Ratiometric Sensing and Imaging of Intracellular pH Using

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Ratiometric sensing and imaging of intracellular pH using polyethyleneimine-coated photon upconversion nanoprobes Tuomas Näreoja, Takahiro Deguchi, Simon Christ, Riikka Peltomaa, Neeraj Prabhakar, Elnaz Fazeli, Niina Perälä, Jessica M. Rosenholm, Riikka Arppe, Tero Soukka, and Michael Schaeferling Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03223 • Publication Date (Web): 15 Dec 2016 Downloaded from http://pubs.acs.org on December 18, 2016

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Analytical Chemistry

Ratiometric sensing and imaging of intracellular pH using polyethyleneiminecoated photon upconversion nanoprobes Tuomas Näreojaae+* Takahiro Deguchi,a+ Simon Christ,b Riikka Peltomaa,b Neeraj Prabhakar,c Elnaz Fazeli,a Niina Peräläb, Jessica M. Rosenholm,c Riikka Arppe,b Tero Soukka,b, Michael Schäferling*d a

Laboratory of Biophysics, Institute of Biomedicine and Medicity research laboratories, University of Turku, Tykistökatu 6A, 20520 Turku, Finland b Department of Biochemistry/Biotechnology, University of Turku, Tykistökatu 6A, FI-20520 Turku, Finland c Pharmaceutical Sciences Laboratory, Faculty of Science and Engineering, Åbo Akademi University, Tykistökatu 6A, 20520 Turku, Finland d BAM – Federal Institute of Materials Research and Testing, Division 1.10 Biophotonics, RichardWillstätter-Straße 11, 12489 Berlin, Germany e Department of Neurosciences, Karolinska Institutet, von Eulers väg 3, 17177 Stockholm, Sweden + Equal contribution *Corresponding authors email: [email protected], [email protected]; Keywords: upconverting nanoparticles, pH-measurement, polyethyleneimine

live

cell

imaging,

endocytosis,

Abstract Measurement of changes of pH at various intracellular compartments has potential to solve questions concerning the processing of endocytosed material, regulation of the acidification process and also, acidification of vesicles destined for exocytosis. To monitor these events the nanosized optical pH probes need to provide ratiometric signals in the optically transparent biological window, target to all relevant intracellular compartments, and to facilitate imaging at subcellular resolution without interference from biological matrix. To meet these criteria we sensitize the surface conjugated pH sensitive indicator via an upconversion process utilizing an energy transfer from the nanoparticle to the indicator. Live cells were imaged with a scanning confocal microscope equipped with a low energy 980 nm laser excitation, that facilitated high resolution and penetration depth into the specimen, and low phototoxicity needed for long-term imaging. Our upconversion nanoparticle resonance energy transfer based sensor with polyethylenimine-coating provides high colloidal stability, enhanced cellular uptake and distribution across cellular compartments. This distribution was modulated with membrane integrity perturbing treatment that resulted into total loss of lysosomal compartments and a dramatic pH shift of endosomal compartments. These nanoprobes are well suited for detection of pH changes in in vitro models with high biological background fluorescence and in in vivo applications, e.g. for the bioimaging of small animal models. Introduction Lanthanide-doped photon upconversion nanoparticles (UCNPs) exhibit many advantages compared to conventional Stokes-shifted luminescent probes such as organic dyes and quantum dots (QDs). Due to ACS Paragon Plus Environment


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the upconversion process, i.e. the conversion of NIR radiation into shorter wavelength light, the limitations of photobleaching, autofluorescence and low penetration depths into tissue shown by classical fluorescent probes absorbing in the UV/VIS range are avoided. This makes UCNPs particularly useful for applications in complex samples occurring in bioanalysis, biomedicine and imaging. The typical excitation wavelength of upconversion materials (around 800 to 980 nm corresponding to the lanthanide dopants) matches the biological window where the background absorption and scattering of biological material is low. Consequently, UCNPs have been utilized as luminescent probes in bioassays1 and sensor films2,3,4,5. Several improvements regarding size and surface modifications enabled applications to intracellular and in vivo imaging6,7,8. The major improvements included advances in synthesis9, control of size10 and surface modulation and modification via different coating techniques11. Several applications in diagnostic biomedical imaging have evolved, e.g. UCNPs were used to visualize tumors or vascularization in living animals.12,13 Recently, also more and more studies deal with the functionalization of UCNPs for chemical sensing14,15,16. Sensing of intracellular pH is of particular interest for the study of membrane dynamics and intracellular trafficking. Structure and function of biomolecules strongly depend on the concentration of protons in their environment. Cells are highly compartmentalized, with each compartment having a different pH, providing distinct conditions for optimal operation of each metabolic pathway17. Therefore, stringent control and maintenance of the right pH in every compartment is crucial to the survival of the organism. Many cells rely on endocytosis in signaling18, acquisition of nutrients and metabolites19, but membrane trafficking is involved also in production of secreted proteins20. Furthermore, proliferation and migration of cells is controlled by intracellular pH21. Endocytic pathways typically involve acidification of vesicle lumen as a part of processing of the cargo, and loss of pH regulation indicates a dysfunction such as cancer or apoptosis22,23. Endocytic pathways can be utilized in treatment of cancer19,24 and gene therapy25, but these approaches require release of cargo from the endosomes26. Pioneering work in the field of intracellular nanoprobes was done by the group of Kopelman et al. with the development of so called PEBBLEs (probes encapsulated by biologically localized embedding). They incorporated two fluorophores (one pH-sensitive and one reference dye) into a nanoparticular polyacrylamide matrix27. Since then a huge variety of different ratiometric referenced nanosized approaches has been realized including QDs28,29,30, dual-labeled31,32,33 and triplelabeled34 polymeric NPs, a DNA nanomachine35, nanogel36, carbon nanodots37 and core/shell silica particles38. The whole research area of fluorescent nanoprobes for intracellular pH-sensing was reviewed recently39,40. However, most of those attempts suffer from the general problem of short wavelength excitation and emission that do not overlap with the biological window. These properties generate interfering background (autofluorescence of biological matrix) and light scattering, and as a result penetration depths are rather low, or subcellular localization of the signal is lost. Additionally, most organic dyes lack the required photostability and are only applicable in a limited pH range (typically of 3 pH units). A promising upconversion pH nanoprobe was described by Arppe et al. using a resonance energy transfer from hexagonal NaYF4: Yb3+, Er3+ nanocrystals to the pH-sensitive fluorophore pHrodoTM Red41. The nanocrystals were coated with a thin shell of aminosilane with several nanometer layer thickness for coupling of the pHrodoTM Red NHS ester.

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For detailed characterization of membrane trafficking and cargo processing several intracellular compartments need to be examined simultaneously, and therefore, a pH probe should achieve an uptake rate of >10 particles per cell. Furthermore, colloidal stability of the probes at neutral pH needs to be high to facilitate quantal uptake and efficient utilization of all endocytosis mechanisms. Here we describe an improved pH nanoprobe based on NaYF4:Yb3+,Er3+ particles coated with polyethyleneimine (PEI) and covalently attached to pHrodoTM Red as the fluorescent molecular pH probe. The highly branched polymer PEI is a polycationic macromolecule that is well known for its ability to facilitate cellular uptake. It has primary, secondary and tertiary amino groups (in the ratio of 1:2:1) and the basic unit of the backbone consists of one nitrogen atom followed by two carbon atoms. Despite the fact that free PEI is reported to be cytotoxic, the effect is greatly reduced in the surfacebound state42. Cell viability was shown to be insensitive to PEI-coated UCNPs and even after two weeks no negative effects were observed43. Another feature of the branched PEI is the high number of positive charges at neutral pH which results in zeta potentials up to 50 mV in deionized water, but of course the zeta-potentials depend strongly on the ratio of particle to PEI and the pH44. Since a high surface charge is an important tool to prevent particles from aggregation and to enable cellular uptake it is desired to have zeta potentials of ≥ 30 mV on the particles surface. Therefore, UCNPs coated with PEI are a valuable tool for intracellular imaging44,45,26. We used branched PEI coating for the following purposes: (a) to generate high positive surface charge to enhance cellular uptake of the UCNP based probes, (b) to improve their colloidal stability to further increase uptake and to ensure individual probes are available for uptake, and (c) to provide a high number of reactive primary amino groups for the conjugation of the pH sensitive dye on the surface. The pH-sensitive pHrodoTM Red rhodamine dye, which has a broad dynamic range from pH 8 to pH 4 with a pKa around 6.5 was chosen as acceptor for the resonance energy transfer process and covalently attached to the surface of PEI-coated UCNPs. The absorption of the indicator perfectly matches the green upconversion luminescence (UCL) emission of the UCNP around 525 – 550nm and fluorescence emission centered at 590 nm is increased at lower pH values upon protonation. Using this upconversion resonance energy transfer (UC-RET) system, ratiometric pH determination is achieved by measuring the pH-dependent fluorescence from the indicator around 590 nm and the green emission of the UCNP around 550 nm as reference signal. Experimental Section Reagents Citric acid monohydrate, disodium phosphate (Na2HPO4 x 7H2O), dimethyl sulfoxide (DMSO), 2-(Nmorpholino)ethanesulfonic acid (MES), Trizma base (TRIS), 4-(2-hydroxyethyl)piperazine-1ethanesulfonic acid (HEPES), N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide (Sulfo-NHS), polyethylenimine (PEI, branched, average Mw 25,000) were purchased from Sigma-Aldrich (St. Louis, MO). Tween-20 was from Merck Millipore (Germany). The indicator dye pHrodoTM Red (M = 650 g mol-1, ε = 65 000 cm-1) was purchased from Molecular Probes (Carlsbad, CA). 100 K / 30 K Macrosep Advance centrifugal devices were from Pall Life Sciences (Omega, Ann Arbor, MI) and Thermo Scientific Nunc Maxisorp microtiter wells from Thermo Scientific (Waltham, MA). Synthesis of the UCNP-PEI-pHrodo™ Red-conjugate ACS Paragon Plus Environment


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Hexagonal NaYF4:Yb3+,Er3+ (XYb 0.17, XEr 0.03) nanocrystals were synthesized via high-temperature co-precipitation method in oleic acid and 1-octadecane46. Average size of the nanocrystals was determined by transmission electron microscopy (TEM) using a JEM-1400+ TEM with 80 kV (JEOL, Japan) and Formvar/Carbon film (300 mesh Cu) from Tecalemit Flow (Finland). Oleic acid ligands on the surface were removed by incubation with 0.1 M HCl (pH 1) overnight in rotation. After washing, particles were resuspended in citric acid (30 mg mL-1). Incubation in rotation for 2 h was followed by three washing steps. Finally, the particles were resuspended in MES (20 mM, pH 6.1). To activate carboxylic acid groups, 15 mg UCNPs (in 400 µL MES) were incubated with 25 mg EDC and 2.5 mg Sulfo-NHS (both in 50 µL MES) and then coupled to 7 mg PEI (in 50 µL MES) for 2 h in rotation. The PEI-coated particles were purified by centrifugation with a 30 K Macrosep Advance centrifugal device and dissolved in water three times. The indicator dye pHrodo™ Red was bound covalently to the amino groups of the PEI-coated UCNPs in the concentration of 67 nmol dye per mg UCNP. For this purpose, 2 mg PEI-UCNP in 250 µL NaHCO3 (100mM, pH 8.5) was mixed with 43.3 µL of pHrodo™ Red succinimidyl (NHS) ester in DMSO (5 µg µL-1) and incubated overnight in rotation at room temperature and protected from light. The UCNP-PEI-pHrodo™ Red-conjugate was washed twice with 500 µL of 10mM TRIS (pH 8.5, 0.1% Tween-20) using the 100 K Macrosep Advance centrifugal device. Finally, it was suspended in 500 µL of the same buffer and stored at 4 °C. The synthesis and of aminosilane-coated UCNP conjugates (UCNP-pHrodo™ Red) using tetramethylorthosilicate and (N-(3-trimethoxysilyl)-propyl)ethylene diamine for preparation of the silane layer and their characterization have been described previously41. Characterization of the pH-nanoprobe The pH-sensitive UCNP-PEI-pHrodo™ Red-conjugate was characterized regarding the yield of conjugation, spectroscopic properties, colloidal stability and zeta potential. The yield of conjugation was determined by measuring fluorescence intensity at 590 nm of a series of pHrodo™ Red standard solutions (25 nM to 45 nM) in citrate buffer (100mM, pH 3.0) upon excitation at 563 nm with a Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies, Santa Clara, CA). The UCNP-PEIpHrodo™ Red-conjugate was measured in two different dilutions and the amount of dye loaded to UCNPs was calculated from regression equation. The upconversion emission spectrum of the UCNP and the sensitized emission spectrum of the pHrodo™ Red dye were measured in phosphate/citrate-buffer (200 mM, pH 3.0, 5.0, 7.0) with Cary Eclipse Spectrophotometer upon IR-excitation with 980 nm laser diode (95mW). The emission spectra were recorded using 0.1 mg mL-1 of the UCNP-PEI-pHrodo™ Red-conjugate. The PMT-voltage was set to 1000 V. A total sample volume of 200 µL was measured in 10x1 quartz-cuvettes (Hellma, Müllheim, Germany). Colloidal stability was investigated by dynamic light scattering (DLS) with a Malvern Zetasizer Nano ZS (Malvern Instruments, UK), as well as zeta potentials. Particles were measured in concentrations of 0.03 mg mL-1 in HEPES buffer (25 mM, pH 4.0, 5.3, 7.2) and 0.02 mg mL-1 in phosphate/citrate-buffer (50 mM, pH 3.0, 5.0, 7.0). Samples were ultrasonicated with a Covaris Acoustic Ultrasonicator (Corvaris, Woburn, MA) for 5 min and measured immediately and 60 min after sonication. Calibration of UCNP-PEI-pHrodo™ Red UCNP-PEI-pHrodo™ Red-conjugate was diluted to 0.01 mg mL-1 in phosphate/citrate-buffer (200 ACS Paragon Plus Environment

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mM). The pH was measured using a SevenEasy pH-meter (Mettler Toledo, Columbus, OH) equipped with an InLab® 423 Combination pH Micro Electrode (Mettler Toledo). The sensitized emission was measured at 590 ± 17 nm (Chroma Technology Corp, Bellows Falls, VT) and reference emission was measured at 535 ± 10 nm with 10-fold attenuation (OD1 filter, Thorlabs, Newton, NJ) using a modified Plate Chameleon™ reader (Hidex Oy, Finland) equipped with a 980 nm laser diode (Roithner Lasertechnik, Austria)47. Samples in 0.01 mg mL-1 concentration were measured in volumes of 200 µL in 96 microwell plates. A second calibration was performed in Dulbecco’s Modified Eagle’s Medium (DMEM) (Life Technologies) with 10% fetal calf serum (FCS), 100 IU mL-1 penicillin, and 100 µg mL-1 streptomycin. The UCNP-pHrodo™ Red-conjugate was used in concentration of 0.01 mg mL-1 and pH was adjusted with glycine. Fluorescence imaging of cells MDA-MB-231 cells (Human breast adenocarcinoma) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 2mM L-glutamine, and 1% penicillin– streptomycin (v/v) in a 8 well glass chamber dish (Ibidi GmbH, Germany) to 50% confluence. The UCNP-PEI-pHrodo™ Red-conjugates were added in clear DMEM 2% FCS at particle concentration 10 µg mL-1 and incubated further at 37 °C for 2-16 hours. For in situ calibration of the pH nanoprobes, intracellular pH was adjusted to that of the extracellular cell culture medium. For this purpose, cells were treated with 20 µM nigericin (Calbiochem, #481990) at 37 °C for 30 min before imaging according to previously described protocol48. Cellular imaging was performed with a Leica TCS SP5 STED/MP laser scanning microscope (Leica Microsystems GmbH, Germany) with a Leica 63x/1.20 water objective and Spectra-Physics Mai Tai HP Ti:Sapphire NIR/IR laser (Newport, Santa Clara, CA) set to 980 nm. The laser intensity was calculated to 53 W mm-2. Optionally, the pHrhodoTM Red was directly excited with a 561 nm diode laser. A GaAs-hybrid detector (Leica) was used for the sensitized UC-RET signal and a standard PMT (Leica) for the reference upconversion signal. The pH response was analyzed by excitation at 980 nm and ratiometric dual wavelength detection using the 550 nm (525 ± 25 nm) emission of the UCNP as reference signal and sensitized emission of the pH indicator at 590 nm (585 ± 20 nm) as sensor response. Images were acquired at 10 Hz without averaging. For visualization the data was processed with 2 pixels mean filter. Quantitation of imaging data Intracellular circular regions of interest (ROI) encompassing a single observed puncta were assigned based on the reference UCNP-signal. Mean signal intensity of UCNP and UCNP-pHrodo FRET was measured using ImageJ (Fiji 2.0.0-rc-43/1.51g) and the paired values were used to calculate I590/I550 – ratios for the observations. These observations were then plotted in ascending order to reveal three different ratio ranges and two main slope changes. This data was the plotted into a probability density function by binning the observations into 0.01 unit bins and using a moving average of 0.03 units. On these curves a function with multiple (1-5) Gaussian peaks were fitted, a fit with lowest number of peaks was selected when it reached adjusted R-square > 0.9. Results and discussion Preparation and characterization UCNPs



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Hexagonal nanoparticles of NaYF4:Yb3+,Er3+ with an average diameter of 23 x 27nm (Figure S-1) were synthesized according to a previously described procedure46. The PEI-coating leads to a positive zeta potential on the nanocrystal surface. The zeta potential is raised from approximately -22 mV for aminosilane-coated UCNPs41 to 25 mV for PEI-coated UCNPs (in pH 7.2) due to more positively charged amino groups on the surface. A zeta potential of 37 mV for free PEI in aqueous solution was reported in literature43, but it strongly depends on the molecular weight and the degree of branching. The UCL spectra of the constructed nanoprobe were measured in different pHs to proof the efficiency of the energy transfer from UCNPs to the indicator dye (Figure 1). Sensitized emission of the rhodamine dye is observed around 590 nm, whereas upconversion emission around 660 nm remains constant. The sensitized acceptor emission shows a sufficient intensity when 67 nmol dye is coupled to 1 mg UCNP. It is clearly distinguishable from the UCL emission around 550 nm. When we measured the pH-dependent emission spectra of UCNP-PEI without the indicator dye the emission at 590 nm was absent (Figure S-3). Weaker UC-RET efficiencies were achieved for lower loadings and self-quenching for higher loadings of the fluorescent dye. The sensitized emission is higher in acidic conditions due to protonation of the rhodamine dye. Therefore, particles treated with 67 nmol pHrodo™ Red dye per mg UCNP were used for further experiments. A yield of 80 % was obtained for this reaction (54 nmol bound dye per mg UCNP). The amount of bound dye was calculated from its fluorescence intensity upon direct excitation at 560 nm and comparison with a standard dilution series of the free dye (see Supporting Information, Figure S-2). Accordingly, the amount of dye molecules on the particles surface was calculated to ~2800 molecules per particle. Compared to ~2400 molecules per particle in case of the aminosilane-coated particles of the same size41, this gives a significant increase of conjugated dyes. Since the same amount of indicator dye was used in both cases for conjugation, it can be assumed that the increased amount of bound dye is due to the higher amount of reactive amino groups provided by the PEI coating compared to the aminosilane-coating. Colloidal stability is an important factor if nanoparticles are applied in aqueous media. Therefore, a highly positive charged coating material such as PEI should support colloidal stability and prevent aggregation in acidic environment. We measured a zeta potential of 25 mV at pH 7.2 directly after ultrasonication for the dye-conjugated particles. The zeta potentials of the conjugate at different pHs are listed in Table 1. Decreasing zeta potentials occurring at lower pH values could indicate the adsorption of anions to the particle coating leading a to slight decrease in colloidal stability at acidic pH. The aggregation of particles is also indicated by a decrease of the zeta potentials measured one hour after ultrasonication. Generally, zeta potentials between -30 mV and +30 mV do not provide sufficient electrostatic stabilization of colloidal systems. Thus, these particles tend to aggregate quite fast in strongly acidic environment where the zeta potentials drop to < +10 mV (pH 4). This can be confirmed by DLS measurements. The Z-average size (intensity weighted distribution) of the conjugates was found to be 186 nm at pH 7.2. This value was relatively constant in time (Table S-1). The average particle size increased to 466 nm at pH 5.8 and 1158 nm at pH 4.0. At acidic pH, a strong additional growth of the particle size was observed one hour after sonication of the sample, with a dramatic increase also of the standard deviations. DLS data and zeta potentials of PEI coated particles and blank uncoated UCNPs are listed in Table S-2. Due to protonation of the free amino groups of PEI the zeta


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potentials become more positive with decreasing pH (16 mV at pH 3.6), which is accompanied by increasing colloidal stability. The comparison at pH 7.2 reveals the impact of the positively charged pHrodo dyes on the surface properties of the PEI coated particles. A zeta potential near zero of the PEI coated particles leads to a tendency to form aggregates. Table 1. Zeta potentials of the nanoprobe at different pHs. pH UCNP-PEI-pHrodo after ultrasonication UCNP-PEI-pHrodo 1 h after ultrasonication

4.0 7.8 mV 6.3 mV

5.8 ±0.6 15.3 ±0.5 mV ±0.6 14.2 ±0.6 mV

7.2 24.9 mV 20.7 mV

±1.4 ±0.8

The upconversion photoluminescence decays of the 550 nm emission of the PEI-conjugated UCNPs and of the UCNP-PEI pHrodo nanoprobes were measured to confirm the UC-RET mechanism. The emission was collected after a 2000 µs excitation pulse of 980 nm wavelength. The data was analyzed by fitting the obtained time-resolved signals with a biexponential decay function. The main component of the lifetime of the 550 nm emission of UCNP-PEI was 102.6 ± 0.5 µs before and 88.3 ± 0.5 µs after conjugation of the pHrodo dye (Figure S-4). This represents only a small decrease in luminescence lifetime of 14% which is lower than in case of the conjugate with the aminosilane-coated particles.41 However, the decrease in lifetime indicates that UC-RET is at least partly involved in the sensitization of the indicator dye. Other mechanisms such as photon re-absorption cannot be excluded, but much larger decrease in the apparent decay time of UCNP emission is not actually expected, since the energy storage and migration at multiple excited Yb3+ -ions within UCNP causes continuous re-excitation of Er3+ -ions and prolongs the apparent lifetime of their emission independent of the UC-RET. The second components were 627 µs (1.8 % relative amplitude) and 624 µs (2.5 % relative amplitude), respectively, and appear not to be involved in the UC-RET process. The measurements were carried out at pH 7.0. The lifetimes did not change in the range between pH 7.0 and pH 3.0. The results of the lifetime measurements imply that the main reason for an enhanced sensitized emission compared to the aminosilane coated particles is the higher loading of the pH-sensitive dye. A more efficient energy transfer between UCNP and indicator could not be observed in the lifetime measurements, which are comparable to uncoated and silica-coated particles41,50. Thus, it can be assumed that the strong upconversion luminescence quenching by water molecules51 is the limiting factor in both cases. Its elimination could significantly improve the intensity of the sensitized indicator emission of the pHnanoprobes, since the energy transfer to the surface-attached indicator is most efficient from the Er3+ ions near the surface of the UCNP that are also most affected by the quenching. Calibration of the pH-nanoprobe The pH-nanoprobe was calibrated in the range between pH 3.0 and 7.0 by measuring the sensitized emission of the pHrodo™ Red at 590 nm and the insensitive upconversion emission of the UCNP at 550 nm and calculating the ratio I590/I550 (Figure S-5). The green emission was attenuated 10-fold using an optical density filter (OD1) making the ratio more responsive to changes in the emission of the indicator. The sensor shows a linear ratiometric response in pure phosphate/citrate buffer. It is known that pH-indicators are affected by the ionic strength52. Therefore, a calibration in cell culture medium ACS Paragon Plus Environment


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was carried out to mimic the conditions inside cells (Figure 2). In this way, it is ensured that the nanoprobe is subject to similar interferences than in case of intracellular pH measurements. Although, PEI has an intrinsic buffer capacity particularly in saline solution45, a good linear fit was found in the range between pH 7 and pH 5, which represents the relevant region for intracellular measurements. The intracellular measurements correlate with the calibration data, but due to different amplification of signals between instruments the slope of calibration curve was lower41. The sensitivity of the pHnanoprobe is significantly increased using a PEI-coating instead of an aminosilane-coating (Figure S-6). Particularly, the ratiometric sensor response is much more sensitive in the near neutral pH range from pH 7.0 to 6.5, where the response of the aminosilane coated nanoprobes is very weak. Cellular uptake of PEI-coated pH nanoprobes Confocal scanning microscopy with 980 nm excitation was used to study cellular uptake and to test the applicability of pH-nanoprobes for in vitro applications. Signals of reference upconversion luminescence at 550 nm and sensitized emission of pHrodo™ Red at 590 nm were detected without any crosstalk. We studied the cellular uptake efficacy of the nanoprobes and determined to which type of microenvironment the UCNPs were targeted into. After 16 h, the UCNP-pHrodo™ Red nanoprobes were internalized by all observed cells when they were added in a concentration of 10 µg mL-1. No cytotoxic effects were observed for the PEI-coated UCNPs over a period of 24 h. As compared to the previous UCNP-construct using an aminosilane coating, cellular uptake was markedly improved (Figure 3a, Figure S-7). The improved uptake is likely due to high positive zeta potential of PEI-coated particles (Table 1) that adhere to negatively charged plasma membrane26. Furthermore, the high charge reduces aggregation of nanoprobes (Figure 3, Figure S-7). Within the cells some particle clusters were visible, and this is most likely due to intracellular compartmentalization in endosomes (Figure 3b)51. Previous research has provided evidence that PEI not only facilitates cellular uptake, but also promotes endosomal escape via the proton sponge mechanism that delays acidification of endosomal compartments53,54. We conducted ratiometric detection of the pH at 16 h after internalization and found that 5% of the nanoprobes resided in neutral microenvironment indicating an escape from endosomes, while majority of them were in acidified compartments (Figure 4 and Table 2). Hence, we conclude that the UCNP-PEI-pHrodo™ nanoprobes mainly remain in endosomes in which they are taken up or in ones that are further processed to late endosomes, multivesicular bodies or lysosomes. Furthermore, the acidification of these compartments appeared unperturbed, matching previous reports45. These properties facilitate tracking of endosomal cargo over long periods of time. The combination of high retention in endosomes and robust uptake due to PEI-coating is likely a result of the colloidal form and high density of the UCNP-material, and matches our previous observations51. Table 2. Intensity ratio 590/550 nm in different cellular compartments and corresponding pH ranges before and after treatment with 20 µM nigericin. The observed nanoprobes are divided into three categories cytoplasmic (7.2-7.5), endosomal (6.0-7.2) and lysosomal (< 6.0) based on the recorded ratios. Untreated cells Nigericin treated cells Number of events Percentage of Number of events Percentage of pH Ratio total population

7.2 – 7.5 6.0 – 7.2.

< 0.1 0.1 – 0.4

41 637

5% 77%


total population

12 119

9% 91%

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< 6.0

> 0.4

144

17%

0

0%

Intracellular distribution of pH-nanoprobes The superimposed image shows ratiometric signal of the pH-probes with proportionally stronger red signal at lower pHs (Figure 4). Green areas indicate a more alkaline environment (cytosol, pH 7.2-7.5), the white mildly acidic endosomal compartments (pH 6.0-7.2) either the ones they were originally endocytosed, ones merged into late endosomes or multivesicular bodies or ones newly synthetized in order to exocytose the nanoprobes, and magenta areas indicate more acidic compartments (lysosomes, 4.0-6.0) (Figure 4a-d). Signals from the insets in Figure 4d are shown magnified in Figure 4e. To control the pH some UCNPs were added to the wells right before imaging. These particles reside outside of the cells in the culture medium (DMEM) at pH 7.4 (Figure 4d,e). Three types of localization were detected, as visualized by distinct slopes when plotting ratiometric signal against observations in ascending order (Figure 4f). Populations of UCNP-probes detonated by these slopes can be defined from the calibration curve measured with a plate reader to approximately match the aforementioned groups (Table 2 and Figures 2, 4 and 5). The exact pH determination of an intracellular compartment from any calibration curve measured in buffer is not possible due to differences caused by gradual proton production and their absorption to PEI-coating versus buffer mediated pH change. This is because in endosomes and lysosomes material becomes incrementally protonated through proton pumping vacuolar ATPase (V-ATPase), and that does not stop at a given pH, but maintains a steady state with a counterion transporters (e.g. Cl– -channels and antiporters) that would also help to dissipate the positive charge induced by unidirectional proton transport54. The process of counterion movement may be an important element in organellar pH-regulatory mechanisms and account for the diversity of acidic compartments that are acidified by V-ATPase. The mechanisms of this pH regulation are poorly understood, especially at a quantitative level. Hence, with the small volumes in question, any probes measuring the pH also affect this steady state by absorbing protons. That is why any absolute value always depends on the cargo and does not serve to identify the vesicle type. In this case the pHnanoprobe overestimates the compartment pH, i.e. gives a higher ratio than predicted, especially in lysosomes. Hence, measuring a pH in one compartment does not mean that any biologically similar vesicle would have the same pH. Furthermore, we have previously shown small differences in the signal amplification between instruments41. The ratios were then plotted into a probability density function with 0.01 increments and Gaussian distributions were then fitted into the data (Figure 5). Gaussian distributions are fitted to the data and peak maxima were found at ratios of 0.03±0.04 (cytoplasmic) and 0.18±0.002 (endosomal) for nigericin treated, and 0.01±0.06 (cytoplasmic), 0.29±0.002 (endosomal) and 0.55±0.02 (lysosomal) for untreated cells. The main Gaussian distribution contained 77% of total observations, and the mildly acidified environment is characteristic for endosomes (Figures 4 and 5, Table 2). Moreover, the endosomal population can be perceived to contain multiple Gaussian subsets, however, these could not be resolved as the pH values are relatively close to one another, again indicative of the various intracellular endosomal compartments. Furthermore, a separate, clearly resolved distribution 17% is observed at higher ratios is likely localized in lysosomes. We presume that the slight variations from the calibration curve are due to the proton sponge –effect that is able to slightly buffer pH changes in small intracellular compartments that gradually produce protons through catalyzed ATP hydrolysis. To confirm that the high ratiometric signals are emanate



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from acidified compartments, we conducted a pharmacological control experiment where we added 20 µM nigericin to destabilize lysosomes and other acidic compartments (Figure 4d-e and 5). The nigericin treatment created small pores in intracellular compartments and disrupted the acidification of endosomal compartments and lysosomes. As expected, result of such treatment was the abolishment of the lysosomal peak in the probability density function and a shift of the endosomal peak to lower ratios and its redistribution to a narrow range (Figure 5 and Table 2). 3. Conclusion In summary, the PEI-coated nanoprobes are distributed across different cellular compartments (Figures 4 and 5) and serve as a convenient sensor to distinguish changes in membrane trafficking and as probes to study compartmentalization, compartment acidification and processing of endocytosed material. Observing the changes in distribution of pH-values across all intracellular compartments will provide a useful tool in studies on regulation of vesicle acidification. The UCNP-PEI-pHrodo™ nanoprobes provide high colloidal stability, enhanced cellular uptake and distribution across cellular compartments. From these compartments cytoplasmic, endosomal and lysosomal populations were distinguished. A majority of PEI-coated pH sensors remained in endosomes. Nigericin treatment resulted into total loss of lysosomal compartments and a dramatic pH change of endosomal compartments. Live cells were imaged with a confocal scanning microscope using 980 nm excitation wavelength that facilitates high resolution and penetration depth into the specimen, and low phototoxicity needed for long-term imaging. These probes are well suited for detection of pH changes in in vitro models with high biological background fluorescence and might be also utilizable to in vivo applications. Acknowledgements The EU CMST COST Action CM1403 “The European Upconversion Network” is gratefully acknowledged. M.S. thanks furthermore the DFG (Deutsche Forschungsgemeinschaft) for a Heisenberg-Fellowship and financial support by the research grant SCHA 1009/17-1. Academy of Finland (project #260599) (JMR) is acknowledged for financial contribution. Supporting information Figure S-1 shows the uniform size of uncoated up-converting NaYF4:Yb3+,Er3 nanoparticles in an electronmicrograph. Figure S-2 and the supplementary methods for quantification of pHrodo™ Red conjugation present the coating efficacy of the PEI-coated UCNPs. Figure S-3 shows the pH-dependent spectra of UCNP-PEIs comparable to Figure 1, but without the acceptor dye and no emission at 590 nm or emission reduction at 550 nm. Figure S-4 shows the reduced lifetime of the UCNPs donor fluorescence indicating energy transfer and the fitting parameters used to extract the results. Figure S-5 shows the pH calibration in citrate buffer. Figure S-6 presents a comparison of calibration curves in DMEM to an aminosilane-coated UCNP pH-probe41, and demonstrates high sensitivity of the developed pH-probe. Figure S-7 shows a comparison of uptake efficacy of the coating variants demonstrating the impact PEI-coating has in bioapplicability. Table S-1 presents pH-dependent colloidal stability of the nanoprobes, indicating that in normal growth medium conditions they resist aggregation. In Table S-2 we compare the pH-dependent polydispersity index and zeta-potential of uncoated and PEI-coated UCNPs demonstrating, with Table 1, the impact each component in the coating has for nanoprobe properties. This material is available free of charge via the Internet at ACS Paragon Plus Environment

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Figure captions Figure 1. Emission spectra of UCNP-PEI-pHrodo™ Red in different pHs; excitation = 980 nm. Insert: pH dependent sensitized emission at λmax = 590 nm. Figure 2. Ratiometric pH response of UCNP-PEI-pHrodo™ Red in cell culture medium (DMEM); excitation = 980 nm. Fitting parameters: slope = -0.37, intercept = 2.86, R2 = 0.98 Figure 3. Uptake and distribution of pH-nanoprobes in MDA-MB-231 cells. All healthy looking cells (A) had taken up multiple nanoprobes that were distributed across the cell (B). Nanoprobes were visualized by direct excitation of the pHrodo™ Red dye at 561 nm with a large field of view (A) and high magnification (B). Cell outline are visualized with transmission detector. Scale bar 50 µm. Figure 4. Ratiometric imaging of pH probes reveals their localization in three types of microenvironment. Panel (A) shows localization of UCNPs detected using 980 nm excitation, (B) sensitized UC-RET emission from pHrodo™ Red, (C) shows outlines of the cell in transmitted light and (D) shows an overlaid ratiometric image of pH-nanoprobes with different ratio depending on the localization. Scale bar 10 µm. The enlarged insets (E) show different ratios in extracellular (ctrl), small endosome, large endosome and lysosome. Brightness of insets is increased by 10 gray level units from the overlaid image (D) for better visibility. Plotting observed I590/I550 – ratios in ascending order (F) on logarithmic x-axis revealed three different slopes for untreated cells (black squares) and two slopes for nigericin treated cells. Switch of slope indicates a dramatic change in the nanoprobe microenvironment while a steady slope reflects a normal distribution of observations. Figure 5. In nigericin treated cells (blue solid curve) bimodal distribution of pH values (fitted Gaussian distributions orange dotted) is measured while lysosomal, cytoplasmic and multiple unresolved populations (fitted Gaussian distributions green dotted) of endosomal microenvironments are seen in untreated cells (red solid curve). For plotting ratios are grouped in 0.01 unit increments and noise is reduced by calculating a 0.03 unit moving average. Gaussian distributions are fitted to the data and peaks were found at ratios 0.03±0.04 and 0.18±0.002 for nigericin treated, and 0.01±0.06, 0.29±0.002 and 0.55±0.02 for untreated. Range is standard error of the fit.

For TOC only



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Figure 1. Emission spectra of UCNP-PEI-pHrodo™ Red in different pHs; excitation = 980 nm. Insert: pH dependent Figure 1 288x201mm (300 x 300 DPI)



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Figure 2. Ratiometric pH response of UCNP-PEI-pHrodo™ Red in cell culture medium (DMEM); excitation = 980 nm. Fitting parameters: slope = -0.37, intercept = 2.86, R2 = 0.98 Figure 2 284x199mm (300 x 300 DPI)


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Figure 3. Uptake and distribution of pH-nanoprobes in MDA-MB-231 cells. All healthy looking cells (A) had taken up multiple nanoprobes that were distributed across the cell (B). Nanoprobes were visualized by direct excitation of the pHrodo™ Red dye at 561 nm with a large field of view (A) and high magnification (B). Cell outline are visualized with transmission detector. Scale bar 50 µm. Figure 3 68x35mm (300 x 300 DPI)



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Figure 4. Ratiometric imaging of pH probes reveals their localization in three types of microenvironment. Panel A shows localization of UCNPs detected using 980 nm excitation, B sensitized UC-RET emission from pHrodo™ Red, C shows outlines of the cell in transmitted light and D shows an overlaid ratiometric image of pH-nanoprobes with different ratio depending on the localization. Scale bar 10 µm. The enlarged insets (E) show different ratios in extracellular (ctrl), small endosome, large endosome and lysosome. Brightness of insets is increased by 10 gray level units from the overlaid image (D) for better visibility. Plotting observed I590/I550 –ratios in ascending order (E) on logarithmic x-axis revealed three different slopes for untreated cells (black squares) and two slopes for nigericin treated cells. Switch of slope indicates a dramatic change in the nanoprobe microenvironment while a steady slope reflects a normal distribution of observations. Figure 4 181x302mm (300 x 300 DPI)


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Figure 5. In nigericin treated cells (blue solid curve) bimodal distribution of pH values (fitted Gaussian distributions orange dotted) is measured while lysosomal, cytoplasmic and multiple unresolved populations (fitted Gaussian distributions green dotted) of endosomal microenvironments are seen in untreated cells (red solid curve). For plotting ratios are grouped in 0.01 unit increments and noise is reduced by calculating a 0.03 unit moving average. Gaussian distributions are fitted to the data and peaks were found at ratios 0.03±0.04 and 0.18±0.002 for nigericin treated, and 0.01±0.06, 0.29±0.002 and 0.55±0.02 for untreated. Range is standard error of the fit. Figure 5 208x159mm (300 x 300 DPI)


Ratiometric Sensing and Imaging of Intracellular pH Using

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