BASIC SCIENCE Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1585 – 1596

Original Article

nanomedjournal.com

Nanoprobing the acidification process during intracellular uptake and trafficking Simone Lerch, PhD a, 1 , Sandra Ritz, PhD a, b, 1 , Karina Bley a, 1 , Claudia Messerschmidt, PhD a , Clemens K. Weiss, PhD a , c , Anna Musyanovych, PhD a , d , Katharina Landfester, PhD a , Volker Mailänder, PhD a , e ,⁎ a

Max-Planck-Institute for Polymer Research, Mainz, Germany Institute of Molecular Biology (IMB) gGmbH, Mainz, Germany c University of Applied Sciences Bingen, Bingen, Germany d Fraunhofer ICT-IMM, Mainz, Germany e 3rd Department of Medicine (Hematology, Oncology, and Pneumology), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany Received 21 January 2015; accepted 14 April 2015 b

Abstract Many nanoparticular drug delivery approaches rely on a detailed knowledge of the acidification process during intracellular trafficking of endocytosed nanoparticles (NPs). Therefore we produced a nanoparticular pH sensor composed of the fluorescent pH-sensitive dual wavelength dye carboxy seminaphthorhodafluor-1 (carboxy SNARF-1) coupled to the surface of amino-functionalized polystyrene NPs (SNARF-1-NP). By applying a calibration fit function to confocal laser scanning microscopy (CLSM) images, local pH values were determined. The acidification and ripening process of endo/lysosomal compartments containing nanoparticles was followed over time and was found to progress up to 6 h to reach an equilibrium pH distribution (maximum pH 5.2 [± 0.2]). The SNARF-1-NP localization in endo/lysosomal compartments was confirmed by transmission electron microscopy (TEM) and quantitative co-localization analysis with fluorescent endolysosomal marker Rab-proteins by confocal laser scanning microscopy (CLSM). The herein described nanoparticular pH-sensor is a versatile tool to monitor dynamic pH processes inside the endolysosomal compartments. From the Clinical Editor: In this interesting article, the authors elegantly designed a nanoparticular pH sensor with fluorescence probe with the capability to measure intracellular and intravesicular pH changes. The application of this method would enable the further understanding of nanoparticle uptake and intracellular physiology. © 2015 Elsevier Inc. All rights reserved. Key words: Nanoparticle; Intracellular trafficking; Acidification; pH responsiveness; Rab-family

For the last decade, polymeric nano-sensors have been developed as probes for real-time imaging and dynamic monitoring of various ions, such as H +, Ca 2+, Mg 2+, K +, Na +, and Cl −, which are important for cellular metabolism. 1-4 Nanoparticle (NP) based sensor systems have several advantages in comparison to highly invasive methods, like e.g. microelectrode probing or the use of

Conflict of interest: The authors declare that no conflict of interest exists. Support for research: This work was supported by the Deutsche Forschungsgemeinschaft (DFG SPP 1313: Biological Responses to Nanoscale Particles. LA1013/13-1; MA 3271/3-1 and the SFB1066: Nanodimensional polymeric Therapeutics for the Treatment of Tumours) and the Max Planck Graduate Center with the Johannes Gutenberg University Mainz (MPGC). ⁎ Corresponding author at: Max-Planck-Institute for Polymer Research, Mainz, Germany. E-mail address: [email protected] (V. Mailänder). 1 Authors contributed equally.

unconjugated fluorescent probes: i) due to their small size and inert material they are physically and chemically less invasive than macroscopic probes, ii) the local concentration and therefore signal strength of the chemical probe can easily be tuned due to the high surface-to-volume ratio 5 and iii) surface functionalization with targeting agents may guide them towards specific sites at or in cells. Surface properties as well as multiple functionalization, such as additional incorporation of a drug, 6 magnetic resonance imaging active inorganic labels 7 or antibodies, 8 e.g. as delivery targets, can be introduced without difficulty. NPs have emerged as promising tools to study mechanisms innate to cells such as endocytotic uptake machineries as well as to function as novel delivery systems for drug transport and for addressing specific cell or tissue types. Most NPs are taken up by cells via various endocytotic mechanisms and follow the endo/lysosomal pathway. 9 The pH of these compartments is lowered during the maturation of the vesicles – from early to late endosomes to lysosomes – to trigger the release of receptor-bound

http://dx.doi.org/10.1016/j.nano.2015.04.010 1549-9634/© 2015 Elsevier Inc. All rights reserved. Please cite this article as: Lerch S, et al, Nanoprobing the acidification process during intracellular uptake and trafficking. Nanomedicine: NBM 2015;11:1585-1596, http://dx.doi.org/10.1016/j.nano.2015.04.010

1586

S. Lerch et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1585–1596 10

ligands, and to digest debris or nutrients. This acidification can be used to trigger the degradation of biocompatible polymeric nanocapsules and to subsequently release incorporated markers or drugs. 11 Intracellular pH changes also occur in the development of tumor cells, e.g. the pH of some tumors is lowered due to an increased P-glycoprotein activity. 12 Since changes in local ion concentration, such as H +, play an important role in metabolic processes, ion transport, cell signaling and cell growth, 13 it is essential to have probes to distinguish between different pH values with a high spatial resolution. So far, knowledge on the accurate interactions of NPs with endosomal compartments and the time scales of NPs trafficking in cells is sparse. 14 Understanding the acidification kinetics in the endo/lysosomal system is important to tailor nanoscale systems for pH-triggered drug release. Currently, some studies attempt to combine a novel sensor-NP with an ability for pH-sensitive drug delivery and the quantitative monitoring of local NPs in endocytotic vesicles. 15-18 Here we present the synthesis of pH-sensitive NPs and their use as nano-sensors for intracellular and intravesicular pH monitoring. Amino-functionalized polystyrene NPs were synthesized by an emulsifier free emulsion copolymerization approach. 19,20 With this technique, additional functionalization 21 as well as the incorporation of markers and drugs 22 is well established. The dual-wavelength pH-sensitive dye carboxy SNARF-1 (seminaphthorhodafluor-1) was bound to the particle surface by a succinimidyl ester (NHS)-mediated coupling reaction. Using this strategy leads to NPs with an accessible probe dye on the particle surface. Ratiometric imaging of the protonated and deprotonated forms of SNARF-1 (emission maxima at λpr = 580 nm and λdep = 640 nm) enables that the readout of the NP pH-sensor is not sensitive to local dye concentration. However, the surface charge of NPs and distance between dye molecules on the surface could influence the measurable pH range, as has been shown. 23 Additionally, contact with the intracellular environment may influence the fluorescence properties of dye-conjugated NPs. 24 Therefore, the ratiometric signal of the dye-coupled NPs was carefully calibrated in cells. Based on the ratiometric pH quantification, a mode for the acidification process of SNARF-1-NPs after endocytosis was established and visualized as pseudo-colored confocal laser scanning microscopy (CLSM) images showing the pH distribution in different endosomal structures. It was also readily possible to distinguish between intra- and extracellularly located NPs. The localization of SNARF-1-NPs in different endo/lysosomal structures was mapped by transmission electron microscopy (TEM) imaging and colocalization studies with Rab proteins.

Materials and methods Materials Styrene (Merck, Darmstadt, Germany) was purified using a nitrogen pressured alumina flash column. All other chemicals were used without further purification: 2-aminoethyl methacrylate hydrochloride (AEMH, Sigma-Aldrich, St. Louis, USA; 90%), 2,2'-azobis(2-(2-imidazolin-2-yl)propane) dihydrochloride (VA-044, Wako Chemicals, Neuss, Germany), pH-sensitive dye 5´ (and 6´) carboxy-10-dimethylamino-3-hydroxy-spiro[7H-

benzo[c]xanthene-7,1´(3´H)-isobenzo-furan]-3´-one (carboxy SNARF-1), SNARF-1 acetate succinimidyl ester (SNARF-1-NHS), SNARF-1 acetoxymethyl ester acetate (SNARF AM ester; all Invitrogen, Karlsruhe, Germany), fluorescamine (Sigma Aldrich, St. Louis, USA; N 98%), sodium borate (Merck, Darmstadt, Germany) and hexylamine (Sigma Aldrich, St. Louis, USA; 99%). Demineralized and ultrapure water was used throughout the experiments. Synthesis of amino-functionalized polymeric NPs Positively charged amino-functionalized particles were synthesized using soap-free emulsion copolymerization in the presence of the co-monomer AEMH following the procedure of Ganachaud et al. 19 Prior to polymerization, 200 mL of ultrapure water was degassed with argon for 30 min under magnetic stirring in a three-necked round bottom flask equipped with a condenser and septa. 15 mL of styrene was added and stirred for 5 min before 682.5 mg (4.1.10 −3 mol) AEMH dissolved in 2 mL ultrapure water was added with a syringe. After additional 5 min 284.4 mg (8.8.10 −4 mol) of the initiator VA-044 dissolved in 3 mL ultrapure water was added. The polymerization proceeded under argon atmosphere and continuous stirring at 500 rpm for 24 h at 55 °C. After synthesis, the particle dispersion was dialyzed (molecular weight cut-off [MWCO] 14,000 g∙mol −1, Carl Roth, Karlsruhe, Germany) for 3 days under repeated exchange of demineralized water. It was further purified with demineralized water via repetitive centrifugation/redispersion at 22,000 min −1 for 45 min. Characterization of amino-functionalized polymeric NPs After purification, the colloids’ average particle size and size distribution were measured by angle-dependent dynamic light scattering (ALV/CGS3 compact goniometer system with a He/Ne laser [632.8 nm]) at 20 °C. The ζ-potential measurements were performed in KCl solution (1∙10 − 3 mol∙L − 1) using a Zeta Nanosizer (Malvern Instruments, UK). The amount of amine groups on the particle surface was determined with fluorescence titration following the procedure published by Ganachaud et al. 19 In brief, the calibration curve was plotted from hexylamine solutions of given concentrations in sodium borate buffer (pH = 9.5, 0.1 mol∙L −1) and freshly prepared fluorescamine solution in acetone (0.3 g∙L −1). 25 μL of the colloidal dispersions with a solid content of 1 weight% (wt.%) was added to 725 μL of borate buffer together with 250 μL of fluorescamine solution. After 30 s vigorous mixing, 100 μL of the dispersion was placed in a 96-well plate (Corning Incorporated 3603) and fluorescence emission of the fluorescamine at λem = 470 nm was followed by a Tecan Infinite M1000 Plate Reader (Tecan Group Ltd., Maennedorf, Switzerland) using an excitation wavelength of λex = 410 nm. The amount of amine groups was found to be 1.5.10 −5 mol∙mL −1 or 2.5 amine groups per nm 2. Coupling the pH-sensitive dye onto the particles The pH-sensitive dye SNARF-1-NHS was coupled onto amino-functionalized polystyrene colloids forming an amide bond between the amine group of the nanoparticle and the carboxylic acid function of the dye (Figure 1, A). 0.11 mg of SNARF-1-NHS dissolved in 440 μL DMSO (c = 0.25 mg∙mL −1) was added to a dispersion of amino-functionalized colloids with a

S. Lerch et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1585–1596

1587

Figure 1. Schematic representation of SNARF-1 coupling and function. (A) Coupling of pH sensitive dye carboxy SNARF-1-NHS onto amino functionalized nanoparticles and cleavage of protecting ester groups under acidic conditions or in the presence of esterases. (B) Switching between deprotonated (A −) and protonated (HA) form of the dye molecules coupled onto particles (SNARF-1-NPs) by shifting the pH value from basic to acidic. (C) SNARF-1-AM ester in the lactone form used for intracellular calibration.

solid content of 0.4 wt.% and stirred for 24 h. An excess of 3000× of SNARF-1-NHS with regard to the total amount of determined surface amine groups was used. After coupling and prior use for

further experiments, the colloids were purified by dialysis (MWCO 14,000 g∙mol −1, Carl Roth, Karlsruhe, Germany) under repetitive exchange of demineralized water for 3 days and then washed with

1588

S. Lerch et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1585–1596

ultrapure water via repetitive centrifugation/redispersion in the same amount of water at 22,000 min −1 for 45 min until no fluorescence intensity was detected in the supernatant. Fluorescence emission spectra of the free carboxy SNARF-1-dye (c = 1 μg∙mL −1) and SNARF-1-NPs (c = 100 μg∙mL −1) were recorded using an excitation wavelength of λex = 514 nm at different pH values to evaluate dye functionality after NP coupling with the M1000 plate reader (Tecan Group Ltd., Maennedorf, Switzerland). The measurements were performed in black clearbottom polystyrene 96-well plates (Corning Inc., New York, USA). Probing of nanoparticle pH environment in HeLa cells Cell culture HeLa cells were incubated in DMEM (Gibco) with 10% fetal calf serum (FCS, Gibco), 100 U·mL − 1 penicillin and 100 μg·mL − 1 streptomycin in a humidified incubator at 37 °C and 5% CO2. For CLSM, cells were seeded at a concentration of 1.5·10 4 cells per cm 2 and grown on 8-well cover glass Lab-Teks (Nunc, Langenselbold, Germany). Intracellular calibration with SNARF-1 AM ester and SNARF-1-NPs SNARF dyes are known to alter their fluorescence properties in the presence of cellular systems, 2 thus the pH-sensor system was calibrated in cells with SNARF-1 AM ester and SNARF-1-NPs. A pH calibration curve was obtained by loading cells with SNARF-1 AM ester or SNARF-1-NPs and probing buffers with different pH values (Table S1) in the extracellular environment. The intra- and extracellular pH was adjusted by applying a combination of ionophores (nigericin and valinomycin) as described in the following. Before imaging cells were allowed to grow for 24 h on 8-well cover glass Lab-Teks. SNARF-1 AM ester (0.5 μg), dissolved in 1 μl DMSO (0.5 g∙L − 1), was incubated with 10 μl of FCS for 5 min to improve the water solubility and loading efficiency. 36 Before loading the HeLa cells with SNARF-1 AM ester, the cells were washed with HBSS buffer (LifeTechnologies), then the mixture was added to the cells together with 500 μl HBSS buffer. The esterase induced cleavage of the dye was performed in a humidified incubator at 37 °C and 5% CO2 for 30 min. After dye loading, the HBSS buffer was replaced by a buffer with the desired pH value (Table S1). Alternatively, for calibration measurements with dye-NP conjugates, cells were loaded with 0.075 g∙L − 1 of SNARF-1-NP for 24 h, then washed and measured in 500 μl buffer with different pH values (Table S1). The pH equilibration was performed by adding the ionophores nigericin (5 μg) and valinomycin (2.5 μg), dissolved in 7.5 μL DMSO to the 500 μL HBSS buffer. To minimize the toxic effects to the cells, the ionophores were added to the cells immediately before the measurements and measured within 5 min. All buffers used are potassium-rich and carbonate-free to ensure good pH equilibration by K + ionophores and long-time pH stability in the presence of air, respectively. Table S1 shows the buffers used for intracellular calibration. Buffer chemicals are dissolved in 500 mL demin. H2O. After pH adjustment with 0.1 M HCl or NaOH, buffers are sterile filtered and stored at 4 °C. CLSM measurements were performed using a TCS SP5 (Leica), a 100 × oil plan apochromatic objective (1.4 numerical aperture), and a tunable argon laser with λex = 514 nm. Emission

range was set to λem = 550-610 nm (protonated form) and λem = 640-750 nm (deprotonated form). Signals were detected by photomultipliers with fixed gain values. For calibration, 5-10 images were collected per pH value and the mean intensity of the cytosol (SNARF-AM ester) or vesicular structures (SNARF-NPs) was quantified. It was taken care that the ratios of the fluorescence intensities of the protonated (HA) and deprotonated (A −) form of SNARF-1 are acquired with the same instrumental setup and settings in all experiments. To generate a calibration fit, the ratios of protonated and deprotonated fluorescence intensities were plotted in dependence to the associated pH value and fitted. The obtained polynomial fit (y = − 2.31 + 1.52x − 0.25x 2 + 0.01x 3) was used as an equation for calibration of CLSM ratio images. Probing of nanoparticle pH environment To determine the environmental conditions of NPs after cellular endocytosis, SNARF-1-NPs were incubated with HeLa cells for different time intervals with a concentration of 75 μg·mL − 1 and imaged, as described above, by CLSM (without the addition of ionophores). Image processing (pH calibration) Pseudo-colored images, which display the local pH value per pixel were created and quantified using the image processing software Volocity 6.1.2 (Perkin Elmer) employing the following steps. a) Creation of a ratio image (protonated/deprotonated form) and application of a lookup table (rainbow LUT, range 0.2-0.7) that displays pseudo-colored images. b) Selection of objects by thresholding round objects (N 0.02 μm 2; SNARF-1-NP) or whole cells (N 0.5 μm 2 SNARF-1-AM). Touching objects were separated. c) Measurement of mean intensity and standard deviation of the objects. The data were further processed in Excel and Origin. Co-localization analysis For the co-localization analysis of SNARF-1-NPs with fluorescent fusion-proteins of the Rab-family (endosomal markers), HeLa cells (1.5·10 5 cells per mL) were transfected in suspension with the help of FugeneHD (Roche) using a plasmid-DNA/Fugene HD ratio of 1 μg:3 μL in 100 μl serum free medium. After addition to cell culture medium, cells were seeded in 8-well cover glass Lab-Teks and cultivated for 48 h, washed twice with cell culture medium to remove left-overs of the transfection agent, and cultured again 24 h before adding the NPs. The fluorescence fusion proteins EGFP-Rab7wt (Addgene plasmid 12605) and EGFP-Rab9wt (Addgene plasmid 12663) were created by Richard E. Pagano. 25 EGFP-hRab5a and EGFP-hRab4a were a generous gift from Marino Zerial. 26 Image processing (co-localization) Object based co-localization analysis of NPs and endosomal structures was performed with a self-written Fiji/ImageJ plug-in (see Supporting Information). Briefly, CLSM images of the respective fluorescence channels (green: endosomal structure, red: NPs) were semi-automatically filtered; structures were

S. Lerch et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1585–1596

selected by a contrast based threshold and converted into binary masks (black and white) (Supporting Information Figure S2) displaying either the 'NPs' or 'endosomal structures'. The binary masks were multiplied with each other to create a result mask displaying only 'NP containing endosomal structures'. The three binary masks (NPs, endosomal structures, NP containing endosomal structures) were analyzed regarding number of objects and area. The percentage of co-localization is the area ratio of 'NP in endosomal structure' divided by 'NPs'. Transmission electron microscopy (TEM) To visualize SNARF-1-NPs at high resolution in their cellular environment, we used TEM on HeLa cells treated with 300 μg mL − 1 of SNARF-1-NPs for a 1 h and 3 h incubation period. Before treatment, cells were cultured for 48 h after seeding onto 3 mm Ø sapphire discs at a density of 30,000 cells·cm − 2 in a 24-well plate. At the end of the incubation period, cells were fixed by means of high pressure freezing using a Compact 01 HPF machine (Wohlwend GmbH, Switzerland). Subsequent freeze-substitution was conducted using a Leica EM AFS 2 device (Leica Microsystems, Germany). The substitution medium contained acetone p.a., 0.2% osmium tetroxide, 0.1% uranyl acetate and 5% water. The mixture was pre-cooled to − 90 °C before the samples were added. After freeze-substitution, the samples were washed twice with acetone p.a. and finally embedded into EPON 812 resin. Ultrathin sectioning of the embedded samples was performed using a Leica Ultracut UCT (Leica Microsystems, Germany) equipped with a diamond knife. Examination of thin sections was conducted using a FEI Tecnai F20 transmission electron microscope (FEI, USA) operated at an acceleration voltage of 200 kV. Bright field images were acquired using a Gatan US1000 slow scan CCD camera (Gatan Inc., USA).

Results Synthesis of pH-sensor nanoparticles Positively charged amino functionalized polystyrene nanoparticles were synthesized using the emulsifier free emulsion copolymerization approach. 19 The average particle diameter was determined by dynamic light scattering (d = 122 nm ± 8 nm) (see SI Figure S1). The amino-functionalization resulted in a positive ζ-potential of 45 ± 6 mV and the fluorescamine assay 19 measured an average of 2.5 amine groups per nm 2 or 1.5.10 −5 mol∙mL −1. Next, the amino-functionalized NPs were functionalized with the pH-sensitive fluorescent dual-wavelength dye carboxy SNARF-1-NHS, with a pKa of ~ 7.5. 24 The dye was coupled onto the NP surfaces by creating an amide bond between the carboxylic group of the dye and the amine group on the particle surface (SNARF-1-NP) as shown in the reaction scheme (Figure 1, A). The ζ-potential of the dye coupled SNARF-1-NPs decreased from 45 ± 6 mV to 19 ± 5 mV, which confirms the presence of less charged surface groups on the particle surface. The determination of remaining amine groups on the particle surface after coupling with SNARF-1-NHS by fluorescamine assay was not possible because the fluorescence emission of the

1589

fluorescamine at λem = 470 nm is absorbed by the SNARF-1 dye molecules (λex = 488 nm and λex = 514 nm) resulting in no detectable signal. The particle size after dye coupling was measured again with angle-dependent dynamic light scattering resulting in an average particle diameter increase of 2 nm. During CLSM imaging we did not observe noticeable aggregation either in cell culture medium or on the glass bottom slide. To ensure that the fluorescent properties of the dye are not altered by the coupling and washing procedures, the fluorescence spectra of free dye carboxy SNARF-1 and of SNARF-1-NPs were measured in buffers with different pH values (pH 4.5-pH 8.5, Table S1). Upon excitation, the protonated form in acidic environment emits light at a lower wavelength than the deprotonated form in basic environment (Figure 1, B). Thus the pH value can be calculated from the intensity ratio (HA/A-) of the two emission wavelengths of the protonated (HA-) and deprotonated (A-) form. Figure 2 (A and B) displays the pH dependent emission spectra and Figure 2 (C and D) shows the ratios of the protonated (HA) and deprotonated (A-) emission peaks obtained by integrating the spectra over a fixed wavelength. The ratios (HA/A-) of both free dye and dye-coupled nanoparticle conjugates are equal and linear in the range from pH 6.8 to pH 8.0. However, emission of the free dye differs from the NP-coupled dye for pH values below pH 6.8. This behavior has been reported previously by other groups. 23 Nevertheless we can conclude that carboxy SNARF-1 still preserved its pH responsiveness after the coupling process. Intracellular pH calibration with SNARF-1-AM ester and SNARF-1-NP An intracellular pH calibration was performed by CLSM cell imaging, because the fluorescent properties of carboxy SNARF-1 may be altered in the presence of proteins or other cellular components. 24 Therefore, the cells were loaded with SNARF-1-AM ester or SNARF-1-NPs and treated with buffers of different pH values (Table S1). The hydrophobic AM esters of the fluorescent dye SNARF-1 are typically non-fluorescent because the extent of the delocalization in the aromatic structure is reduced by esterifying the naphtol-OH (Figure 1, C). But the AM ester is able to cross the cell membrane. Once the molecule is inside the cytoplasm, the ester bond is cleaved by unspecific esterases. The polar free form of the dye is not able to cross the membrane barrier and trapped inside the cell. The extra- and intracellular pH values were equilibrated with the ionophores nigericin and valinomycin, 27-29 which permeabilize the cell membrane to H +/K +. However, it is reported that the intracellular pH does not fully adjust to the extracellular pH values in nigericin containing high K + saline buffer, but deviates by − 0.12 ± 0.02, 30 which must be considered as an additional calibration error. The intracellular pH was found to be stable after the addition of ionophores within approximately 100 s. To prevent the results to be influenced by severe toxic effects of the ionophores to the cells, which may lead to the cell membrane rupture and release of the trapped dye into the medium, measurements were performed within 5 min after the addition of ionophores and stabilization of the intracellular pH. A positive

1590

S. Lerch et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1585–1596

Figure 2. Calibration curves for free SNARF-1 dye and SNARF-1-NPs obtained by fluorescence spectroscopy. Fluorescence emission spectra of (A) carboxy SNARF-1 dye and (B) SNARF-1-NPs at different pH values, λex = 514 nm. (C, D) Ratios of emission integrals obtained from fluorescence spectroscopy of protonated (λem HA = 550-610 nm) and deprotonated (λem A- = 640-720 nm) emission range for the (C) SNARF-1 dye and (D) SNARF-1-NPs (λex = 514 nm).

control of dye-loaded cells provided an intracellular pH of approximately 7.2, which is consistent with the previously published values for HeLa cells. 31 The free SNARF-1-AM ester was found to be homogeneously distributed in the cytoplasm of the cells (Figure 3, A), whereas the SNARF-1-NPs were exclusively located in vesicular structures (Figure 3, B). The pH dependent emission ratios (HA/A-) of the free SNARF-1-AM ester and SNARF-1-NPs are plotted in Figure 3, C and D. The calibration values of SNARF-1-NPs were fitted by a polynomial fit (Figure 3, D), and the resulting equation was used to calibrate all further images. The particles were non-toxic for the cells as evaluated by a cell viability test (data not shown). Probing of intracellular nanoparticle pH environment The time-dependent nanoparticle localization in living cells and their normal pH environment was monitored by applying the calibration fit function to CLSM images acquired after different incubation times (Figure 4). Pseudo-colored CLSM images show the pH values of the particles in vesicular compartments of HeLa cells incubated with SNARF-1-NPs for 1, 2, 4, 6, 8 and 24 h (Figure 4, A). Only after 30 min the signal intensity was high enough for reliable detection. At first, few SNARF-1-NPs loaded compartments were observed (1 h); with increasing time, the number and size of vesicular compartments increased until

equilibrium is reached after 4-6 h. For early time points (1-2 h) 'blue spots' (pH ≥ 7.4) confirmed the location of the SNARF-1-NPs on the outside of the cell. Intracellular vesicular compartments with green, yellow and red pseudo-color have an acidic pH between pH 5.5 and 7, respectively, and are supposed to be endo/lysosomal structures. According to this, quantitative analysis of the pseudo-colored vesicular structures revealed a bimodal pH distribution with maxima around pH 6.8 and pH 6 for early time points (5.8 ± 0.2, 1-2 h), which progressed to an equilibrium of pH 5.2 ± 0.2 after 4-6 h (Figure 4, B and C). This pH equilibrium was constant for 48 h (time course of the experiment). The time course of the gradual acidification is plotted in Figure 4, C. Data represent the mean value of 10 images per time point comprising roughly 5 cells per image and all vesicular structures in these cells. Intracellular fate of the SNARF-1-NPs The intracellular vesicular localization of the SNARF-1-NPs as well as the time-dependent pH drop indicated a location in endosomal/lysosomal compartments. In order to confirm this, we performed localization studies by TEM and life-cell imaging with fluorescent endosomal marker proteins of the Rab-family. Electron micrographs visualized the SNARF-1-NPs uptake by c-shaped membrane ruffles typical for a macropinocytosis driven mechanism (Figure 5, A, Supporting Information Figure

S. Lerch et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1585–1596

1591

Figure 3. Intracellular calibrations for SNARF-AM and SNARF-1-NPs by fluorescence confocal laser scanning microscopy (CLSM) in HeLa cells. HeLa cells were loaded for 24 h with SNARF-1-NPs (0.075 g∙L −1) or for 30 min with SNARF-AM (0.5 g∙L −1), treated with buffers of different pH values and permeabilized with ionophores to equilibrate the extra- and intracellular pH values. (A) Representative pseudo-colored pH ratio images of the free SNARF-AM ester in the cytosol (bar scales 50 μm). (B) SNARF-1-NPs located in vesicular structures (bar scales 5 μm). (C, D) Intracellular ratios of emission integrals obtained from CLSM imaging of protonated (λem AH = 550-610 nm) and deprotonated (λem A- = 640-750 nm) emission range for the (C) SNARF-AM ester and for (D) SNARF-1-NP conjugates (λex = 514 nm). The polynomial fit function served as a calibration curve for intracellular measurements.

S3). No clathrin-dense structures were observed. After 1 h, single SNARF-1-NP or packages of ≤ 5 NPs surrounded by phospholipids, dark contrasted with osmium tetroxide, were localized in the cytoplasm close to the plasma membrane (Figure 5, B, Supporting Information Figure S4). Contemporaneously, NP packages (~ 5-10 NPs) were located in perinuclear regions (Figure 5, C). After 3 h, NPs were identified in endosomal/lysosomal vesicles and multivesicular bodies (MVB) densely filled with NPs (N 10 NPs) (Figure 5, D, Supporting Information S5). To confirm that the vesicular structures observed in electron micrographs belong to endosomal structures, we investigated the co-localization of internalized SNARF-1-NPs with GFP-labeled small Rab GTPases known to occur in distinct endosomal compartments. The Rab family of small GTPases is a major protein family regulating intracellular trafficking and fusion of endosomal

structures: Rab4a is present in early endosomes and regulates recycling to the plasma membrane. 31 Rab5a is a marker for vesicle trafficking from the plasma membrane to early endosomes and homotypic fusion of early endosomes. 32,33 Rab7 accompanies transport from early endosomes via multivesicular bodies to late endosomes/lysosomes. 34 Rab9 is a marker for cycling between late endosomes and trans-Golgi network. 35 Live cell imaging was performed with HeLa cells that were transiently transfected with the fluorescent Rab proteins 4a, 5a, 7 and 9, before supplying SNARF-1-NPs (75 μg·mL −1) for 1 h. Representative fluorescence images are shown in Figure 6, A (see also supporting info Figure S2). Quantitative image analysis revealed that after 1 h of incubation with nanoparticles, most of the SNARF-1-NPs are on the way from early to late endosomes and lysosomes indicated by co-localization with Rab7 (70% ± 9.5%) and Rab 9 (62% ± 18%) stained structures. Fewer particles are on the route from the plasma-membrane

1592

S. Lerch et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1585–1596

Figure 4. Time course of uptake and pH measurements with SNARF-1-NP by fluorescence live cell CLSM in HeLa cells. HeLa cells were loaded with SNARF-1-NPs and imaged after the indicated time points (λex = 514 nm, λem HA = 550-610 nm, λem A = 640-750 nm). (A) Representative pseudo-colored pH ratio images of SNARF-1-NPs located in vesicular compartments (bar scales 10 μm). (B) Representative pH distribution in intracellular vesicular compartments calculated from one ratio image (ca. 5 cells) per time point. (C) Time dependent pH measurements of SNARF-1-NPs uptake. Mean value and standard deviation calculated from pH mean values from at least 10 ratio images per time point applying the calibration fit function.

to early endosomes (co-localization with Rab5a [14% ± 3%]) or on the way backwards to the plasma membrane (co-localization with Rab4a [6% ± 1%]).

effects which are hard to control in living cells due to dynamic changes during cell uptake and division. As an 'internal standard' is present in such a dye, the knowledge about the absolute concentration is not necessary, as it is the case for single-wavelength pH-sensitive dyes (e.g. fluorescein).

Discussion Fluorescent particle reporters for intracellular sensing were so far mainly established for static and not for dynamic measurements. Here we described the covalent coupling of a fluorescent pH indicator onto the surface of polystyrene nanoparticles (d = 122 ± 8 nm, ζ-potential 19 ± 5 mV) for local endosomal pH measurements in living cells by fluorescence microscopy. SNARF-1 is a widely used, commercially available dual-wavelength emitter. The ratiometric imaging between protonated and deprotonated emission ranges of the dye prevents failures occurring from photo bleaching, or concentration dependent

pH dependent properties of SNARF-1-NPs The pH dependent emission of SNARF-1 was critically assessed after coupling to NP by creating pH calibration curves of SNARF-1-NPs in the range of pH 4.5 to pH 8.5 and comparing those to i) free SNARF-1 dye in buffer (fluorescence spectrometer) and ii) SNARF-1-AM ester in living cells (CLSM). Therefore, the intracellular pH was equilibrated with an adjusted extracellular pH by using ionophores as has been done for other applications of the free dye. 27,36 The intracellular calibration and the derived

S. Lerch et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1585–1596

1593

The dye molecules preserve their pH sensitive dual-wavelength emission after NP coupling. However, while the curve linearity of the integrated intensities is comparable between the free and NP-coupled dye in the range from pH 6.8 to pH 8.0, the intensities below pH 6.8 differ. Possible reasons could be the repulsion of H + ions from the positively charged particle surface lowering the local pH as has been shown in more detail by others groups. 23 Förster resonance energy transfer (FRET) may also occur between protonated and deprotonated SNARF-1 on the NP surface: due to immobilization of SNARF-1 on the NP surface, the average distance between dye molecules is in the nanometer scale (potentially down to 2.5 molecules per nm 2, as this is the density of reactive amine groups), which may allow energy transfer between protonated und unprotonated forms. Furthermore, the photophysical properties of SNARF-1 may be altered through the presence of cellular components. 24 All these effects are taken care by calibrating the fluorescence of the SNARF-1-NPs and performing the calibration in the cellular environment. Acidification scheme of pH sensor particles after cellular uptake Thirty to 60 min after uptake into cells, acidification of the NP environment was detected by quantifying the local pH with a maximal pH distribution of 5.8 ± 0.2, which is in a typical range for early endosomes (~ pH 6). 37,38 An acidification equilibrium with a pH maximum of 5.2 ± 0.2 was reached after 4-6 h, which is in the range of lysosomal pH. 39 The proposed acidification process of SNARF-1-NPs after their uptake into HeLa cells was correlated with the co-localization of Rab familiy members by CLSM and by TEM imaging (Figure 6, C). Before endocytosis, the majority of surface bound carboxy SNARF-1 was deprotonated (magenta stars). During maturation from early to late endosomes and lysosomes, the ratio of protonated (purple stars) to deprotonated (magenta stars) SNARF-1-NPs increased with the decrease of the pH, which is caused by the vacuolar type of H +-ATPases (V-ATPases). 40 The results demonstrate that the acidification process of the endosomal compartments can be monitored in a temporally and spatially resolved manner, which is an important information for further development of nanocarriers, which may use low pH for controlled release of payload. Localization of NPs after uptake into cells

Figure 5. Transmission electron microscopy (TEM) images of SNARF-1-NP uptake in HeLa cells. HeLa cells were loaded with SNARF-1-NPs (0.3 g∙L−1) and prepared for TEM imaging after 1 h and 3 h incubation time. MVBs (multivesicular bodies). Bar scales for 300 nm.

polynomial fit function allowed for detection of pH-dependent emission of SNARF-1-NPs in the range of pH 5.0 to pH 8.0. The fit function also enables automatic image acquisition and processing on a pixel to pixel level. As this detectable pH range lies well in the range of pH changes seen in both the extracellular milieu and endo/lysosomal compartments, SNARF-1-NPs were considered to be an ideal sensor for acidification of NPs after endocytotic uptake into cells.

TEM imaging supported the endo/lysosomal localization of SNARF-1-NPs by displaying a distinct phospholipid layer around the particles. Cluster of NP seen in the TEM images indicated that the fluorescence signals in the CLSM images occurred mostly from more than one NP. In addition, the number of particles in a cluster increased with the incubation time and with progression of particles from early endosomes to lysosomes and MVBs. This suggests a fusion of vesicular compartments containing NPs during the ripening process. The acidification time course of SNARF-1-NPs in HeLa cells was supported by co-localization studies with fluorescent endo/lysosomal Rab family proteins. After 1 h, the main fraction of SNARF-NPs was spread between early and late endosomes, MVBs and lysosomes, and only few were present in early or recycling endosomes exclusively stained by Rab4a. Similar trends

1594

S. Lerch et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1585–1596

Figure 6. Co-localization of SNARF-1-NPs with endo/lysosomal marker proteins inside HeLa cells (1 h). (A) Fluorescence live cell images of HeLa cells expressing fluorescent-labeled proteins of the Rab small GTPase family (green): EGFP-Rab4a (early and recycling endosomes), EGFP-Rab5a (early endosome), EGFP-Rab7 (early endosome, late endosome, MVB, lysosome), EGFP-Rab9 (late endosome/lysosome/trans Golgi-network). Cells were loaded with SNARF-1-NPs (red, 0.075 g∙L −1) and imaged after 1 h incubation. Scale bar measures10 μm. (B) Quantitative analysis of SNARF-1-NPs co-localizing with the indicated Rab proteins after 1 h uptake in HeLa cells. Object based co-localization is expressed as percentage area of NP overlapping with endosomal marker protein in relation to all SNARF-NPs. Mean value and standard deviation calculated from N = 5 images per time point (see supporting information S3). (C) Time dependent acidification scheme for positively charged polystyrene SNARF-1-NPs (122 ± 8 nm, ζ-potential 19 ± 5 mV) in HeLa cells as we measured it in our model system. The time dependent pH changes were correlated with the localization found by the co-localization analysis with the Rab proteins (CLSM) and TEM imaging. SNARF-1 (stars) coupled to amino-functionalized polystyrene NP (green sphere). With increasing time the deprotonated SNARF-1 (blue star) is gradually protonated (orange star).

for intracellular co-locations with Rab family members were recently observed by Sandin et al. 41 Nanoparticle uptake mechanisms are influenced by the physico-chemical properties of the NPs (charge, functionalization, size, material) and the protein corona, which is subsequently formed by biomolecules upon contact with biological fluids (cell culture medium, blood) as well as the targeted cell type. The direct interaction of NPs with the cell membrane is still a matter of debate and can be described either by an (i) active, energy-dependent endocytosis processes, like macropinocytosis, clathrin mediated endocytosis, caveolae-mediated endocytosis and

mechanisms independent of clathrin and caveolin, or by (ii) passive membrane translocation processes, like energy dependent formation of nanoscale membrane holes 42 or energy-independent membrane translocation. 43,44 There are excellent studies by Tiago dos Santos et al, 45 Dausend et al, 46 Rejman et al 47 or reviews 9 dissecting the uptake mechanisms of polystyrene NPs, either plain polystyrene NPs or with positive or negative surface functionalization. In the current study we used a positive charged amino-functionalized PS-NPs with a hydrodynamic diameter of 122 ± 8 nm, a ζ-potential of 19 ± 5 mV and HeLa cells as model system. These conditions

S. Lerch et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1585–1596

are very close to a previous study, where we showed that energy dependent uptake mechanism with an involvement of dynamin and F-actin leads to an uptake of PS-NPs (110-120 nm) in HeLa cells, either with amino-functionalized positive or with carboxyfunctionalized negative surface charge. 46 Macropinocytosis (Na +/K + pump blocking by EIPA treatment) has been described to be the main uptake mechanism for positively charged polystyrene NPs with a size of 113 nm. The role of lipid rafts could not unambiguously solved, because cholesterol reduction (filipin, methyl-β-cyclodextrine) does not inhibit NP uptake, whereas the inhibition of lipid raft-associated proteins (receptorassociated tyrosine specific protein kinase inhibited by genistein, folate receptor inhibited by indomethacin), showed an effect. 46 Taken together macropinocytosis was determined to be the main process of uptake and therefore all our studies here represent the acidification process for this route of uptake. Conclusion Nanoparticle-conjugated pH sensors are potential tools for monitoring local pH changes in endo/lysosomal compartments. Their application will improve the understanding of basic cell-physiological processes, like infection cycle of viruses or NP uptake and pH triggered drug release. Additionally, they may be applied for drug screening of pH affecting compounds like ionophores, or as diagnostic tools for pH affected diseases like cancer or Alzheimer. The presented concept of SNARF-1 coupled to aminofunctionalized NPs could be further developed towards a multifunctional sensor NP, e.g. by coupling additional ion sensors. The fluorescent spectrum of SNARF-1 can easily be distinguished from the Ca 2+ sensor fura-2 or the Na + sensor SBFI, facilitating simultaneous measurement of H +, Ca 2+ and Na + concentrations in cells. 48 This study increases the understanding of cellular trafficking of nanoscale particles and will provide a valuable tool for future mechanistic studies in the fields of cell biology and nanotechnology but may also be applied to other fields where pH is measured on a submicrometer scale. Acknowledgements We gratefully acknowledge Marino Zerial for the donation of EGFP-hRab5a and EGFP-hRab4a, as well as Richard E. Pagano for the supply of EGPF-Rab7wt and EGFP-Rab9wt to addgene. The authors thank Christoph Sieber for excellent TEM sample preparation. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.nano.2015.04.010.

References 1. Clark HA, Hoyer M, Parus S, Philbert MA, Kopelman R. Optochemical nanosensors and subcellular applications in living cells. Microchim Acta 1999;131:121-8.

1595

2. Kreft O, Javier AM, Sukhorukov GB, Parak WJ. Polymer microcapsules as mobile local pH-sensors. J Mater Chem 2007;17:4471. 3. Park EJ, Brasuel M, Behrend C, Philbert MA, Kopelman R. Ratiometric optical PEBBLE nanosensors for real-time magnesium ion concentrations inside viable cells. Anal Chem 2003;75:3784-91. 4. Almdal K, Sun HH, Poulsen AK, Arleth L, Jakobsen I, Gu H, et al. Fluorescent gel particles in the nanometer range for detection of metabolites in living cells. Polym Adv Technol 2006;17:790-3. 5. Koo Y-EL, Fan W, Hah H, Xu H, Orringer D, Ross B, et al. Photonic explorers based on multifunctional nanoplatforms for biosensing and photodynamic therapy. Appl Opt 2007;46:1924-30. 6. Singh R, Lillard JW. Nanoparticle-based targeted drug delivery. Exp Mol Pathol 2009;86:215-23. 7. Na H Bin, Song IC, Hyeon T. Inorganic nanoparticles for MRI contrast agents. Adv Mater 2009;21:2133-48. 8. Kumar S, Aaron J, Sokolov K. Directional conjugation of antibodies to nanoparticles for synthesis of multiplexed optical contrast agents with both delivery and targeting moieties. Nat Protoc 2008;3:314-20. 9. Iversen TG, Skotland T, Sandvig K. Endocytosis and intracellular transport of nanoparticles: present knowledge and need for future studies. Nano Today 2011;6:176-85. 10. Maeda K, Kato Y, Sugiyama Y. pH-dependent receptor/ligand dissociation as a determining factor for intracellular sorting of ligands for epidermal growth factor receptors in rat hepatocytes. J Control Release 2002;82:71-82. 11. Tannock IF, Rotin D. Acid pH in tumors and its potential for therapeutic exploitation. Cancer Res 1989;49:4373-84. 12. Keizer HG, Joenje H. Increased cytosolic pH in multidrug-resistant human lung tumor cells: effect of verapamil. J Natl Cancer Inst 1989;81:706-9. 13. Khramtsov VV. Biological imaging and spectroscopy of pH. Curr Org Chem 2005;9:909-23. 14. Benjaminsen RV, Sun H, Henriksen JR, Christensen NM, Almdal K, Andresen TL. Evaluating nanoparticle sensor design for intracellular pH measurements. ACS Nano 2011;5:5864-73. 15. Sokolova V, Kozlova D, Knuschke T, Buer J, Westendorf AM, Epple M. Mechanism of the uptake of cationic and anionic calcium phosphate nanoparticles by cells. Acta Biomater 2013;9:7527-35. 16. Peng J, He X, Wang K, Tan W, Wang Y, Liu Y. Noninvasive monitoring of intracellular pH change induced by drug stimulation using silica nanoparticle sensors. Anal Bioanal Chem 2007;388:645-54. 17. Pallaoro A, Braun GB, Reich NO, Moskovits M. Mapping local pH in live cells using encapsulated fluorescent SERS nanotags. Small 2010;6:618-22. 18. Chen KJ, Chiu YL, Chen YM, Ho YC, Sung HW. Intracellularly monitoring/imaging the release of doxorubicin from pH-responsive nanoparticles using Förster resonance energy transfer. Biomaterials 2011;32:2586-92. 19. Ganachaud F, Sauzedde F, Elaïssari A, Pichot C. Emulsifier-free emulsion copolymerization of styrene with two different amino-containing cationic monomers. I. Kinetic studies. J Appl Polym Sci 1997;65:2315-30. 20. Holzapfel V, Musyanovych A, Landfester K, Lorenz MR, Mailänder V. Preparation of fluorescent carboxyl and amino functionalized polystyrene particles by miniemulsion polymerization as markers for cells. Macromol Chem Phys 2005;206:2440-9. 21. Crespy D, Landfester K. Miniemulsion polymerization as a versatile tool for the synthesis of functionalized polymers. Beilstein J Org Chem 2010;6:1132-48. 22. Landfester K, Musyanovych A, Mailaender V. From polymeric particles to multifunctional nanocapsules for biomedical applications using the miniemulsion process. J Polym Sci A 2010;48:493-515. 23. Zhang F, Ali Z, Amin F, Feltz A, Oheim M, Parak WJ. Ion and pH sensing with colloidal nanoparticles: influence of surface charge on sensing and colloidal properties. ChemPhysChem 2010;11:730-5. 24. Owen CS, Carango P, Grammer S, Bobyock S, Leeper DB. pHdependent intracellular quenching of the indicator carboxy-SNARF-1. J Fluoresc 1992;2:75-80.

1596

S. Lerch et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1585–1596

25. Choudhury A, Dominguez M, Puri V, Sharma DK, Narita K, Wheatley CL, et al. Rab proteins mediate Golgi transport of caveola-internalized glycosphingolipids and correct lipid trafficking in Niemann–Pick C cells. J Clin Invest 2002;109:1541-50. 26. Zeigerer A, Gilleron J, Bogorad RL, Marsico G, Nonaka H, Seifert S, et al. Rab5 is necessary for the biogenesis of the endolysosomal system in vivo. Nature 2012;485:465-70. 27. Takahashi A, Camacho P, Lechleiter JD, Herman B. Measurement of intracellular calcium. Physiol Rev 1999;79:1089-125. 28. Seksek O, Bolard J. Nuclear pH gradient in mammalian cells revealed by laser microspectrofluorimetry. J Cell Sci 1996;109(1):257-62. 29. Reijngoud DJ, Tager JM. Effect of ionophores and temperature on intralysosomal pH. FEBS Lett 1975;54:76-9. 30. Nett W, Deitmer JW. Simultaneous measurements of intracellular pH in the leech giant glial cell using 2’,7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein and ion-sensitive microelectrodes. Biophys J 1996;71:394-402. 31. Tafani M, Cohn JA, Karpinich NO, Rothman RJ, Russo MA, Farber JL. Regulation of intracellular pH mediates Bax activation in HeLa cells treated with staurosporine or tumor necrosis factor-alpha. J Biol Chem 2002;277:49569-76. 32. Bucci C, Wandinger-Ness A, Lütcke A, Chiariello M, Bruni CB, Zerial M. Rab5a is a common component of the apical and basolateral endocytic machinery in polarized epithelial cells. Proc Natl Acad Sci U S A 1994;91:5061-5. 33. Gorvel JP, Chavrier P, Zerial M, Gruenberg J. rab5 controls early endosome fusion in vitro. Cell 1991;64:915-25. 34. Vonderheit A, Helenius A. Rab7 associates with early endosomes to mediate sorting and transport of Semliki forest virus to late endosomes. PLoS Biol 2005;3:1225-38. 35. Lombardi D, Soldati T, Riederer MA, Goda Y, Zerial M, Pfeffer SR. Rab9 functions in transport between late endosomes and the trans Golgi network. EMBO J 1993;12:677-82. 36. Thomas JA, Buchsbaum RN, Zimniak A, Racker E. Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry 1979;18:2210-8.

37. Rybak SL, Murphy RF. Primary cell cultures from murine kidney and heart differ in endosomal pH. J Cell Physiol 1998;176:216-22. 38. Cain CC, Sipe DM, Murphy RF. Regulation of endocytic pH by the Na +, K+-ATPase in living cells. Proc Natl Acad Sci U S A 1989;86:544-8. 39. Geisow MJ, Evans WH. pH in the endosome. Measurements during pinocytosis and receptor-mediated endocytosis. Exp Cell Res 1984;150:36-46. 40. Marshansky V, Futai M. The V-type H+−ATPase in vesicular trafficking: targeting, regulation and function. Curr Opin Cell Biol 2008;20:415-26. 41. Sandin P, Fitzpatrick LW, Simpson JC, Dawson KA. High-speed imaging of Rab family small GTPases reveals rare events in nanoparticle trafficking in living cells. ACS Nano 2012;6:1513-21. 42. Hong S, Hessler JA, Banaszak Holl MM, Leroueil P, Mecke A, Orr BG. Physical interactions of nanoparticles with biological membranes: the observation of nanoscale hole formation. J Chem Health Saf 2006;13:16-20. 43. Trabulo S, Cardoso AL, Mano M, de Lima MCP. Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery systems. Pharmaceuticals 2010;3:961-93. 44. Koren E, Torchilin VP. Cell-penetrating peptides: breaking through to the other side. Trends Mol Med 2012;18:385-93. 45. Dos Santos T, Varela J, Lynch I, Salvati A, Dawson KA. Quantitative assessment of the comparative nanoparticle-uptake efficiency of a range of cell lines. Small 2011;7:3341-9. 46. Dausend J, Musyanovych A, Dass M, Walther P, Schrezenmeier H, Landfester K, et al. Uptake mechanism of oppositely charged fluorescent nanoparticles in HeLa cells. Macromol Biosci 2008;8:1135-43. 47. Rejman J, Oberle V, Zuhorn IS, Hoekstra D. Size-dependent internalization of particles via the pathways of clathrin- and caveolaemediated endocytosis. Biochem J 2004;377:159-69. 48. Han J, Burgess K. Fluorescent indicators for intracellular pH. Chem Rev 2010;110:2709-28.

Nanoprobing the acidification process during intracellular uptake and trafficking.

Many nanoparticular drug delivery approaches rely on a detailed knowledge of the acidification process during intracellular trafficking of endocytosed...
2MB Sizes 0 Downloads 9 Views