Article pubs.acs.org/Biomac
Mitochondrial Routing of Glucose and Sucrose Polymers after Pinocytotic Uptake: Avenues for Drug Delivery Rafi Rashid,†,‡,§,◆ Sebastian Beyer,†,§,¶ Anna Blocki,†,§,∥,+ Catherine Le Visage,⊥ Dieter Trau,§,# Thorsten Wohland,‡,▽ and Michael Raghunath*,§,∥,○ †
NUS Graduate School for Integrative Sciences & Engineering, National University of Singapore, 117456 Singapore NUS Centre for Bioimaging Sciences (CBIS), National University of Singapore, 117557 Singapore § Department of Biomedical Engineering, Faculty of Engineering, National University of Singapore, 117575 Singapore ∥ NUS Tissue Engineering Programme (NUSTEP), Life Science Institute, National University of Singapore, 117510 Singapore ⊥ Inserm, U698, Cardiovascular Bio-Engineering Group, Paris 75654, France # Department of Chemical & Biomolecular Engineering, Faculty of Engineering, National University of Singapore, 117576 Singapore ▽ Departments of Biological Sciences & Chemistry, Faculty of Science, National University of Singapore, 117543 Singapore ○ Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 117597 Singapore ‡
S Supporting Information *
ABSTRACT: Mitochondria are key organelles organizing cellular metabolic flux. Therefore, a targeted drug delivery to mitochondria promises the advancement of medicine in fields that are associated with mitochondrial dysfunction. However, successful mitochondrial drug delivery is limited by complex transport steps across organelle membranes and fast drug efflux in cases of multidrug resistance. Strategies to deliver smallmolecular-weight drugs to mitochondria are very limited, while the use of complex polymeric carriers is limited by a lack of clinical feasibility. We show here that clinically established macromolecules such as a sucrose copolymer (Ficoll 70/400 kDa) and polyglucose (dextran 70/500 kDa) are micropinocytosed swiftly by mesenchymal stem cells and subsequently routed to mitochondria. The intracellular level of Ficoll appears to decrease over time, suggesting that it does not persist within cells. After coupling to polysucrose, the low-molecular-weight photodynamic drug Rose Bengal reached mitochondria and thus exhibited an increased destructive potential after laser excitation. These findings support new opportunities to deliver already clinically approved drugs to mitochondria.
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INTRODUCTION Targeted drug delivery to mitochondria promises the advancement of medicine in fields that are associated with mitochondrial dysfunction such as neurodegenerative diseases,1 cancer,2 cardiovascular diseases,3 diabetes,4 and potentially other metabolic diseases. Current strategies to achieve mitochondrial targeting of macromolecules are limited to a few small molecules such as triphenylphosphonium (TPP)5 and dequalinium6 as well as peptide targeting sequences derived from mitochondrial proteins.7 Peptides that target quantum dots to mitochondria have also been reported.8 Current approaches come with several limitations that prevent their clinical application. Often, materials and polymers with unknown short- and long-term effects on human physiology are used as carriers. In addition, covalently linking target sequences and moieties could lead to an accumulation and retention of the carrier material within cells, with unknown effects on cellular function. Materials that might undergo © 2014 American Chemical Society
degradation in lysosomal compartments might not decay in other cellular compartments such as mitochondria.9 Currently, the only piece of evidence for mitochondriotropism of a material without specific targeting is copolymer micelles made from poly(caprolactone)-b-poly(ethylene oxide) (PCL-b-PEO) block copolymer with tetramethylrhodamine-5carbonyl azide (TMRCA) covalently attached to the PCL end of the polymer. These micelles were shown to enter the mitochondria, Golgi apparatus, endoplasmic reticulum, and lysosomes.7 Astonishingly little is known about the intracellular fate of carbohydrate polymers, although they have been in clinical use for a long time as plasma expanders or for the assessment of physiological parameters such as kidney function,10 where their Received: February 17, 2014 Revised: May 3, 2014 Published: May 8, 2014 2119
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Dextran 70 kDa and dextran 500 kDa have hydrodynamic radii of 6.49 and 15.90 nm, respectively.18 Pullulan 200 kDa has a hydrodynamic radius of 11.1 nm.19) PVP−TRITC was prepared as follows: PVP 360 kDa was conjugated to TRITC via nitrene chemistry. The protocol involves the following steps: (1) TRITC dye was functionalized with ethylene diamine to introduce a free amine group. (2) PVP was treated with a photo-cross-linker called 5-azido-2-nitrobenzoic acid Nhydroxysuccinimide ester and exposed to ultraviolet light for up to 1 min. (3) Amine-functionalized TRITC dye was mixed with the PVP− photo-cross-linker conjugate in an equimolar ratio. (4) TRITCconjugated PVP360 was purified using a spin-column (MWCO = 100 000 kDa). Pullulan−RBITC was prepared as follows. One g of pullulan was dissolved in 9 mL of anhydrous DMSO, then mixed with 1 mL of RBITC in DMSO solution (100 mg RBITC in 1 mL of DMSO) after the addition of 20 mg of dibutyltin dilaurate. The reaction was performed at 95 °C for 2 h. For purification, the reaction solution was transferred to 35 mL of cold 100% ethanol in a 50 mL of Falcon tube, mixed, and centrifuged at 4000 rpm for 10 min at 4 °C. Ethanol supernatant containing free RBITC was removed, and the step was repeated until ethanol supernatant was clear (four to five times). After the last centrifugation, pullulan−RBITC was solubilized in 50 mL of distilled water, and the solution was transferred to a dialysis tubing (MWCO 12 000−14 000 kDa) and dialyzed at 4 °C for 3 days with a daily change of water. The purified solution was freeze-dried. Ficoll− RB conjugates were prepared as follows: Dry DMSO, ideally stored over molecular sieves, must be used for this protocol, and ambient measures to account for the hydrophilicity of CDI must be applied. The conjugation can be carried out using 2 mL of capped plastic tubes and pipettes with disposable tips. A conjugation ratio of 1 RB molecule per 50 monomer units of Ficoll polymer was assumed to be a reasonable drug/carrier ratio. The molecular weight of one Ficoll monomer unit was defined to be that of the condensation product between epichlorohydrin and sucrose. Batches of 50 mg Ficoll were used for each preparation and dissolved in 200 μL of dry DMSO. 40− 60 mg each of RB and CDI was weighed and separately dissolved in dry DMSO to yield a concentrated stock solution. Volumes that contained the equimolar amount of CDI and 10× excess of RB with respect to a conjugation ratio of 1 RB to 50 monomer units for 50 mg of Ficoll were mixed together and allowed to react for 20 min. The CDI-activated RB was then mixed with the Ficoll solution and allowed to react for 3 h at 70 °C. The reaction was stopped by the addition of a large excess of water into the 2 mL tube. Purification was performed by repeated dissolution and precipitation cycles of the Ficoll−RB conjugate using water and isopropanol in a ratio of 1:15 by volume until the isopropanol supernatant appeared to be visually free of nonbound RB. The Ficoll−RB precipitate was dried from isopropanol for storage. Cell Culture. hMSCs (Lonza) were seeded on eight-well Lab-Tek chamber slides with a borosilicate bottom (NUNC), at 10 000 cells per well in low-glucose Dulbecco’s modified Eagle medium (LGDMEM, 5.6 mM glucose, Gibco-Life Technologies) with 10% fetal bovine serum (FBS, Gibco-Life Technologies) and penicillin−streptomycin. Cells were incubated at 37 °C in 5% CO2. After 16 h, cells were separately incubated with fluorophore- or drug-tagged polymers for 30 min or 1 h in serum-free and antibiotic-free medium and then thoroughly washed three times with Hank’s balanced salt solution (HBSS). Then, phenol red-free and serum-free LGDMEM was added to the hMSCs in the Lab-Tek chamberslides. Cells were subsequently imaged with a confocal microscope. Confocal Imaging. Cells were viewed with a FV300 laser scanning confocal microscope (Olympus, Japan). The excitation beam from a 543 nm HeNe ion laser (MellesGriot, Singapore) was focused by a water immersion objective (60×, NA1.2, Olympus) into the fluorescent sample. For organelle colocalization studies, an additional 488 nm Ar laser (MellesGriot, Singapore) was used to excite fluorescent organelle labels. Images were acquired with the Olympus FV300 software. The laser power used was 25 μW for both the 488 and 543 nm laser lines. Polymer Washout Experiments. hMSCs were incubated with each TRITC-tagged polymer for 1 h, then were washed four times with
size would be an important parameter for measuring the glomerular filtration rate. We therefore investigated the intracellular routing of different sizes of sucrose copolymers (Ficoll), glucose polymers (dextran), and a maltotriose polymer (pullulan). Sucrose copolymer has been used under the trade name “Ficoll” (GE Healthcare Biosciences AB) as a cell fractionation agent for blood and bone marrow preparations (buffy coat) in a clinical setting for at least 30 years11 and was recently approved as a freezing media supplement for embryos.12 We have used Ficoll as a cell culture additive to exert macromolecular crowding effects that drive extracellular microenvironment formation.13 For the current study, we chose bone-marrow-derived human mesenchymal stem cells (hMSCs) because we had studied the extracellular effects of Ficoll on this cell type extensively and sought to investigate its intracellular effects. To understand the fate of Ficoll and dextran, we compared their uptake and routing with that of (1) polyvinylpyrrolidone (PVP), a polymer that once had been used in clinical practice as a plasma expander but was subsequently phased out due to its notorious long-term retention in bone marrow cells,14 and (2) pullulan, a polymer of the trisaccharide, maltotriose, which consists of three glucose subunits. Recently, the novel use for PVP as a macromolecular crowder for stem-cell proliferation and differentiation has been reported.15 The surprising observation that Ficoll is swiftly micropinocytosed and routed to mitochondria without the need for a targeting sequence prompted us to couple Ficoll with the low-molecular-weight drug Rose Bengal (RB), a fluorescent compound that is used for photodynamic therapy, which is not known to enter mitochondria.16 In addition to visualizing the intracellular routing of the Ficoll−RB conjugates and their effects on mitochondria, we also compared the cellular toxicity of the conjugates with that of the drug alone. It is envisioned that this study will open novel avenues for delivery, to the mitochondria, of clinically relevant formulations of established low-molecular-weight drugs, which can either be payload-coupled or complexed with polymers of sucrose or glucose.
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EXPERIMENTAL SECTION
Materials. Unlabeled Ficoll 70 and 400 were purchased from GE Healthcare (Uppsala, Sweden), and unlabeled pullulan 200 kDa was purchased from Hayashibara (Okayama, Japan). Unlabeled dextran 70, dextran 500, PVP 360 kDa, tetramethylrhodamineisothiocyanate (TRITC), rhodamine B isothiocyanate (RBITC), RB, carbonyldiimidazole (CDI), dibutyltin dilaurate, methylbetacyclodextrin (MBCD), chlorpromazine (CPZ), amiloride (Am), and monensin (Mon) were purchased from Sigma-Aldrich (St. Louis, MO). TRITCconjugated Ficoll 70, Ficoll 400, dextran 70, and dextran 500 were purchased from TdB Consultancy AB (Uppsala, Sweden). Dimethyl sulfoxide (DMSO) was purchased from Thermo Fisher Scientific (Singapore). Lysotracker Green, Mitotracker Green, ER Tracker Green, and NBD C6-ceramide were purchased from Invitrogen (Singapore). The CellTiter Aqueous One solution cell proliferation assay system was purchased from Promega (Singapore). Methods. Fluorescent Polymer Preparation and Ficoll−Drug Conjugation. As previously stated, we purchased TRITC−Ficoll 70, 400, dextran 70, and dextran 500 from TdB Consultancy, and their conjugation protocol is as follows.17 Dextran (MW = 70 000; 1 g) was dissolved in methyl sulfoxide (10 mL) containing a few drops of pyridine. Isothiocyanato-fluorescein (0.1 g) was added, followed by dibutyltin dilaurate (20 mg), and the mixture was heated for 2 h at 95 °C. After several precipitations in ethanol to remove free dye, the FITC−dextran was filtered off and dried in vacuo at 80 °C. (Ficoll 70 and Ficoll 400 have hydrodynamic radii of 4 and 8 nm, respectively.13c 2120
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Figure 1. Uptake of TRITC-tagged PVP, Ficoll 70 (Fc70), and Ficoll 400 (Fc400) into human mesenchymal stem cells and modulation of its uptake by pinocytosis inhibitors. (A) Cells were pulsed for 1 h with 1 μM of each polymer and monitored for 20 h. (B) Cells exposed for 1 h to pinocytosis inhibitors methyl-beta-cyclodextrin (MβCD) (10 mM), chlorpromazine (CPZ) (28 μM), amiloride (Am) (300 μM), and monensin (Mon) (10 μM) were incubated for a further 1 h with a mixture of each inhibitor and each TRITC-labeled polymer (scale bar = 20 μm). phenol- and serum-free LGDMEM. The supernatant from the fourth wash was retained and set aside as a baseline measurement at the 1 h time point. For the 5, 10, and 20 h time points, cells were incubated in phenol red-free and serum-free LGDMEM, and the supernatant removed for measurement at the corresponding time points. The fluorescence intensity of each sample was measured using a PheraStar fluorimeter (BMG Instruments, Offenburg, Germany). All measurements were performed in triplicate. Pinocytosis Inhibition Studies. hMSCs (Lonza) were preincubated for 1 h with 10 mM MBCD, 28 μM CPZ, 300 μM Am, and 10 μM Mon in serum-free LGDMEM. The cells were then incubated for a further 1 h with a mixture of each inhibitor and each TRITC-labeled polymer in serum-free LGDMEM. Organelle Colocalization Studies. hMSCs were incubated with each TRITC-labeled polymer for 1 h in phenol red-free and serum-free LGDMEM and were then colabeled with the following fluorescent organelle labels for a further 15 min: Lysotracker (50 nM) for lysosomes, Mitotracker (100 nM) for mitochondria, ER Tracker (1 μM) for the endoplasmic reticulum, and NBD C6-ceramide (1 μM) for the Golgi apparatus. Drug Uptake Studies. hMSCs were labeled with Mitotracker (100 nM), ER Tracker (1 μM), or NBD C6-ceramide (1 μM) for the Golgi apparatus for 15 min and then were incubated either with 1 μM of Ficoll 70−RB or with Ficoll 400−RB in phenol red-free and serumfree LGDMEM for 30 min or 1 h. Separately, hMSCs were labeled with Mitotracker (100 nM), ER Tracker (1 μM), or NBD C6-ceramide (1 μM) for the Golgi apparatus for 15 min and then were incubated with 1 μM of RB alone in phenol red-free and serum-free LGDMEM for 1 h. To determine the effect of Ficoll−RB conjugates on cell viability, hMSCs were treated for 30 min with Ficoll 70-RB, Ficoll 400-
RB, free RB, Ficoll 70, and Ficoll 400 and then illuminated by a 543 nm laser for 30 s to photoactivate the RB. (This illumination wavelength is close to the maximum excitation wavelength of RB at 535 nm.20) The dose of light per unit surface area that was used to achieve mitochondrial fragmentation as visualized by confocal imaging was 250 mW/cm2. After illumination, the drug solution was washed off and replaced with a solution of 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) to assess viability. Cell Viability Assay. The CellTiter Aqueous One solution cell proliferation assay kit was used to assess the effect of Ficoll−RB conjugates and free RB on the viability of hMSCs. The effect of nonRB-conjugated Ficoll molecules on the viability of hMSCs was also assessed. The CellTiter 96 Aqueous One solution reagent contains a novel tetrazolium compound (MTS) and an electron coupling reagent (phenazine ethosulfate (PES)). A working solution of the MTS solution was prepared by mixing one part of the stock MTS solution with five parts of phenol red-free Dulbecco’s modified Eagle medium (DMEM) supplemented with pyruvate and Glutamax. After a 45 min incubation, the absorbance of the formazan product released by the cells was read on a Magellan Tecan plate reader at a measurement wavelength of 490 nm and a reference wavelength of 630 nm.
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RESULTS Polymer Uptake into Cells. When hMSCs were exposed to TRITC-tagged PVP360, Ficoll 70, and Ficoll 400, uptake occurred swiftly within 1 h (Figure 1A). PVP360 showed a granular distribution whose fluorescence intensity persisted for 20 h with a peripheral redistribution of the observed pattern. 2121
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Figure 2. Tracking of intracellular routing of TRITC-tagged PVP, Ficoll 70 (Fc70), and Ficoll 400 (Fc400) in human mesenchymal stem cells. (A) Lysosomal Tracking: cells were incubated with polymer (red fluorescence) for 1 h and for a further 15 min with Lysotracker (green fluorescence). Superimposition of images and resulting yellow mix color indicate colocalization. (B) Mitochondrial tracking: Cells were incubated with polymer for 1 h and for a further 15 min with Mitotracker (green fluorescence). (C,D) Micropinocytosed TRITC-tagged PVP, Fc70−TRITC, and Fc400− TRITC into human mesenchymal stem cells do not enter the Golgi apparatus or the endoplasmic reticulum. Cells were incubated with each TRITClabeled polymer for 1 h and for a further 15 min with ER tracker and NBD C6-ceramide to selectively visualize the endoplasmic reticulum and Golgi apparatus, respectively (scale bar = 20 μm).
active for particles up to 100 nm in size. Methyl β-cyclodextrin (MβCD) is a cholesterol-extracting agent that interferes with caveolae-dependent pinocytosis. It completely inhibited uptake of all three polymers (Figure 1B). CPZ, an inhibitor of clathrindependent pinocytosis, greatly reduced the presence of all three polymers in vesicular structures but trapped Fc in the labyrinthlike compartments. Am, an inhibitor of clathrin-independent pinocytosis, attenuated uptake of polymers across the board but without a noticeable difference in intracellular distribution patterns. Mon, a carboxylic ionophore that disrupts receptormediated endocytosis,21 did not inhibit internalization as such but arrested polymer-laden vesicles in the periphery of the cells,
The granular pattern suggests localization to endosomal/ lysosomal compartments. The Ficolls, particularly Ficoll 400, showed an uptake into labyrinth-like compartments after 1 h. After 20 h, a transition to a granular pattern and a granular peripheral dispersed redistribution with receding fluorescence intensity could be observed. This phenomenon was most pronounced with Fc400 (Figure 1A). The uptake of Fc70 and Fc400 was also tracked during the first 15 min of incubation (Figure S1 in the Supporting Information). Polymer Uptake Occurs via Micropinocytosis. To characterize the cellular uptake mechanism, we employed a variety of inhibitors of micropinocytosis, an uptake mechanism 2122
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Figure 3. Tracking of intracellular distribution kinetics of Rose Bengal (RB) tagged to Ficoll 70 (Fc70) and Ficoll 400 (Fc400) in human mesenchymal stem cells (hMSCs). Superimposition of images and resulting yellow mix color indicate colocalization. White arrows point to mitochondrial fragmentation (scale bar = 20 μm).
preventing either their deeper penetration into the cytoplasm or their recycling toward the cell surface. Colocalization of Polymers with Organelles. To better define the subcellular distribution of Ficoll upon uptake, we employed organelle tracking dyes (Figure 2, Figure S2 in the Supporting Information). Both Ficolls show a clear regional colocalization with mitochondria after 1 h. PVP is taken up immediately into a subset of lysosomes and stays in this location for the duration of the experiment. Unexpectedly, Ficoll, particularly Fc400, moved to the mitochondria early and could still be seen at this subcellular location after 5 h. More lysosomal compartments had also become occupied at this 5 h time-point. A gradually increasing lysosomal presence peaked late at 20 h. Fc70 showed intermediate behavior; it was routed to mitochondria first but was mostly cleared from this compartment after 5 h with a clear shift toward lysosomes. After 20 h, Fc70 content was greatly reduced in hMSCs, with most of the lysosomes cleared from the polymer. As a control, we exposed hMSCs to equimolar amounts of free TRITC for 1 h. This resulted in a faint homogeneous fluorescence throughout whole cells without conspicuous sequestration into mitochondria or any other organelle (Figure S3 in the Supporting Information). Probing for other organelles revealed
no colocalization at any given time point for Ficoll (Figure 2). Traces of PVP appeared to be present in the Golgi apparatus after 1 h, which were absent after 5 h. When we investigated the uptake of dextran and pullulan, we found a fast mitochondrial routing for dextran 500 kDa with kinetics similar to Ficoll 400 kDa (Figures S3 and S4 in the Supporting Information). However, this was not observed for pullulan 200 kDa. Drug Delivery to Mitochondria Using a Polysucrose Vehicle. When free RB was added to hMSC cell culture, it could be seen throughout the cytoplasm but not at the mitochondria (Figure 3). RB is intrinsically fluorescent and in uncoupled form does not colocalize with mitochondria (first row) after 30 min, while Ficoll-conjugated RB reaches mitochondria. During the imaging period of 30 s with a confocal laser beam, no conspicuous cell damage and, in particular, no mitochondrial damage occurred. This changed dramatically when equimolar amounts of RB coupled to Ficoll were added to cell cultures. Bioimaging showed substantial fragmentation of mitochondria, indicating a mitochondriotropic effect of RB after Ficoll coupling (Figure 3). The strong fragmentation of the mitochondria that can be seen at 30 min becomes more extensive at 60 min for Ficoll−RB conjugates. Mitochondrial fragmentation is attributed to photodynamic 2123
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marrow-derived hMSCs. One of the major challenges of drug delivery is to ensure that the carrier is taken up by cells in sufficient amounts. We have overcome this first hurdle by using Ficoll as a carrier for the fluorophore TRITC. From our experiments, we can conclude that micropinocytosis is a major uptake mode for Ficoll and PVP. Because MβCD completely inhibited polymer uptake, the predominant mode of uptake appears to be caveolae-dependent pinocytosis. With the colocalization data in mind, it was interesting to note that CPZ, an inhibitor of clathrin-dependent pinocytosis, greatly reduced the presence of all three polymers (Fc400, Fc70, and PVP) in vesicular structures (Figure 1B). Because the TRITC fluorophore alone did not colocalize with any organelle, we can conclude that the TRITC signal visualized via confocal microscopy (and captured via fluorospectrometry, as shown in Figure 5B) was still attached to sugar moieties from Ficoll. We have observed that Fc70 is cleared more rapidly from mitochondria than Fc400 (Figure 2). The faster mitochondrial clearance rate for Fc70 is likely due to its smaller size (the hydrodynamic radii of Fc70 and Fc400 are 4 and 8 nm, respectively). After observing the colocalization of Ficoll with mitochondria, we decided to use it as a vehicle to deliver a drug molecule to the mitochondria. Mitochondrial drug delivery that includes classical drugs and drugs of the so-called next generation (DNA, RNA, proteins, etc.) holds great potential for treating diseases of metabolism in general and, in particular, for treating diabetes, cardiovascular disease, neurodegenerative disease, and cancer. In light of their role in regenerative medicine, their classical relationship with plasma expanders and cell fractionation agents, and our own experience with them in crowding experiments, we focused on bone-marrow-derived hMSCs as a model. Because all of our mitochondrial colocalization data with Fc−TRITC were obtained with hMSCs, we decided to use this cell type for the drug delivery experiments to ensure comparability with the Fc−TRITC data set. Recently, nanocarriers containing doxorubicin were shown to colocalize with mitochondria and cause cytotoxicity.22 However, no physical damage to the mitochondria was reported. To determine the effect of a drug on mitochondrial structure, we chose RB as an exemplary drug for its intrinsic fluorescence and photodynamic action. This photosensitizing drug has been reported to be an effective therapeutic agent against melanoma23 and, more recently, against sarcoma.24 Photosensitizing drugs work via photochemical reactions that produce toxic products, such as reactive oxygen species, which can bring about cell death.16 The cytotoxicity of illuminated photosensitizers in the presence of oxygen has been known for a long time.25 We have shown that RB conjugated to a carrier such as Ficoll is more effective at killing cells than free RB. The unconjugated RB does not cause any observable mitochondrial fragmentation at the concentration used for imaging. The results of the cell viability assay show that the EC50 of Fc400−RB for cell death is 16 times lower than for RB, while the EC50 of Fc70−RB is 5.5 times lower. The data suggest that Ficoll facilitates the transport of RB to the mitochondria, where it causes fragmentation of this organelle under laser illumination, thus resulting in cytotoxicity. In addition to demonstrating the cytotoxic effects of Ficoll−RB, we also show that the conjugate causes direct damage to the mitochondria. This discovery holds great promise for drug delivery. The cytotoxic effects are the result of photodynamic action and include membrane and DNA damage, interference with metabolism and death, and mutagenesis.26 These effects
radical formation upon excitation of RB with visible light at 543 nm. The yellowing of the superimposed images of disintegrating mitochondria suggested that Ficoll−RB is in close proximity to this organelle. Great care was taken to shield RB-treated cell cultures from ambient light. Light exposure occurred only during the brightfield identification of an area of interest and subsequent scanning of cell cultures with 488 nm (for Mitotracker) and 543 nm (for RB), respectively, for 30 s each, at a laser power of 25 μW. An aqueous solution of RB dye shows a maximum excitation wavelength at 535 nm,20 which means that the confocal scanning was close to the excitation maximum. The mitochondrial destruction was rapid and thorough. No damage could be seen to the endoplasmic reticulum or the Golgi apparatus (Figure S5 in the Supporting Information), thus demonstrating that the destructive effect of Ficoll−RB is specific to the mitochondria. The effect of different concentrations of Fc70−RB, Fc400− RB, and free RB on viability of hMSCs was tested using a cell proliferation assay kit, and the results are shown in Figure 4.
Figure 4. Effect of the phototoxicity of different concentrations of Ficoll−Rose Bengal conjugates and of free Rose Bengal on the viability of human mesenchymal stem cells (hMSCs), as determined by the MTS tetrazolium compound-based cell proliferation assay. The effective concentration at which 50% of cells are killed (EC50) for Ficoll 400−Rose Bengal is 4 μM.
Fc400−RB exerted the most damaging effect and yielded an effective concentration for 50% cell death (EC50) of 4 μM. The EC50 for Fc70−RB was 22 μM, and that for RB alone was 64 μM. Polymer Fate in Cells. We observed over a period of 72 h that PVP remained in lysosomal structures during the whole observation period with undiminished intensity (Figure 5A). In contrast, Ficoll 400 (Fc400) reached the lysosomal compartment after 24 h, but its fluorescent signal faded rapidly (Figure 5A). Both polymers, PVP and Fc400, had been labeled with the same fluorophore, and the samples were treated exactly the same way, which rules out experimental conditions, such as bleaching of the fluorophore, as an explanation. Fluorospectrometric capturing of fluorochrome released into the culture medium confirmed that PVP-associated fluorescence was constant but close to the lower detection threshold. In contrast, Ficoll-associated fluorescence in the medium was strong and increased with time, with a 10 h peak for Fc70 and a still increasing amount for Fc400 (Figure 5B).
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DISCUSSION We report here the unexpected observation that polymers of sucrose and glucose travel to the mitochondria of bone2124
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Figure 5. Tracking the fate of Fc400 and PVP in human mesenchymal stem cells over a 72 h period. (A) Cells were incubated with polymer (red fluorescence) for 1 h and for a further 15 min with Lysotracker (green fluorescence). Superimposition of images and resulting yellow mix color indicate colocalization. (B) Release of fluorochrome over 24 h is smallest with PVP. Cells were pulsed for 1 h with 1 μM of TRITC-tagged PVP, Fc70, and Fc400, respectively, and release of fluorescence into culture medium was monitored via fluorescent spectrometry from 5 to 20 h (scale bar = 20 μm).
with a photoactivated NHS ester and ethylenediamine as linker. The only bond that might possibly be subjected to cleavage under physiological conditions is a carbamate bond at the crosslinker side or a thiourea bond at the fluorophore side. Ficoll fluorophore conjugates were also produced using isothiocyanate-functional fluorophores that form carbamate bonds upon reaction with Ficoll. Therefore, there is no different conjugation chemistry that could account for the different release behavior. Of note, Ficoll can be autoclaved at neutral pH but hydrolyses spontaneously under acidic conditions. It is conceivable that after routing into the acidic environment of lysosomes, Ficoll starts to degrade into sucrose moieties. Work is currently in progress to determine the extent of degradation and whether the theoretically conceivable final degradation products (glucose, fructose, glycerol) are shunted into cellular metabolism or are released by the cells.
result from the generation of singlet oxygen and superoxide anion.27 As a result of our findings, the potential for delivering Fc−RB to the mitochondria of disease cells looks promising. Another challenge of drug delivery is ensuring that the carrier does not accumulate in cells and cause toxicity. Our follow-up work on the fate of the materials within cells and organisms indicates that Ficoll can be degraded and washed out of the cells. This is unlike PVP, which, according to previous observations, is retained in the body and causes subsequent failure of tissues after intravenous administration (so-called PVP storage disease).28 The retention of PVP in lysosomes that we have observed could be due to PVP being much less susceptible to degradation within the acidic lysosomal environment than Ficoll (information from the manufacturer29). This greater susceptibility to degradation within an acidic environment would result in the more rapid clearance of Ficoll. The isothiocyanate-functional fluorophore was conjugated to PVP 2125
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Present Addresses
Ficoll has been used worldwide and extensively as a cell fractionation agent for blood and bone-marrow preparations. Dextrans have enjoyed a long history of safe use, and this has allowed them to be employed as additives to food and chemicals and in pharmaceutical and cosmetics manufacturing.30 In addition, some investigations have been carried out to determine whether dextrans can be used for the targeted and sustained delivery of drugs, proteins, enzymes, and imaging agents.31 There is no account of adverse effects of Ficoll, and, in our hands, Ficoll supplementation of cell culture media has never shown toxicity. PVP was originally developed as a plasma expander, but PVP with a molecular weight greater than 20 000 cannot be excreted by the kidneys. It remains in the circulation and is phagocytosed and permanently stored in the reticular endothelial system14b and in bone marrow, causing PVP storage disease.28 Our experiments underline the diverging intracellular fates of PVP and Ficoll and explain their current clinical and approval status. Because the polymer chemistry of polysucrose is based on a relatively straightforward synthesis, it would be conceivable to use similar techniques as described in Ting et al.32 to build polysucrose-based materials that would be targeted to mitochondria, as shown here with TRITC and RB, both of which normally would not reach this compartment in their free form. Our data contribute to current international efforts toward developing biologically safe and targeted vehicles for drug delivery to mitochondria and point toward the possibility of using clinically established polymers for this purpose.
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R.R.: Singapore Centre on Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University SBS-01n-27, 60 Nanyang Drive, Singapore 637551. ¶ S.B.: Singapore MIT Alliance for Research and Technology (SMART) − BioSystems and Micromechanics (BioSym) Integrative Research Group, 1 CREATE Way, #04−13/14 Enterprise Wing, #B-10, Singapore 138602. + A.B.: Translational Molecular Imaging Group (TMIG), Singapore Bioimaging Consortium (SBIC), Biomedical Sciences Institute, 11 Biopolis Way, #02-02 Helios, Singapore 138667. Author Contributions
R.R., S.B., T.W., and M.R. conceived and designed the project. R.R. performed the bulk of the experimental work. S.B. prepared the PVP-TRITC, Fc70-RB, and Fc400-RB conjugates. C.L.V. provided the Pul200-RBITC conjugate. The data were compiled by R.R. and M.R. and were analyzed by all authors. All authors discussed the results and took part in producing the manuscript. Notes
The authors declare no competing financial interest.
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CONCLUSIONS Carbohydrate polymers with hydrodynamic radii in the nanometer range can be used to deliver drugs to the mitochondria. Polysucrose and polyglucose molecules have opened up new avenues of delivering small molecules such as drugs to the mitochondria. Our study brings us a step closer to achieving efficacious treatment of diseases caused by mitochondrial dysfunction.
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ASSOCIATED CONTENT
S Supporting Information *
Fluorescence intensity measurements by confocal microscopy of TRITC−Ficoll 70 and TRITC−Ficoll 400 uptake into hMSCs during the first 15 min of incubation. Separate green and red channel images for mitochondrial tracking of Fc70− TRITC and Fc400−TRITC within human mesenchymal stem cells. A mesenchymal stem cell that was incubated for 1 h with tetramethylrhodamine isothiocyanate (TRITC) dye alone and then incubated for a further 15 min with Mitotracker Green. Tracking of intracellular routing of TRITC-tagged dextran 70 and dextran 500 and RBITC-tagged pullulan 200 in human mesenchymal stem cells. Uptake of TRITC-tagged dextran 70 (Dex70) and dextran 500 (Dex500) and RBITC-tagged pullulan 200 (Pul200) into human mesenchymal stem cells. The Ficoll−Rose Bengal conjugates have no damaging effect on the endoplasmic reticulum and Golgi apparatus. This material is available free of charge via the Internet at http://pubs.acs.org.
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REFERENCES
(1) Beal, M. Mitochondrial dysfunction in neurodegenerative diseases. Biochim. Biophys. Acta 1998, 1366, 211−223. (2) Modica-Napolitano, J.; Singh, K. Mitochondrial dysfunction in cancer. Mitochondrion 2004, 4, 755−762. (3) Ballinger, S. Mitochondrial dysfunction in cardiovascular disease. Free Radical Biol. Med. 2005, 38, 1278−1295. (4) Lowell, B.; Shulman, G. Mitochondrial dysfunction and type 2 diabetes. Science 2005, 307, 384−387. (5) (a) Adlam, V.; Harrison, J.; Porteous, C.; James, A.; Smith, R.; Murphy, M.; Sammut, I. Targeting an antioxidant to mitochondria decreases cardiac ischemia-reperfusion injury. FASEB J. 2005, 19, 1088−1095. (b) Sheu, S.; Nauduri, D.; Anders, M. Targeting antioxidants to mitochondria: a new therapeutic direction. Biochim. Biophys. Acta 2006, 1762, 256−265. (c) Muratovska, A.; Lightowlers, R.; Taylor, R.; Turnbull, D.; Smith, R.; Wilce, J.; Martin, S.; Murphy, M. Targeting peptide nucleic acid (PNA) oligomers to mitochondria within cells by conjugation to lipophilic cations: implications for mitochondrial DNA replication, expression and disease. Nucleic Acids Res. 2001, 29, 1852−1863. (d) Theodossiou, T.; Sideratou, Z.; Tsiourvas, D.; Paleos, C. A novel mitotropic oligolysine nanocarrier: Targeted delivery of covalently bound D-Luciferin to cell mitochondria. Mitochondrion 2011, 11, 982−986. (e) Marrache, S.; Dhar, S. Engineering of blended nanoparticle platform for delivery of mitochondria-acting therapeutics. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 16288−16293. (6) (a) Weissig, V. From serendipity to mitochondria-targeted nanocarriers. Pharm. Res. 2011, 28, 2657−2668. (b) D’Souza, G.; Rammohan, R.; Cheng, S.; Torchilin, V.; Weissig, V. DQAsomemediated delivery of plasmid DNA toward mitochondria in living cells. J. Controlled Release 2003, 19, 189−197. (7) Breunig, M.; Bauer, S.; Goepferich, A. Polymers and nanoparticles: intelligent tools for intracellular targeting? Eur. J. Pharm. Biopharm. 2008, 68, 112−128. (8) (a) Hoshino, A.; Fujioka, K.; Oku, T.; Nakamura, S.; Suga, M.; Yamaguchi, Y.; Suzuki, K.; Yasuhara, M.; Yamamoto, K. Quantum dots targeted to the assigned organelle in living cells. Microbiol. Immunol. 2004, 48, 985−994. (b) Delehanty, J.; Blanco-Canosa, J.; Bradburne, C.; Susumu, K.; Stewart, M.; Prasuhn, D.; Dawson, P.; Medintz, I. Sitespecific cellular delivery of quantum dots with chemoselectivelyassembled modular peptides. Chem. Commun. (Cambridge, U.K.) 2013, 49, 7878−7880.
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(9) Albanese, A.; Tang, P.; Chan, W. The effect of nanoparticle size, shape, and surface chemistry on biological systems. Annu. Rev. Biomed. Eng. 2012, 14, 1−16. (10) Venturoli, D.; Rippe, B. Ficoll and dextran vs. globular proteins as probes for testing glomerular permselectivity: effects of molecular size, shape, charge, and deformability. Am. J. Physiol.: Renal, Fluid Electrolyte Physiol. 2005, 288, F605−F613. (11) Harada, M.; Aoyagi, N.; Kondo, K.; Ishino, C.; Odaka, K.; Matsue, K.; Yoshida, T.; Hattori, K. Allogeneic bone marrow transplantation from a major ABO-incompatible donor. Infusion of hemopoietic stem cells isolated by Ficoll-Hypaque method. Transplantation 1983, 35, 191−192. (12) File # K092107. www.fda.gov (accessed April 9, 2014). (13) (a) Lareu, R. R.; Subramhanya, K. H.; Peng, Y.; Benny, P.; Chen, C.; Wang, Z.; Rajagopalan, R.; Raghunath, M. Collagen matrix deposition is dramatically enhanced in vitro when crowded with charged macromolecules: the biological relevance of the excluded volume effect. FEBS Lett. 2007, 581 (14), 2709−2714. (b) Lareu, R. R.; Arsianti, I.; Subramhanya, H. K.; Yanxian, P.; Raghunath, M. In vitro enhancement of collagen matrix formation and crosslinking for applications in tissue engineering: a preliminary study. Tissue Eng. 2007, 13 (2), 385−391. (c) Chen, C.; Loe, F.; Blocki, A.; Peng, Y.; Raghunath, M. Applying macromolecular crowding to enhance extracellular matrix deposition and its remodeling in vitro for tissue engineering and cell-based therapies. Adv. Drug Delivery Rev. 2011, 63 (4−5), 277−290. (d) Zeiger, A.; Loe, F.; Li, R.; Raghunath, M.; van Vliet, K. Macromolecular crowding directs extracellular matrix organization and mesenchymal stem cell behavior. PLoS One 2012, 7, e37904. (e) Ang, X.; Lee, M.; Blocki, A.; Chen, C.; Ong, L.; Asada, H.; Sheppard, A.; Raghunath, M. Macromolecular crowding amplifies adipogenesis of human bone marrow-derived MSCs by enhancing the pro-adipogenic environment. Tissue Eng., Part A 2014, 20 (5−6), 966−981. (14) (a) Dunn, P.; Kuo, T.; Shih, L. Y.; Wang, P. N.; Sun, C. F.; Chang, M. J. Bone marrow failure and myelofibrosis in a case of PVP storage disease. Am. J. Hematol. 1998, 57, 68−71. (b) Huang, W.; Chang, C.; Tsai, C. Polyvinylpyrrolidone storage disease presenting as pathologic fracture and anemia: report of a case with imprint cytology. Diagn. Cytopathol. 2012, 40, 69−72. (15) Rashid, R.; Lim, N.; Chee, S.; Png, S.; Wohland, T.; Raghunath, M. Novel use for polyvinylpyrrolidone as a macromolecular crowder for enhanced extracellular matrix deposition and cell proliferation. Tissue Eng., Part C 2014, DOI: 0.1089/ten.tec.2013.0733. (16) Panzarini, E.; Inguscio, V.; Dini, L. Timing the multiple cell death pathways initiated by Rose Bengal acetate photodynamic therapy. Cell Death Dis. 2011, 2, e169. (17) de Belder, A.; Granath, K. Preparation and properties of fluorescein-labelled dextrans. Carbohydr. Res. 1973, 30, 375−378. (18) Armstrong, J.; Wenby, R.; Meiselman, H.; Fisher, T. The hydrodynamic radii of macromolecules and their effect on red blood cell aggregation. Biophys. J. 2004, 87, 4259−4270. (19) Nishinari, K.; Kohyama, K.; Williams, P.; Phillips, G.; Burchard, W.; Ogina, K. Solution properties of pullulan. Macromolecules 1991, 24, 5590−5593. (20) Kapoor, R.; Jain, M.; Mishra, V. Fluorescence and absorption spectra of Rose Bengal dye in the presence of surfactants. J. Lumin. 1981, 22, 429−439. (21) (a) Thirion, J.; Wattiaux, R. Effect of monensin on fluid phase and receptor mediated endocytosis by rat hepatocyte monolayers. Biochem. Biophys. Res. Commun. 1988, 152, 1275−1281. (b) Chen, X.; Wang, Z. Regulation of epidermal growth factor receptor endocytosis by wortmannin through activation of Rab5 rather than inhibition of phosphatidylinositol 3-kinase. EMBO Rep. 2001, 2, 842−849. (22) Theodossiou, T.; Sideratou, Z.; Katsarou, M.; Tsiourvas, D. Mitochondria delivery of doxorubicin by triphenylphosphoniumfunctionalized hyperbranched nanocarriers results in rapid and severe cytotoxicity. Pharm. Res. 2013, 30, 2832−2842.
(23) Thompson, J.; Hersey, P.; Wachter, E. Chemoablation of metastatic melanoma using intralesional Rose Bengal. Melanoma Res. 2008, 18, 405−411. (24) Tan, C.; Neuhaus, S. Novel use of Rose Bengal (PV-10) in two cases of refractory scalp sarcoma. Aust. N. Z. J. Surg. 2013, 83, 93. (25) Martin, J. J.; Burch, P. Production of oxygen radicals by photosensitization. Methods Enzymol. 1990, 186, 635−645. (26) Halliwell, B.; Gutteridge, J. Free Radicals in Biology and Medicine; Clarendon: Oxford, U.K., 1985. (27) Cunningham, M.; Krinksy, N.; Giovanazzi, S.; Peak, M. J. Superoxide anion is generated from cellular metabolites by solar radiation and its components. Free Radical Biol. Med. 1985, 1, 381− 385. (28) Maeda, K.; Iseki, S.; Urushibata, I.; Uesugi, Y.; Sato, S. 2 cases of PVP storage disease. Naika 1966, 17, 159−166. (29) Ficoll PM 70, Ficoll PM 400; GE Healthcare Bio-Sciences and Amersham Biosciences AB: Uppsala, Sweden, 2007; pp 1−6. (30) Kato, I. Fragrance J. 2005, 33, 59−64. (31) Mehvar, R. Dextrans for targeted and sustained delivery of therapeutic and imaging agents. J. Controlled Release 2000, 69, 1−25. (32) Ting, S. R.; Gregory, A. M.; Stenzel, M. H. Polygalactose containing nanocages: the RAFT process for the synthesis of hollow sugar balls. Biomacromolecules 2009, 10, 342−352.
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