferred to the TEM instrument. Z-contrast images were acquired using a high-angle annular dark-field detector in scanning mode. Scanning transmission electron microscopy micrographs coupled with EDX elemental mapping were acquired with an applied sample drift correction using Bruker Esprit software in order to investigate the spatial distribution of the constituent elements within a sample. Finally, the samples were measured on a copper-silicon matrix. The average size of the GdQDs and the size distribution were determined by quasielastic DLS with a Malvern Zetasizer (NANO-ZS, Malvern Instruments Ltd., Worcestershire, UK). First, 1.5 mL of an aqueous solution of GdQDs (2 mM) was added to a polystyrene latex cell and measured at a wavelength of 633 nm, a detector angle of 173°, a refractive index of 0.30, real refractive index of 1.59 and a temperature of 25°C. FTIR spectra were measured over a wavenumber range of 4,000 - 550 cm-1 using a Thermo Scientific Nicolet iS5 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an iD5 Diamond ATR accessory. Magnetic measurements were carried out on a Quantum Design MPMS-3 EverCool magnetometer (Quantum Design, San Diego, CA, USA). A solid sample of GdQDs (35.1 mg) was hermetically sealed in foil and attached to a stick. The DC moment (H) curve was measured over a range of -7 to +7 T. Differential pulse voltammetric measurements were performed at 5°C on a 663 VA Computrace instrument equipped with a three-electrode system. A hanging mercury drop electrode with a drop area of 0.4 mm2 was used as the working electrode. A platinum electrode and an Ag/AgCl/3 M KCl electrode were used as the auxiliary and reference electrodes, respectively. The analysed


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samples were deoxygenated prior to the measurements by purging with argon (99.999%) saturated with water for 15 s. The Brdicka supporting electrolyte containing 1 mM [Co(NH3)6]Cl3 and 1 M ammonia buffer (NH3(aq) + NH4Cl, pH = 9.6) was used in this experiment. The parameters of the measurement were as follows: initial potential of −0.7 V, end potential of −1.8 V, pulse period of 0.80 s, sample period of 10 ms, pulse width of 30 ms, step potential of 2 mV, modulation amplitude of −5 mV, sweep rate of 2.5 mV·s−1, Eads of 0 V, tads of 120 s and stirring speed of 1,000 revolutions.min−1. Molecular modelling All calculations were performed with the Gaussian 09 suite of programs. Natural population analyses were performed under the Natural Bond Orbitals formalism with the NBO3.1 program as supplied with the aforementioned suite. Density Functional Theory based methods were employed for the electronic structure calculations with the M06-2X functional22 and the LANL2DZ basis set which includes a quasi–relativistic pseudopotential for heavy atoms (second row and beyond),23 together with a relativistic pseudopotential for the electronic structure of Gd in conjunction with the corresponding basis set24. The crystal cell for CdTe was retrieved from the Cambridge Crystallographic Data Center, under identifier number 900884025 and the corresponding surfaces were created with Mercury 3.5.1. All orbital and molecular electrostatic potential images were created with GaussView5.0; rendering of optimized geometries were obtained with QuteMol.26 Separately, the Gd3+ complex geometry was optimized at the same level of theory starting from the crystallographic conformation obtained herein. Consistently with the magnetic properties observed a multiplicity of 8, corresponding to 7 unpaired electrons on Gd, was employed to


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obtain a stable wave function. The detail on the NBODel procedure is given in supporting information. Synthesis of the peptides Hecate (FALALKALKKALKKLKKALKKAL) and all other peptides were synthesized using a Liberty Blue peptide synthesizer (CEM, Matthews, NC, USA). The Fmoc protecting group was deblocked with 20% piperidine v/v in N,N-dimethylformamide. Coupling was achieved using N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium

hexafluorophosphate,

N,N-

diisopropylethylamine and DMF. The side-chain protecting groups were cleaved by treating the peptide resin with 95% trifluoroacetic acid v/v, 2.5% H2O v/v and 2.5% triisopropyl-propylsilane v/v for 30 min at 38°C under microwave irradiation. Detection of plasma membrane damage on cells using GdQDs PC-3 cells (established from a grade-4, androgen-independent and unresponsive prostatic adenocarcinoma from a 62-year-old Caucasian male and derived from the metastatic site in bone) were purchased from the American Type Culture Collection (ATTC, Salisbury, UK). The cells were cultured in Ham's F12 medium supplemented with 10% foetal bovine serum, penicillin (100 U/mL) and streptomycin (0.1 mg/mL). All cell lines were incubated in 5% CO2 and 90–100% relative humidity at 37°C in an incubator (Galaxy 170 R, Eppendorf, Hamburg, Germany). The cells (5000 cells/well) were seeded into a 96-well plate and incubated for 24 h prior to treatment. The membrane-damaging peptide, Hecate (1; 25 and 50 µM in medium) was applied to the cells for 15 min. Then, the cells were washed thrice with PBS buffer to remove the peptide. Next, the cells were incubated with GdQDs (50 µM) for 20 sec at 25°C and washed thrice with the PBS buffer. Finally, 100 µL of the medium was added to each of the wells, and the cells were visualized by an Olympus IX 71S8F-3 fluorescence microscope (emission filter


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700 nm) (Olympus, Tokyo, Japan) equipped with an Olympus UIS2 series objective, LUCPlanFLN 40× (N.A. 0.6, WD 2.7 – 4 mm, F.N. 22).The labeling protocol was further exploited for visualization of damage caused by membrane puncture (done by Eppendorf Transferman NK2 micromanipulator, Eppendorf, Hamburg, Germany), sonication (60 sec, Bandelin Sonoplus mini20 ultrasonic needle, Bandelin Electronic GmbH, Berlin, Germany) and exposure to carboplatin and cisplatin (both 10 µM for 12 h). The images were acquired with an Olympus DP73 camera (Olympus) and processed with Stream Basic 1.7 software (Olympus Soft Imaging Solutions GmbH, Münster, Germany). The treated and untreated PC-3 cells were stained with FDA, PI and Hoechst 33342. Cryo-electron microscopy Prior vitrification, cells were seeded onto Quantifoil grids R2/1 (Micro Tools Ltd., Tefen, Israel) in 6-well plates (50 000 cells/well). Vitrification was carried out in an automated vitrification robot (FEI Vitrobot Mark IV, FEI, Company, Hillsboro, OR, USA) using liquid ethane as cryogen. After vitrification, the grids were stored in liquid nitrogen for analyses. Cryo-FIB-SEM visualization of samples was done with FEI Versa3D equipped with a Quorum cryo stage and transfer station (FEI Company). Cryo-TEM visualization of samples was done with Tecnai F20 microscope equipped with a 4k CCD camera FEI Eagle (FEI Company). Fragmentation of GdQDs-bound proteins by MALDI-TOF MS A solution of a fine fraction of GO (5 cm3, 2.38 mg/mL) was prepared (see Supporting Information) and dispensed on a glass coverslip (24×24 mm), which was placed in the centre of a petri dish (diameter, 90 mm) to completely submerge all the corners of the coverslip. The water from the solution was evaporated very slowly in an oven at 50°C so that the coverslip was covered with a continuous and regular film of the fine fraction of GO. After drying, the coverslip


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was completely covered with an aqueous solution of GdQDs (2 mM). After further evaporation, the GdQDs-modified coverslip was used to bind the specific peptides from the samples. The modified coverslip was immersed in the sample solution for 20 sec and subsequently washed three times as described before. Then, the coverslip was allowed to dry at 25°C. Finally, all the solid matter was carefully collected from the coverslip by scraping and subjected to MALDITOF MS analysis. The mass spectrometry experiments were performed on a Bruker ultrafleXtreme MALDI-TOF MS instrument (Bruker Daltonik GmbH, Germany) equipped with a laser operating at a wavelength of 355 nm and at an accelerating voltage of 25 kV (cooled with nitrogen) and a maximum energy of 43.2 µJ with a repetition rate of 2000 Hz in linear and positive mode. Data acquisition and processing were carried out with specialized FlexControl software, version 3.4, and flexAnalysis software, version 2.2. The 2,5-dihydroxybenzoic acid matrix (Bruker, Germany) was prepared in TA30 (30% acetonitrile, 0.1% trifluoroacetic acid solution). The mixture was thoroughly vortexed and ultrasonicated in a Bandelin 152 Sonorex Digital 10P ultrasonic bath (Bandelin electronic GmbH) for 2 min at ambient temperature. Solutions of the matrix and sample were mixed in a ratio of 1:1 for the analysis. After obtaining a homogeneous solution, 1 µL was applied on the target and dried under atmospheric pressure at 25°C. The MS spectra were typically acquired by averaging 20 sub spectra from a total of 500 shots of the laser (Smartbeam 2, version: 1_0_38.5). The MALDI-TOF MS/MS spectra were recorded in LIFT mode for fragmentation. The LIFT mass spectra were acquired in positive ion mode, where 200 laser shots for the parent and 1600 laser shots for the fragments were summed. The MS/MS data were searched using Mascot software (Matrix Science, London, UK). Immunofluorescence of NHE-RF2 in non-permeabilized cells


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For detection of NHE-RF2, cells pre-incubated in Hecate, were fixed with 4% formaldehyde for 10 min and blocked for 20 min in PBS with 1% (w/v) BSA. Fixed and BSA-blocked cells were incubated sequentially with anti-NHE-RF2 (D3A5) rabbit mAb (cat. no. ♯9568, Cell Signaling Technology, Danvers, MA, USA) and goat anti-rabbit IgG conjugated to Alexa Fluor 488 (cat. no. ab 150077, Abcam, Cambridge, UK) for 30 min each. All antibodies were diluted in PBS containing 10% (w/v) BSA and all steps were carried out at 4°C. Labelled cells were suspended in 10% (w/v) BSA-PBS and examined immediately under Olympus IX 71S8F-3 fluorescence microscope (Olympus). Results Characterization of GdQDs In the present study, an aqueous solution of CdTe QDs was prepared using microwave irradiation. Subsequently, the surface of the QDs was doped using Gd-SB. The schematic representation of the synthesis of Schiff base [(2-[(E)-2-pyridylmethyleneamino]-N-[2-[(E)-2pyridylmethylene-amino]ethyl]ethanamine)] and its gadolinium complex (upper panel) are shown in figure S1a. The proposed binding of gadolinium complex with CdTe via carboxylate bridges of MSA is shown figure S1b. Figure 1a shows the absorbance and fluorescence spectra of the GdQDs. The fluorescence emission maximum of the GdQDs was observed at λ = 630 nm. Under UV transillumination (λ = 312 nm), the GdQDs solution displayed a dark red colour. The highest fluorescence quantum yield for the prepared GdQDs was 29.5%. The average particle size and the particle size distribution of the prepared GdQDs were analysed by high-resolution transmission electron microscopy (HR-TEM, Figure 1b and 1c) and dynamic light scattering (DLS, Figure 1d). The HR-TEM micrographs revealed the crystallinity of GdQDs, in which the distinct planes of the crystal structure can be observed. The inter-planar distance was found to be


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0.31 nm, which is in accordance with the inter-planar spacing of CdTe QDs.27 The diameter of the GdQDs (11±2 nm) was clearly larger than the diameter of the CdTe QDs (7±2 nm), which is in agreement with the assumed surface doping with Gd-SB. The modification also led to a change in the ζ potential from -35±0.17 mV (for CdTe QDs) to -43.6±0.13 mV (for GdQDs), reflecting the higher stability of GdQDs. The prepared GdQDs showed good stability in aqueous solution for several months.


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Figure 1. (a) Absorption (i) and fluorescence (ii) spectra of the GdQDs. Photographs of the GdQDs under visible and UV (λ = 312 nm) light are shown in the inset. (b) and (c) HR-TEM micrographs of the GdQDs; scale bars are 20 and 5 nm, respectively. (d) DLS measurements of the GdQDs. Column charts indicate the hydrodynamic diameter of the GdQDs. (e) FT-IR spectra of the GdQDs. (f and g) Magnetic characteristics of the GdQDs: (f) magnetic field dependence of the DC moment (T = 2 K) and (g) thermal dependence of the DC moment (H = 1 kOe) (ᴑ experimental points, ̶ simulated curves). (h) Comparison of the electrochemical signals of MSA (i), CdTe QDs (ii), Gd-SB (iii) and GdQDs (iv). RS2Co: typical signal from Brdicka electrolyte;


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MSA1 and 2: signals from MSA; Cd: signal from Cd2+ in the QDs (CdTe or GdQDs); Gd-SB1 and 2: signals from Gd-SB. The Fourier-transform infrared (FT-IR) spectrum of the GdQDs indicate the medium intensity peaks near 652 cm-1 and 842 cm-1 are characteristic of thiol-containing compounds and attributable to the ν(C-S) vibration of mercaptosuccinic acid (MSA) on the GdQDs surface (Figure 1e). The very intense peaks near 1566 and 1381 cm-1 are attributed to νas(COO) and νs(COO) vibrations, respectively; however, the peak near 1566 cm-1 probably overlaps with the ν(C=N) vibration of the Schiff base. A weak peak near 1250 cm-1 is attributed to the ν(C-N) vibration of heterocyclic rings.28 CdTe QDs were chosen because of their very high fluorescence as well as their high stability in solution. The prepared GdQDs are stable at mentioned conditions for months and they do not lose fluorescent properties, i.e. the intensity of fluorescence remains the same after at minimum two months. High stability is also confirmed by very high zeta potential (-43.6 mV) which was not changed with time. We also sought to demonstrate the presence of gadolinium in the sample and to explore its possible utilization for magnetic resonance imaging. The DC moment (H) curve measured in the range -7 to +7 T is presented in Figure 1f (open circles). Its shape is reminiscent of the presence of paramagnetic species in the measured sample, and its course is reproduced well by the simulated curve (blue line) based on equations 1-3, including the Brillouin function BS for S = 7/2, the Landé factor g = 1.95 (expected for the almost isotropic character of GdQDs) and a scaling factor of 0.047. The DC moment (T) curve (Figure 1g) is reproduced well by Curie’s law (eq. 4-5), including a term for the diamagnetic contribution (foil and intrinsic diamagnetism of


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the material) and the relevant scaling factor. These results suggest that the Gd3+ ions are incorporated in the GdQDs material. DC moment ( ) ~ = () () =

coth

(eq. 1)

− coth ( )

= /

(eq. 2) (eq. 3)

DC moment ( ) ~ χ =

"#$# %

−

&'#( %

)*+* ~ 0.1252( + 1)

(eq. 4) (eq. 5)

where N is the Avogadro constant, g is the Landé factor, S is the spin, β is the Bohr magneton, H is the magnetic field, k is the Boltzmann constant, T is the temperature, and para and diam are the paramagnetic and diamagnetic contributions, respectively, to the measured magnetic moment. Energy dispersive X-ray (EDX) analysis confirmed the presence of all elements forming the GdQDs (Figure S2, Supporting Information). Furthermore, the electrochemical responses of the GdQDs were studied by using the Brdicka reaction. The strongest electrochemical signals of the CdTe QDs or GdQDs are attributed to their surface modification by MSA (characteristic peaks MSA1 and MSA2, Figure 1h-I, Figure S3 and S4, Supporting Information). After surface passivation of the CdTe QDs with MSA, peak MSA2 (−1.55 V) disappeared (Figure 1h-ii), indicating that the –SH groups of MSA had become electrochemically inactive. The voltammogram of Gd-SB (no –SH moieties) contains two relatively weak signals (Gd-SB1 and Gd-SB2, Figure 1h-iii). Notably, Gd-SB modification is likely to cause the -SH moieties of MSA to become electrochemically active, which was observed as an increase in signal at -1.55 V (Figure 1h-iv). The Cd peak of the CdTe QDs shifted from −0.83 V to −0.80 V after modification with Gd-SB.


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Molecular Modelling of GdQDs structure Upon optimization, it was found that the Gd3+ complex is not planar but rather resembling a dextrorotatory helicene. Calculation of Wiberg bond indices (WBI)29 for the Gd complex strongly indicate that the cation is held in the cavity by electrostatic interactions; the highest WBI for Gd3+ found were 0.16; 0.21 and 0.17 with the neighboring N atoms (a covalent single bond would result in a value closer to 1.0). The molecular electrostatic potential mapped onto the isodensity surface is shown in figure 2a. The HOMO (Figure 2b) in Gd-SB is mainly composed of electron lone pairs 2sp3 orbitals on the amino groups and π orbitals on both pyridine rings; whereas the LUMO (Figure 2c) consists mainly of the empty 5d orbital on Gd. Only those orbitals corresponding to the alpha density are shown in the figures for the sake of clarity. MSA molecule was placed on the 100 face of a 2x2x1 CdTe cell and was optimized at the M062X/lanl2dz level of theory while keeping all Cd and Te atoms frozen in order to assess their coordination to the surface both in their neutral (Figure 2d) and anionic (Figure 2e) form, with the latter being the relevant deprotonation scheme at physiological pH. In their neutral state, MSA binds to CdTe through a single carboxylic group whereas in the anionic form both carboxylate groups bind to CdTe leaving the mercapto- moiety to interact through available electron lone pairs with two Cd atoms on the surface, which is indicative of its Lewis acidity. In both cases, the sulfur atom interacts closely with the surface through its basic electron lone pairs. The complete system under study: a frozen layer of CdTe (100 face), two MSA molecules and the pre-optimized Gd complex was further optimized with the PM6 semiempirical Hamiltonian30 and the final geometry is observed in figure 2f. Both MSA anions retain their original binding towards the CdTe surface and retain the Gd complex exclusively through electrostatic


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interactions which allow for a strong binding of the complex on the surface yet easily accessible by a polar medium such as water for release. .

Figure 2. (a) Optimized structure of Gd-SB at the M06-2X/lanl2dz level of theory with the molecular electrostatic potential mapped onto the isodensity surface (ρ = 0.02 e Å-3). Hydrogen atoms are omitted for clarity. Frontier orbitals HOMO (b) and LUMO (c) for Gd-SB calculated at the M06–2X/lanl2dz–SDD(Gd) level of theory. An MSA in neutral (d) and anionic form (e) optimized on the surface of a 2×2×1 cell of CdTe interacting through the 100 face (large pink circles = Te; small pink circles = Cd). (f) Optimized geometry of Gd-SB–MSA2–CdTe(100) (GdQDs) at the semiempirical PM6 level of theory. As a part of characterization, we further evaluated possible cytotoxic effects of GdQDs using 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide

(XTT)

assay.

Comparing the dose-response viability curves for GdQDs to those obtained after incubating the


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cells with non-modified CdTe QDs, it was found that the formation of the Gd-SB shell resulted in a pronounced increase in the GdQDs cytocompatibility (Figure S5A, Supporting Information). Moreover, Figure S5B shows that Gd-SB complex alone do not induce toxic effects partially observed upon applying gadolinium nitrate. Noteworthy, during 24 h incubation in fully supplemented culture medium, no significant release of Cd from GdQDs was found (Figure S6). These results underpin importance of Gd-SB shell that: i) bind Gd3+ ions and ii) also stabilizes the CdTe core and eliminates release of toxic Cd2+ ions. Gd-SB surface modification makes GdQDs markedly cytocompatible, which is a pivotal prerequisite for applications in living cell cultures. GdQDs fluorescence spatially indicates the plasma membrane damage In the present experiment, Hecate, a well-known cationic lytic peptide17, 31 was used to damage the cell membranes. After pre-incubation of the cells with Hecate, the GdQDs showed the damage on the plasma membrane by fluorescing bright red due to their significant clustering within the region (Figure 3). The extent of membrane injury was observed to increase with increasing concentration of Hecate. This experiment indicates that GdQDs preferentially bind cells with damaged plasma membranes. The fluorescence spectra of 5,000 cells pre-incubated with Hecate and stained with GdQDs are shown in Figure 3. The fluorescence intensity of the GdQDs attached to the damaged plasma membrane increased with increasing concentration of Hecate due to increasing extent of cell membrane damage. The quantitative correlation between the extent of cell membrane damage and fluorescent intensity of aggregated GdQDs are shown in supplementary data (Figure S7). The same experiment was also carried out using CdTe QDs, and we did not detect any fluorescent signal from the cells pre-incubated with Hecate (Figure S8).


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Figure 3. Cells were treated with different concentrations (indicated at the top of each figure) of Hecate to damage the plasma membrane and subsequently stained with GdQDs. Pictures of the cells were taken with a fluorescence microscope. Arrows indicate some of the damaged areas of the plasma membrane. The fluorescence spectra of 5,000 Hecate-untreated and -treated cells (stained by GdQDs) are shown in the fourth row. The blue and red lines indicate the control (without GdQDs staining) and sample (with GdQDs staining) cells, respectively. The membrane integrity of the PC-3 cells was assessed by double staining with fluorescein diacetate (FDA,


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green) and propidium iodide (PI, red). The merged FDA/PI-staining and bright-field images are shown in the fifth row. The length of scale bar is 20 µm. The membrane integrity of the Hecate-untreated and -treated cells was also assessed by double staining with FDA and PI. The untreated cells fluoresced bright green and were thus considered viable, and the red fluorescence emitted by the cells treated with Hecate confirmed the damages to the plasma membrane caused by the treatment. We further analysed the Hecate-induced membrane damage and subsequent binding of GdQDs using Cryo-electron microscopy. Representative Cryo-focused ion beam scanning electron microscopy (FIB-SEM) micrograph of membrane of intact non-treated cell is depicted in Figure 4a. Pre-incubation with Hecate caused obvious signs of membrane disruptions in the size of hundreds of nm (Figure 4b), which corresponds to the size of spots determined by GdQDs labeling. Moreover, Cryo-TEM micrographs (Figure 4c and 4d) revealed a significant clustering of GdQDs within the disrupted regions.


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Figure 4. Cryo-FIB-SEM micrographs of a membrane of (a) control (Hecate-untreated) and (b) Hecate-treated (25 µM) cell showing obvious signs of membrane damages. (c) and (d) CryoTEM micrographs of GdQDs clustering within the membrane damage caused due to Hecate preincubation. To check that the fluorescent signals from the GdQDs did not arise from any influence of the pre-incubation with Hecate, we punctured the cells mechanically with a glass capillary and subsequently stained them with the GdQDs. Figure 5a clearly shows that the GdQDs were attached to the damaged portion of the cell. The bright red fluorescence denotes the exact area of penetration of the cell membrane.


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Figure 5. (a) Micrograph of PC-3 cells that had been mechanically punctured using a glass capillary and stained with GdQDs. The damaged portion of the cells is indicated by an arrow. (b) Micrograph of the debris of PC-3 cells stained by GdQDs. The PC-3 cells were disrupted by sonication without Hecate treatment. (c) PC-3 cells semi-disrupted by osmosis, stained with GdQDs, and counterstained with Hoechst 33342. The length of scale bar is 20 µm. Furthermore, approximately 5,000 cells were completely disrupted by sonication, and the cell debris was then air-dried on a glass slide and stained with the GdQDs (Figure 5b). The bright red fluorescence emitted from the cell debris confirmed that the GdQDs directly binds to almost all fragments of disrupted cells. In a separate experiment, the cells were partially disrupted by osmosis induced by placing the cells in deionized autoclaved water. The cells were stained with the GdQDs and counterstained with Hoechst 33342. The red fluorescence-emitting GdQDs bound strongly to the nuclear membrane and to the cytoplasm (Figure 5c). NHE-RF2 sequence motif GEQGYGFHLHGE is a preferential target for GdQDs binding To investigate which component or biomolecule in the cells was responsible for binding to the GdQDs, a graphene oxide (GO, Figure S9 and S10) film was prepared on a glass coverslip and fully covered by GdQDs. Then, the cell suspension was incubated with the GdQDs immobilized


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on the GO film and analysed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry in LIFT mode (Figure 6a). The results revealed the presence of a GEQGYGFHLHGE (m/z 1329.806 Da) sequence motif belonging to the Na(+)/H(+) exchange regulatory cofactor protein (NHE-RF2, Mass Score 48.6, Swiss-Prot No. SLC9A3R2), which is localized on endomembrane systems, the nucleus, the apical cell membrane and the peripheral membrane (Figure 6b). To confirm the result, we synthesized the peptide GEQGYGFHLHGE and incubated it with GdQDs immobilized on a graphene oxide (GO) film. After washing, MALDI-TOF (Figure S11) was carried out again as mentioned above and we confirmed only the presence of the NHE-RF2 protein motif, showing its specific affinity for GdQDs.

Figure 6. (a) MALDI-TOF spectrum of the peptide (experiment with whole-cell suspensions), part of NHE-RF2 sequence, bound to GdQDs (green line) and spectrum of the peptide (m/z 1329.826 Da) after MALDI-TOF fragmentation. (b) LIFT-TOF/TOF spectrum and peptide mass fingerprint of m/z 1329.826 Da with assignment of the identified sequence: GEQGYGFHLHGE. (c) Peptide sequence that had been manually docked on top of the optimized GdQDs ensemble


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and optimized using molecular mechanics with the UFF, considering all atoms in the GdQDs as fixed and rigid spheres in order to provide a rough description of the interaction between the peptide and the GdQDs. (d) Comparison between post-Hecate pre-incubation GdQDs staining (left) and anti-NHE-RF2 immunostaining (right), which shows similar damage patterns. The length of scale bar is 20 µm. The GEQGYGFHLHGE was further manually docked on top of the optimized GdQDs and optimized using molecular mechanics (Gaussian 09) with the Universal Force Field (UFF) considering all atoms in the GdQDs to be fixed in rigid spheres in order to provide a rough description of the interaction between this peptide and the GdQDs. Both ends of the peptide interacted weakly with the GdQDs surface, whereas the middle portion of the peptide wrapped around the Gd-SB (Figure 6c). The NHE-RF2 sequence motif appears to interact preferentially with the GdQDs through the electron-rich and hydrophobic residues located in the middle, namely Tyr and Phe. Finally, to validate the presence of NHE-RF2 within the membranes injured by Hecate, we conducted immunolabeling of fixed, non-permeabilized cells using anti-NHE-RF2 mAb (Figure 6d). Noteworthy, it was found that both, GdQDs and anti-NHE-RF2 shared a similar fluorescence staining pattern. This provides a direct evidence that GdQDs interact with NHERF2 exposed due to a membrane damage. Moreover, we provide a first evidence that NHE-RF2 could serve as indicator of membrane damage. We are eager to further work on this aspect. GdQDs are applicable for visualization of the status of cell membrane Cells were grown without changing the medium to deplete the nutrients and stained with GdQDs. Under a fluorescent microscope, we observed small injuries to the plasma membrane of the cells, which were probably caused by cells starvation in unfavourable environmental


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conditions (Figure 7a). This information helped us determine the membrane integrity status of the cells. In addition, we pre-incubated the cells with two different conventional chemotherapeutic agents, carboplatin and cisplatin. GdQDs staining revealed significantly different effects of these two chemotherapeutic agents on the plasma membrane (Figure 7b and 7c). Despite the mainstream of investigations focuses on DNA as main target of platinum-based cytostatics, several works are in agreement with our findings that platinum cytostatics can also alter the plasma membranes.32-33 In the light of our results, it should be noted that Rebillard and coworkers revealed that cisplatin triggers generation of ceramide concomitantly with an increase in membrane fluidity.33 This phenomenon is associated with a fast, non-competitive inhibition of Na+/H+ membrane exchanger-1 (NHE-1) that results in hydroelectric imbalances and cytoskeleton alterations.34-35 Since NHE-1 belongs to the same protein family (NHE) as NHERF2, we anticipate that cisplatin could exhibit similar inhibitory activity towards NHE-RF2. Compared to cisplatin, carboplatin differs in structure having a bidentate carboxylate ligand in place of two chloride ligands of cisplatin.36 This results in a markedly lower reactivity, potency and susceptibility to distinct cellular degradation mechanisms. Contrary to cisplatin, carboplatin exposure has not yet been connected with a fast inhibition of NHE-1 at the protein level. Considering all these facts, we anticipate that the increased membrane fluidity enables for interacting between NHE-RF2 and GdQDs in exposed cells and the disparities in GdQDs fluorescence patterns are caused by plausible inhibitory activity of cisplatin to NHE-RF2. Indeed, these results deserve further investigation since inhibitory activity of cisplatin towards NHE-RF2 could bring novel fundamental insights into the non-genomic toxic effects of cisplatin.


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Overall, the above discussed application clearly demonstrates that GdQDs could serve as simple and fast screening indicator of plasma membrane health status of cells exposed to various unfavourable conditions including drugs.

Figure 7. (a) Micrographs of PC-3 cells that had been cultured in medium without exchange for 7 days and stained with GdQDs. The red fluorescent spots indicate the presence of damage on the plasma membrane caused by the depleted medium, in which most of the nutrients had already been consumed by the cells. Micrographs of cells treated with (b) carboplatin (10 µM) or (c) cisplatin (10 µM) for 12 h and subsequently stained with GdQDs. Different amounts of red fluorescence indicate distinct effects of the chemotherapeutic drugs on the plasma membrane. The length of scale bar is 20 µm. Discussion We developed a novel core-shell structure formed from CdTe QDs with Gd-SB surface modification with the capability of real-time spatial imaging of membrane injuries. Our synthetic


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strategy allows for direct synthesis of core-shell GdQDs in an aqueous medium instead of in organic solvents, making the synthesis simple and environmentally friendly. Similar core-shell nanostructured materials have recently received increasing attention owing to their exceptional properties and broad range of biological applications, including in vivo imaging,37 magnetic resonance imaging,38 drug delivery and tissue engineering.39 Nevertheless, to the best of our knowledge, there are no such methods for the visualization of membrane disruptions. Notably, we successfully designed and used GdQDs to visualize membrane damage caused by the Hecate, mechanical puncturing, osmosis, starvation and conventional cytostatics. Thus, we anticipate that GdQDs will be applicable as an inexpensive, versatile and fast tool for studying the integrity of cell membranes. Compared with other existing methods for studying membrane disruptions, including electron and probe microscopy10 or methods utilizing cellular systems with the stable expression of fluorophores,9 our approach has several immediately identifiable advantages. First, no genetic manipulations are required, thus saving money and time. Second, our core-shell nanostructure can be easily exploited through a very simple and fast staining procedure. Third, GdQDs can be used for sensitive real-time spatial imaging using fluorescence microscopy, which is inexpensive compared to electron or probe microscopic techniques. Moreover, we have also shown that the fluorescence signal from the injured portions of membranes can be directly quantified in harvested cells. Plasma membranes contain permanently anchored integral membrane proteins and temporarily attached peripheral membrane proteins.40 During injury to the membrane, the proteins attached to the intracellular portion of membranes become available to interact with the extracellular environment. Indeed, GdQDs are able to widely bind with cellular debris, possibly indicating


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preferential interaction with intracellular (rather than extracellular) proteins. Similarly, after partial disruption and Hoechst counterstaining of nuclei, we found that GdQDs bound very well to nuclear membranes. Hence, the next step was to identify the preferential binding target of GdQDs. A series of experiments using a cell suspension immobilized on a GO film revealed that the GdQDs surface specifically interacts with the sequence motif of the Na(+)/H(+) exchange regulatory cofactor (NHE-RF2), a protein localized on endomembrane systems, nuclei, apical cell membranes and peripheral membranes.41 NHE-RF2 connects plasma membrane proteins with proteins from the ezrin/moesin/radixin family, helping to link these latter proteins to the actin cytoskeleton.42 Moreover, NHE-RF2 also acts as scaffold protein in the nucleus.43 Molecular mechanics confirmed that the middle portion of the GEQGYGFHLHGE sequence of NHE-RF2 wraps around the Gd-SB preferentially through electron-rich and hydrophobic residues (Tyr and Phe). As NHE-RF2 is ubiquitously expressed in all eukaryotic cell types, we believe that it is an excellent target for similar labelling applications44. We also successfully optimized the NHE-RF2 labeling by anti-NHE-RF2 antibodies (Figure 6d), with the results comparable to GdQDs staining. Despite that when comparing the complexity of both methods, immunostaining has several drawbacks, including variability, reliability and cost of primary antibodies, photodegradability of fluorophore conjugated to secondary antibody, or ability to penetrate deeper into the cytoplasm, which results in biased results. Further investigation (using advanced proteomic tools) of the interaction between whole protein composition of the cells and GdQDs is necessary for precise identification of the bound protein. Based on the fluorescence patterns of GdQDs and anti-NHE-RF2 mAb (Figfure 6d), we believe that the binding is confirmed with NHE-RF2 protein. We expect more proteins would be present on the surface of GdQDs. However due to significant affinity of NHE-RF2 toward GdQDs, we expect that it is the


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most abundant protein present; therefore it was simply identified by MALDI-TOF-MS despite its known poor resolution.

Conclusion Our novel GdQDs core-shell nanostructures affords a unique opportunity to spatially assess membrane injuries in a simple, fast, real-time manner, which can be otherwise challenging with other techniques. To explore this opportunity, we successfully evaluated the applicability of these structures to visualizing the membrane disruption caused by various exogenous stresses, including exposure to a CPP, puncturing, starvation and treatment with cytostatics. To the best of our knowledge, this is the first report, demonstrating a flexible and expandable method allowing real-time monitoring of membrane damage and recovery without any special requirements for instrumentation. Taking our results together, we expect that the simple and novel method will be used in versatile field of applications, such as evaluation of CPP activity, determination of cell membrane integrity in various undesirable conditions and recovery from injury, and the observation of host-pathogen interactions.

Supporting Information The following files are available free of charge. Details on cell viability assay, Details on synthesis of fine fractions of large are graphene oxide (GO), Details on the NBODel procedure, Differential pulse voltammograms for GdQDs, Results of XTT cytotoxicity assay, SEM micrographs of GO and AFM micrographs of GO (PDF) Corresponding Author


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Vojtech Adam, Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno, Czech Republic; E-mail: [email protected], Tel.: +420-5-4513-3350; Fax: +420-5-4521-2044 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. (match statement to author names with a symbol) Acknowledgement Financial support from CEITEC 2020 (LQ 1601) of the Ministry of Education, Czech Republic and The Czech Science Foundation (GACR 16-18917S) is acknowledged. We also acknowledge the CF CEITEC - Cryo-electron Microscopy and Tomographysupported by the CIISB research infrastructure (LM2015043 funded by MEYS CR) for their support with obtaining scientific data presented in this paper. Abbreviations QDs, quantum dots; GdQDs, gadolinium-Schiff base-doped quantum dots; CPP, cell-penetrating peptide; Gd-SB, gadolinium-Schiff base complex; HR-TEM, high-resolution transmission electron microscopy; DLS, dynamic light scattering; FT-IR, Fourier transform infrared spectroscopy; MSA, mercaptosuccinic acid; EDX, energy dispersive X-ray analysis; WBI, Wiberg bond index; UFF, universal force field; XTT, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)2H-tetrazolium-5-carboxanilide; PI, propidium iodide; FDA, fluorescein diacetate; FIB-SEM, focused ion beam scanning electron microscopy; GO, graphene oxide; MALDI-TOF, matrixassisted laser desorption/ionization time-of-flight; NHE-RF2, Na(+)/H(+) exchange regulatory cofactor;


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Table of Contents


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Real-Time Visualization of Cell Membrane Damage Using Gadolinium

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