Cell-Penetrating Conjugates of Coproporphyrins ... - ACS Publications

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Cell-Penetrating Conjugates of Coproporphyrins with Oligoarginine Peptides: Rational Design and Application for Sensing Intracellular O2 Ruslan I. Dmitriev,† Honorata M. Ropiak,† Gelii V. Ponomarev,‡ Dmitri V. Yashunsky,‡ and Dmitri B. Papkovsky*,† †

Biochemistry Department, University College Cork, Cavanagh Pharmacy Building, Cork, Ireland Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, Pogodinskaia Ul. 10/2, 119992 Moscow, Russia

‡

S Supporting Information *

ABSTRACT: A panel of phosphorescent oligoarginine conjugates of tetracarboxylic Pt(II)-coproporphyrin I dye (PtCP), monosubstituted with long peptides or tetrasubstituted with short peptides and having different linkers and peripheral groups, is described. Their photophysical properties, cell loading efficiency, and mechanisms of transport into the cell were investigated and compared. The conjugates were seen to rely on endocytotic mechanisms of cell entry, which are different from that of the unconjugated oligoarginine peptide, and show diverse patterns of intracellular distribution. On the basis of this study, the tetra-substituted PtCP conjugate displaying whole cell distribution was selected for the sensing of intracellular O 2. This probe has been tested in biological experiments on a fluorescence plate reader, including the monitoring of in situ oxygenation of respiring cells and their responses to metabolic stimulation. Similar conjugates of the phosphorescent Pd(II)-coprorphyrin and fluorescent coproporphyrin-ketone were also synthesized and assessed for the sensing of low levels intracellular O 2 and ratiometric pH-sensing, respectively. The results produced and the structure−activity relationships determined can facilitate the rational design of new bioconjugates of porphyrin dyes tailored to specific applications.

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In quenched-phosphorescence sensing of molecular oxygen (O2), where Pt(II)- and Pd(II)-porphyrins are commonly used,15 there is a need to have both extracellular, cellimpermeable probes for imaging tissue and vasculature,12,16 and intracellular probes targeted to subcellular compartments, particularly the mitochondria where most of the O2 gets consumed.17 However, hydrophobic tetrapyrrols show poor selectivity of intracellular accumulation and distribution, limited solubility in aqueous media, and high nonspecific binding to various biomolecular and cellular structures. Cationic porphyrins often possess cyto- and genotoxicity (via interaction with the DNA), and therefore, anionic dyes such as coproporphyrin and substituted tetraphenylporphyrin derivatives are more frequently used. However, these structures have low intrinsic ability to penetrate the plasma membrane of mammalian cells.4,18 Aiming to improve intracellular delivery and distribution of the porphyrins, several approaches have been proposed including functionalization with hydrophobic polyethyleneglycols19 or sugars,20 preparation of dendrimers and nanoparticles, or conjugation with other fluorophores such as rhod-

INTRODUCTION Targeted delivery of molecules into the cell is required for many research (probes and effectors), diagnostic (imaging agents), and therapeutic (drugs) purposes. The main strategies to achieve this include direct (passive) translocation across the plasma membrane, facilitated transport through receptor or drug-mediated endocytosis, and the use of liposomes, delivery, and targeting vectors.1−3 To date, significant progress has been achieved with nucleic acid and protein structures,2 whereas the delivery of small molecules such as drugs, fluorophores, and other macrocyclic structures still require a better understanding of the mechanisms of intracellular transport and structure− activity relationships.4−7 Tetrapyrrols represented by numerous natural (porphyrins, heme, and vitamin B12) and synthetic compounds are actively used as drugs (e.g., SOD mimetics8), photosensitizers in tumor photodynamic therapy (PDT),9−11 oxygen-sensitive probes,9,12 and labels.13 For some of these applications, targeted delivery of the tetrapyrrol into the cell, tissue, or a particular subcellular compartment is an essential requirement. Thus, in PDT, the initial focus on the improvement of photophysical properties of drug candidates and distribution between normal and malignant tissue is now shifting toward more specific delivery of sensitizer molecules to a particular location within the cells (e.g., mitochondria rather than lysosomes11,14). © 2011 American Chemical Society

Received: June 21, 2011 Revised: October 24, 2011 Published: October 31, 2011 2507

dx.doi.org/10.1021/bc200324q | Bioconjugate Chem. 2011, 22, 2507−2518

Bioconjugate Chemistry

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Table 1. Description of Conjugates Prepared in This Studya porphyrin dye PtCPTE-MI PtCPTE-PEG850-MI PtCPTE-PEG850-MI PtCPTE-MI PtCP(NHCH2CH2OH)3-PFP PtCP(PFP)4 PdCP(PFP)4 CPK(PFP)4 PtCP(PFP)4 PtCP(PFP)4 PtCP(PFP)4 PtCP(PFP)4 PtCP(PFP)4 a

peptide sequence and conjugation site (underlined)

name of the conjugate

molecular weight

total number of R/ charge

RP-HPLC retention time (min)

CFRRRRRRRRR CFRRRRRRRRR MGRTVVVLGGGISGLAAGCGRRRRRRRRR MGRTVVVLGGGISGLAAGCGRRRRRRRRR NH2-GRRRRRRRRR

PEPP1 PEPP2 PEPP4 PEPP5 PEPP6

2830 3700 5200 4350 2497

9/8 9/8 10/9 10/9 9/8

12 12 15 16 10.5

NH2-RR-amide NH2-RR-amide NH2-RR-amide NH2-RRA-amide NH2-RRS-amide NH2-RGR-amide NH2-RRR NH2-Arg-OMe

PEPP3 PEPP3B PEPP3C T1 T2 T3 T4 T5

2093 2004 1916 2378 2442 2322 2722 1529

8/8 8/8 8/8 8/8 8/8 8/8 12/8 4/4

11 N/D N/D N/D N/D N/D N/D 11.5

Structures of PtCP-conjugates are presented in Figure 1, and initial porphyrin dyes are shown in Supporting Information.

Figure 1. Structures of the PtCP conjugates. All conjugates have the same coproporphyrin core but different side groups. The position of conjugation with the peptide is designated by R. A, conjugates PEPP1 and PEPP5; B,: conjugates PEPP2 and PEPP4; C, conjugate PEPP6; and D, branched conjugates PEPP3 and T1-T5.

amine,16,21−25 the use of escort peptide sequences (such as cyclic RGD),26 or cell-penetrating peptides.19,27 Thus, mono-

substituted conjugates of porphyrins with Arg- and Pro-rich peptides have shown improved cell penetration, intracellular 2508



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accumulation, and retention of the key functionality in oxygen sensing applications or PDT.28−31 However, this strategy requires a more detailed knowledge of structure−activity relationships. Here, we describe a panel of new cell-penetrating conjugates of Pt-coproporphyrin I (PtCP) with oligoarginine peptides. Considering the cell penetrating behavior of oligoarginine peptide vectors32 and the initial studies with monosubstituted PtCP conjugates with relatively long peptides (9−11 amino acids long),28−31 we hypothesized that tetra-substituted (branched) PtCP conjugates with short Arg-rich peptides may also possess cell-penetrating ability. Therefore, we synthesized an extended panel of both mono- and tetrasubstituted PtCP conjugates and evaluated them comparatively to establish structure−activity relationships. We demonstrate that depending on the substitution, linkers, peripheral groups, and hydrophilicity, the resulting conjugates can show different cell penetrating properties and intracellular distribution while retaining the important functional properties of the porphyrin moiety. These results provide the basis for the development of new porphyrin structures with controlled cell permeation and distribution for a range of biological applications.

Discovery DSC-18 20 mL/5 g column (Supelco-Sigma), washed with MeOH/H2O (1:1), and eluted with MeOH/ H2O/CH3COOH (50:48:2). The conjugate fractions were vacuum-dried and reconstituted in water or DMSO. The yield was typically ∼90% with respect to the porphyrin. Purity of the conjugates was confirmed by RP-HPLC (see Supporting Information). The composition, chemical structure, and abbreviations of the conjugates are given in Table 1 and Figure 1. Spectral Measurements. Absorption spectra were recorded on a 8453 UV−vis diode-array spectrophotometer (Agilent) and luminescence spectra on a LS50B luminescence spectrometer (PerkinElmer). Phosphorescence quantum yields were measured in PBS, containing 10% FBS (21% O2) or with additional 5 mg/mL KH2PO4 and 5 mg/mL Na2SO3 (0% O2) using PtCP as a reference.33 Phosphorescence lifetimes of the conjugates were measured on a Cary Eclipse spectrometer (Varian-Agilent) using phosphorescence decay application and single-exponential fit. The pH-sensitive fluorescence of CPK and PEPP3C probes was measured at 0.25 μM concentration in a citrate−phosphate buffer system at pH 3−8 using 406 nm excitation wavelength with fluorescence emission collected at 600−700 nm. For measurement of the fluorescence of free base, the probes were dissolved in DMSO. Cell Culture. Human colon carcinoma HCT116, rat pheochromocytoma PC12, human epithelial carcinoma HeLa, human neuroblastoma SH-SY5Y, human hepatocellular liver carcinoma HepG2, and murine embryonic fibroblasts MEF cells were from ATCC (Manassas, VA, USA). The cells were cultured in standard tissue culture flasks, collagen IV-coated 96well microplates, 35 mm Petri dishes or collagen-poly-D-lysinecoated glass bottom dishes (MatTek, Ashland, MA), and 12well chambers (Ibidi, Martinsried, Germany) using DMEM medium supplemented with 10% FBS (SH-SY5Y, HepG2, MEF, and HeLa cells) or McCoy 5A with 10% FBS (HCT116). PC12 cells were grown and differentiated as described previously.29 For the assessment of cellular uptake efficiency, cells were grown to a confluence 75−100%, incubated with 1 μM of the conjugate for 16 h, washed three times with medium, and measured on the TR-F plate reader. For the analysis by flow cytometry, cells were grown on 35 mm Petri dishes, similarly stained with conjugates, washed with PBS, trypsinized, and then analyzed. Mechanisms of Cellular Uptake Studies. MEF and HCT116 cells were grown on glass-bottom dishes to a confluence 30−50% and incubated with the conjugates (5 μM PEPP3 or 2 μM PEPP4) for 6 h at 37 °C, then washed, and analyzed by fluorescence microscopy. Inhibitors of endocytosis were used as described previously28 at concentrations 50 μM (EIPA, inhibits macropinocytosis), 5 mM (MBCD, inhibits lipid-raft dependent endocytosis), and 10 μg/mL (CPZ, inhibits clathrin-mediated endocytosis) with 30 min of preincubation. The influence of surface proteoglycans on cellular uptake was assessed by 1 h of preincubation and subsequent incubation with the conjugate in the presence of 50 μg/mL heparin sulfate (Sigma H6279). To see the effect of competition with unlabeled oligoarginine, cells were incubated with the probe in the presence of 10 μM GR9 peptide. Temperature effect was studied by incubating the cells for 30 min at 4 °C and then with the conjugate for 6 h at 4 °C. ATP depletion was achieved by preincubating cells in glucose-free medium (DMEM/10% FBS/10 mM Galactose and 20 mM

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EXPERIMENTAL PROCEDURES Materials. The reactive derivatives of coproporphyrin I containing the amino (pentafluorophenyl, PFP)- and thiolreactive (maleimido, MI) modifications (PtCPTE-MI, PtCP(PFP)4, PtCPTE-PEG850-MI, PdCP(PFP)4, CPK(PFP)4, and PtCP(NH-CH2CH2-OH)3-PFP) were synthesized in our lab. Their synthesis procedures and molecular structures are given in Supporting Information. Fluorescent probes MitoTracker Green, MitoTracker Red, LysoTracker Green, LysoTracker Red, and Alexa488-transferrin were from Invitrogen (USA), and phosphorescent probe MitoXpress was from Luxcel Biosciences (Cork, Ireland). Synthetic peptides (purity >85% by HPLC; structures confirmed by mass spectrometry) were from Genscript (Piscataway, NJ, USA). Luminescent cell viability kit CellTiter-Glo was from Promega (Madison, WI, USA), and H2NPEG850NHBoc was from RAPP Polymere (Tubingen, Germany). Standard cell culture 96 well plates from Sarstedt (Wexford, Ireland) were used for growing the cells and for time-resolved phosphorescence measurements. White 96 well plates from Greiner Bio-One (Frickenhausen, Germany) were used for the luminescent ATP assay. All other reagents were from SigmaAldrich Ltd. (Dublin, Ireland). Preparation of Conjugates. The monosubstituted conjugates PEPP1−2, PEPP4−6 were prepared by mixing equimolar quantities (0.1−1.0 μmol scale) of the reactive porphyrin derivative and the corresponding peptide in DMSO (in the presence of triethylamine for amino-coupling), incubating for 24 h at room temperature, and purifying by RP-HPLC on a Discovery C18 column (1 × 25 cm, 5 μm, Sigma) using a gradient of CH3CN in 0.1% TFA. The conjugate fractions were collected, vacuum-dried, reconstituted in DMSO, and stored in small aliquots at −18 °C. Typical yields were 30−90%. The tetra-substituted conjugates PEPP3, PEPP3B, PEPP3C, and T1-T5 were prepared via amino-coupling in DMF in the presence of molar excess of triethylamine, using PtCP(PFP) 4 and peptide or L-Arg methyl ester at a 1:10 molar ratio, and incubating for 6−16 h at room temperature; 1−5 mg scale. Then, the mixture was diluted 1:10 with water, applied on the 2509



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HEPES, pH 7.2) for 1 h 45 min, followed by 15 min of incubation with 10 μM oligomycin, and then incubation with the conjugate for 6 h. To block the function of lysosomes, a 30 min preincubation with 0.25 μM concanamycin A was used. In flow cytometry analysis, loaded cells were trypsinized, washed with Phenol Red-free DMEM with 10% FBS and 20 mM HEPES, pH 7.2, and measured on a Guava PCA-96 flow cytometer (Millipore, Billerica, MA, USA) under 532 nm laser and 675 nm emission filters, as recommended.28 Live cell microscopy was performed on a wide-field fluorescence microscope Axiovert 200 (Carl Zeiss, Goettingen, Germany) equipped with a 390 nm LED and filter cube (ex., 390/40 nm; em., 655/40 nm) for imaging of PtCP, PdCP, and CPK dyes. PEPP3B probe was imaged at decreased (2%) O 2. Staining with organelle-specific markers MitoTracker Green/ Red (50 nM), LysoTracker Green/Red (100 nM), and transferrin-Alexa488 (0.5 μM) was performed for 30 min, followed by one washing. Cell viability was assessed via total ATP levels using a CellTiter-Glo luminescent kit (Promega) and the manufacturer’s protocol. TR-F intensity and lifetime measurements were conducted on a Victor2 reader (Perkin-Elmer) as described previously 34 using D340 excitation and D642 emission filters, counting at two delay times, 30 μs (t1) and 70 μs (t2), gate time 100 μs, and integration time 1 s. For the PEPP3B probe, the D665 emission filter, t1 = 400 μs and t2 = 450 μs, gate time 1200 μs, and counting time 2 s were used. Lifetime was calculated as: τ = (t2 − t1)/ln(F1/F2), where F1 and F2 correspond TR-F readings at delay times t1 and t2. Conjugate uptake efficiency by dPC12, HCT116, HeLa, and MEF cells was assessed by comparing F1 signals for the different probes and conditions. Monitoring of Respiration and icO2. Differentiated PC12 cells were stained with 2 μM PEPP3 (or PEPP3B) for 16 h in RPMI1640 supplemented with 1% HS and 100 ng/mL NGF and washed three times with the same medium then with Phenol Red-free DMEM containing 10 mM Glucose (or 10 mM Galactose instead), 1 mM Na-pyruvate, 20 mM HEPES, pH 7.2, and 100 ng/mL NGF. The cells were incubated for 2 h and then measured on a Victor2 plate reader. After baseline stabilization (∼20−60 min depending on O2), mitochondrial effectors were added to the cells (1/10 volume of 10× stock), and monitoring was resumed. Calibration of PEPP3 phosphorescence lifetime was performed in the hypoxia chamber at 37 °C and different pO2. Cells stained with the probe were treated with 10 μM AntA (to block respiration and local O2 gradients) were monitored kinetically to determine steady-state τ values. These values were plotted against dissolved O2 concentration (200 μM at 20.9% O2, 37 °C35), from which, using Origin 6.0 (Microcal, USA) software, the following analytical function was obtained (r 2 = 0.9807): [O2] = 1551.73− 57.55·τ + 0.5347·τ 2. Data Assessment. The microplate reader results are presented as the mean values with the standard deviation (error bars on the plots) obtained from at least 6 replicates. In cellular uptake experiments, the variability in each group was analyzed by one-tailed t test (significance level 0.05). The microscopy experiments were performed in triplicate. This ensures the consistency of the results.

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RESULTS Design and Synthesis of Cell-Penetrating PtCP Conjugates. Cell-penetrating ability of oligoarginine peptides and their conjugates mostly depends on the total number of Arg residues (7−9 are required for efficient transport), while their sequence and length are less important.32 The conjugates of coproporphyrins demonstrate rather low cell-penetration for tricarboxylic PtCP and improved accumulation for the neutral derivatives (PtCPTE-CFR9, PEPP1); however, the latter structures are quite hydrophobic and show a high degree of nonspecific binding and localization similar to those of lysosomes and endosomes.29 Aiming to overcome these drawbacks, we prepared modified monosubstituted conjugates with (i) an additional hydrophilic PEG850 moiety (PEPP2), (ii) mitochondrial targeting sequence36 with (PEPP4), and (iii) without PEG850 (PEPP5). In order to increase hydrophilicity and reduce nonspecific binding, three ester groups in PtCPTE were also replaced with ethanolamine (PEPP6) (Figure 1). Four propionic acid residues allow simple tetra-substitution of the PtCP moiety. It was shown that branched oligoarginine sequences can also provide efficient intracellular delivery.32,37 Therefore, we prepared tetra-substituted PtCP conjugates with eight (PEPP3), four (T5), and 12 (T4) Arg, and also with additional Ala (T1), Ser (T2), and Gly (T3) residues (Figure 1d). The conjugations were performed by thiol or amino coupling using the reactive MI and PFP derivatives of PtCP, respectively, with purification by RP-HPLC29 (Figure S1, Supporting Information). Some of the peptides were amidated at Ctermini to eliminate zwitterionic structures and retain molecular charge at around +8 (except for T5, PEPP4, and PEPP5; see Table 1). The branched conjugates showed shorter retention times on the C18 column and higher hydrophilicity than the monosubstituted conjugates with a similar charge (e.g., PEPP5 and PEPP3). PEPP6 conjugate with ethanolamine modification was also quite hydrophilic (Table 1). Photophysical Properties. All tetra-substituted conjugates displayed absorption spectra similar to those of free PtCP and its oligonucleotide conjugates.38 For the monosubstituted conjugates, absorption maxima were slightly blueshifted similar to those of protein conjugates,13 which is indicative of J or H stacking interactions of the PtCP moiety 39 (Figure S2A-B, Supporting Information). Excitation and emission spectra remained unaffected for all the conjugates; however, the conjugation with peptides had a significant effect on the emission yield (Figure S2, Supporting Information). In 10% FBS (typical culturing conditions), quantum yields for the monosubstituted conjugates decreased at a higher length of the peptide (e.g., PEPP1 vs PEPP5) and the addition of the PEG spacer. For PEPP6 with three ethanolamine residues, the absorption spectra shift and internal quenching were much greater. Internal quenching, which was also high for tetrasubstituted conjugates, can be largely reduced by the anionic surfactants (0.5 mM SDS), which solubilize the PtCP moiety (for PEPP3 in deoxygenated conditions, the quantum yield was 0.55; not shown). Similar interactions may occur with the conjugate inside the cells (see below). Despite the differences in quantum yields, all the PtCP conjugates displayed similar phosphorescence lifetimes: 20−23 μs in air-saturated and ∼80 μs in deoxygenated media (DMEM with 10% FBS, Figure S2D, Supporting Information), i.e., similar to the other PtCP based probes.34 The PEPP3 conjugate measured in DMEM at 0, 1, 2510



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Figure 2. Uptake of conjugates by different mammalian cell lines. Cells were seeded onto collagen IV-coated 96 well plates, incubated with conjugates (1 μM) for 16 h, washed three times, and measured on a microplate reader.

PEPP6, which was undetectable even at 10 μM and 16 h of incubation. Loading with T2 was marginally lower than that for T1, T3, or PEPP3 (Figure 4) confirming that hydroxyl groups interfere with cell penetrating ability. For T5, loading was detectable only at 10 μM, but with clear signs of toxicity and the appearance of a round cell shape. Similar efficiency and loading patterns were observed with the other cell lines: rather weak for HeLa, moderate for PC12 and HCT116, and high for MEF, HepG2, and SH-SY5Y (data not shown). The differences can be explained by the different shape, size, membrane composition, and mechanisms of transport for these cells. Intracellular Distribution of the Conjugates. Widefield fluorescence microscopy imaging revealed that different conjugates show different intracellular distribution in HCT116, MEF, PC12, SH-SY5Y, and HepG2 cells (see representative data in Figures 3 and S3 (Supporting Information)). Compared to PEPP1, PEPP2 displayed a more diffused cytoplasmic distribution, while PEPP3 gave a more uniform distribution across the cell including the nucleus (Figure 4), with higher accumulation in the nucleus at 10 μM (Figures 3 and S3 (Supporting Information)). PEPP4 and PEPP5 harboring the mitochondrial targeting sequence (with or without PEG 850 linker) displayed a better intracellular accumulation and brightness than PEPP1, but they did not localize in the mitochondria (Figure 4) and had localization similar to that of the endosomal marker transferrin (data not shown). All branched conjugates T1−T4 and PEPP3 showed very similar intracellular distribution, and the addition of Ala (T1), Gly (T3), or extra Arg (T4) had no visible effects (Figures 3, 4, and S3 (Supporting Information)). T2 with the Ser residue gave only a faint punctuated cytosolic pattern, and localization of T5 could not be assessed as the cells quickly died at high probe concentrations (not shown).

10, 50, and 100% FBS showed no changes in phosphorescence emission lifetime (not shown). Uptake of the Conjugates by Mammalian Cells. Cellular uptake of the conjugates was first analyzed in cell populations on the TR-F reader. Most of the conjugates showed efficient cell loading with MEF, HeLa, HCT116, and dPC12 cells at 1 μM and 16 h incubation (Figure 2). Linear and branched conjugates PEPP1 and PEPP3 produced high loading signals in all cell lines, whereas the PEG-modified PEPP2 showed a lower degree of loading. The more hydrophobic PEPP4 and PEPP5 with the mitochondrial targeting sequence displayed cell-specific penetration, while conjugates PEPP6 (ethanolamine-modified) and T5 (four Arg) showed poor accumulation in all cell lines tested. The tetrasubstituted T1−T4 conjugates showed cell-specific loading: high for HCT116, moderate for dPC12, and poor for HeLa cells. The presence of additional amino acids in branched conjugates (Ala in T1 or Gly in T3) did not interfere with cell loading; however, the presence of Ser in T2 reduced it. As expected, the higher number of Arg in T4 did not improve loading. Therefore, we can conclude that 8−9 Arg ensure optimal cell penetration of coproporphyrin conjugates and that hydroxyl groups can decrease it (conjugates T2, PEPP6, and PEG-containing PEPP2 and PEPP4). The above results on cell loading were verified by fluorescence microscopy and flow cytometry, which allow single cell analyses. Flow cytometry was not as sensitive as TRF, and only highly loaded cells were detectable. For such samples, the whole population gave rather uniform fluorescent signals, and unloaded cells (scattering, but no fluorescence) were not seen. Fluorescence microscopy also confirmed the positive loading of HCT116 and MEF cells with the monosubstituted conjugates at 1 and 10 μM concentrations (Figures 3 and S3 (Supporting Information)), except for 2511



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Figure 3. Fluorescence microscopy analysis of HCT116 cells stained with different PtCP conjugates. Cells were seeded onto collagen-poly-D-lysine coated glass bottom dishes at a concentration of 30,000/dish, incubated for 16−18 h with conjugates at specified concentrations, washed, and imaged. Brightfield and fluorescent (390 nm excitation/650 nm emission) images are shown. Scale bar = 50 μm.

The main mechanisms of cell entry reported for oligoarginine, endocytosis and direct translocation,37,40,43,44 can be assessed by analyzing the temperature and ATP dependence of the uptake. For PEPP3 and PEPP4, the loading at 4 °C was almost completely inhibited; however, weak staining of the cell surface for PEPP4 indicates that interaction with the cell membrane still occurs (Figures 5 and S5 (Supporting Information)). Uptake of PEPP3 and PEPP4 by the cells depleted in ATP (olygomycin and absence of glucose) was reduced significantly. We therefore concluded that both branched PEPP3 and linear PEPP4 rely on an endocytosis mechanism of cell entry. Furthermore, inhibition of macropinocytosis by EIPA slightly reduced the uptake of branched conjugate PEPP3 by HCT116 and MEF cells, with the loss of nuclear localization (Figure 5). Inhibition of the clathrin-mediated pathway by CPZ reduced the uptake even more strongly, and inhibition of lipid-raft formation by MBCD had no effect. Noteworthy, 6 h of treatment with EIPA and CPZ still left a high percentage of

Mechanisms of Cellular Uptake for Mono- and TetraSubstituted Conjugates. Knowledge of the mechanisms of cellular uptake and cell entry pathways allows the regulation of transport of the cargo inside the cell. Such studies involve the inhibition of specific uptake pathways with drugs using the shortest treatment time to minimize the disruption of cellular function.40,41 Since the flow cytometry method cannot distinguish between internalized and surface-bound probes, fluorescence microscopy which allows direct observation of the cell phenotype and the qualitative effect of inhibitors, was applied to study HCT116 and MEF cells. Two representative conjugates from each class were chosen for these experiments: PEPP3 (branched) and PEPP4 (linear). PtCP conjugates show rather slow rates of cellular uptake,18,31,32,37,42 which were 4−6 h for PEPP3 and PEPP4 (Figure S4, Supporting Information). The unlabeled oligoarginine did not have a measurable effect on the loading of the conjugates (Figures 5 and S5 (Supporting Information)), thus indicating a different mechanism of uptake. 2512



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Figure 4. High resolution microscopy images of PEPP3−PEPP5 conjugates in HCT116 cells. Cells were loaded with PEPP4, PEPP5 (1 μM), and PEPP3 (5 μM) conjugates for 16 h, washed, and counter-stained with organelle-specific markers: MitoTracker Green (MTG, mitochondria) and transferrin-Alexa488 (TFN, pool of clathrin-dependent recycling endosomes). Scale = 50 μm.

Figure 5. Effects of temperature, ATP depletion, the presence of unlabeled GR 9, and inhibitors of endocytosis on the cellular uptake of PEPP3 and PEPP4. MEF cells were incubated at 37 °C (or at 4 °C) with 5 μM PEPP3 or 2 μM PEPP4 for 6 h in the presence of GR9 peptide and inhibitors or subjected to ATP depletion, then washed, and analyzed. Scale bar = 50 μm. 2513



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Figure 6. Monitoring of icO2 with the PEPP3 probe in dPC12 cells. (A) Effect of PEPP3 (0−10 μM for 16 h, 37 °C) on the viability of dPC12 cells. (B) Kinetics of the loading of dPC12 cells with PEPP3 (0−10 μM for 16 h) measured by TR-F. (C) Average phosphorescence lifetimes for dPC12 cells loaded with PEPP3 (0−10 μM for 16 h). (D) Phosphorescence lifetime (τ) at different O 2 concentrations for dPC12 cells loaded with 2 μM PEPP3 for 16 h and treated with 10 μM antimycin A. (E and F) Metabolic responses (presented in O 2 scale) of PC12 cells loaded with PEPP3 and measured in glucose-free media. (E) Responses to FCCP addition for the cells pretreated with 10 μM oligomycin (OM) at 21% of external O2. (F) The same as E but at 10% of external O2.

somal escape can be inhibited by agents that disturb the function of acidic compartments.45−47 We treated MEF cells with concanamycin A (V-ATPase inhibitor) and observed that PEPP3 lost its nuclear localization, remaining only in the cytoplasm. This drug had no effect on PEPP4 (Figure S5, Supporting Information), thus suggesting that PEPP4 stays in recycling endosomes. Furthermore, the presence of heparin sulfate caused a partial decrease of uptake and perinuclear localization of PEPP3, whereas for PEPP4, the uptake was completely abolished with the formation of visible aggregates (Figure S5, Supporting Information). This suggests that interactions cell surface sugars are important for the monosubstituted conjugates. Monitoring of Intracellular O2 with the PtCP Conjugates. The PEPP3 conjugate was among the best in terms

viable cells, whereas treatment with MBCD changed cell morphology (round shape phenotype) and caused cell detachment. Similarly, treatment with EIPA and CPZ moderately decreased the uptake of monosubstituted PEPP4, and MBCD had no visible effect (Figure.5). Since the uptake of PEPP3 was significantly affected by CPZ, we performed a competition experiment with transferrin (marker of clathrinmediated endocytosis41) and observed a partial (∼30%) reduction of loading of the conjugate (Figure S5, Supporting Information). These results indicate that PEPP3 and PEPP4 utilize both macropinocytosis and clathrin-mediated uptake pathways. This means that after internalization they should remain in recycling endosomes or undergo endosomal escape followed by migration to the cytoplasm and other compartments. Endo2514



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Figure 7. Properties of PEPP3B and PEPP3C conjugates. (A) Structures of the conjugates. (B) Fluorescence microscopy images of HCT116 cells stained with the conjugates (5 μM, 16 h). Scale bar = 50 μm. (C) UV−vis absorption spectra of CPK, PEPP3B, and PEPP3C. (D) Phosphorescence (PEPP3B) and fluorescence (PEPP3C) spectra. (E) Effect of pH on the fluorescence of PEPP3C measured at 0.25 μM in citrate−phosphate buffer. (F) Average fluorescence at 640 nm for dPC12 cells loaded with 0−10 μM PEPP3C for 16 h. (G−H) Metabolic responses (in phosphorescence lifetime scale) of PC12 cells loaded with PEPP3B in glucose-free media at 10% pO 2. (E) Responses to 1 μM FCCP and 10 μM AntA. (F) Responses to 1 μM FCCP for the cells pretreated with 10 μM OM.

respiration (the cell layer is acting as a sink for dissolved O 2 and the medium layer as a diffusion barrier for atmospheric oxygen35). The response had a bell shape, decreasing at high concentration (4 μM) due to FCCP toxicity. Pretreatment of cells with 10 μM oligomycin (complex V inhibitor) decreased basal respiration (higher icO2) and the magnitude of response (Figures 6e and S6, Supporting Information). Antimycin A (complex III inhibitor) increased icO2 to ambient levels (Figures 6f and S6, Supporting Information). These results are in agreement with those of the other icO2 probes.28,29,34 They demonstrate that the PEPP3 conjugate works reliably as an icO2 probe in the measurement of cell oxygenation and metabolic responses of cell populations producing good quality data in a simple format with high sample throughput. Conjugates of the Other Coproporphyrin Dyes. Compared to PtCP, PdCP has a significantly longer unquenched phosphorescence lifetime (∼1 ms) and therefore

of cellular uptake and distribution; therefore, we decided to test it as an O2-sensing probe with dPC12 cells. Optimal loading was at around 2 μM producing minimal cell toxicity, high phosphorescence intensity signals, and stable lifetime values in resting cells (Figure 6a−c). To enable the determination of O 2 concentration, we calibrated PEPP3 in live nonrespiring dPC12 cells35 by measuring its phosphorescence lifetime at different levels of atmospheric pO2 and 37 °C. Calibration curve O2 = f(τ) is shown in Figure 6d. To test probe performance, the cells grown, differentiated, and loaded in standard microplates were treated with metabolic effectors: FCCP, oligomycin, and antimycin A (Figure 6e−f). Prior to stimulation, resting dPC12 cells demonstrated stable baseline signals, which reflect partial deoxygenation of the monolayer compared to the bulk medium. The addition of FCCP (mitochondrial uncoupler) induced a prominent response: a large decrease in cellular oxygen due to increased 2515



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is better suited for the sensing of physiological O2 levels (0−50 μM9). In contrast to the phosphorescent dyes PtCP and PdCP, coproporphyrin-ketone (CPK) shows bright longwave fluorescence, structural and spectral similarity with chlorins, high photostability, and characteristic changes to pH. This makes CPK a promising candidate for use as a ratiometric pH probe 48 or a photosensitizer in PDT.11 On the basis of PdCP and CPK dyes, we synthesized structural analogues of PEPP3 conjugate, PEPP3B and PEPP3C, respectively (Figure 7a), and investigated their properties. PEPP3B and PEPP3C demonstrated absorption and emission spectra similar to those of unconjugated CPK and PdCP (Figure 7c−d). Thus, PEPP3B showed significantly longer lifetimes and higher sensitivity to O2 than the PEPP3 probe, producing rather low phosphorescent signals at 21% O 2 due to strong quenching. PEPP3C exhibited bright fluorescence and pH-dependence, however, compared to those of free CPK, and spectral changes in fluorescence occurred at more acidic pH (pKa < 3), which did not match the cytosolic pH range (7.0−7.4) (Figures 7e and S7, Supporting Information). As expected, both PEPP3B and PEPP3C showed efficient uptake by HCT116 cells and intracellular distribution similar to that of PEPP3 (Figure 7b). In the case of PEPP3C, high fluorescent signals were observed (Figure 7f), suggesting its potential suitability for imaging and plate reader applications within the optimized range of working concentrations (2−10 μM, similar to PtCP-based branched conjugates). The behavior of the PEPP3B conjugate in the experiments with dPC12 cells on a microplate reader performed at 10% O2 (Figure 7g,h) was seen to be similar to PEPP3. This conjugate produced similar signal patterns: basal respiration levels produced phosphorescence lifetimes of ∼500−600 μs, which dropped to ∼400 μs in oligomycin and antimycin treated cells and increased to ∼700 μs upon mitochondrial uncoupling with FCCP. This indicates that PEPP3B can be used for monitoring of icO2 under hypoxia. Other conjugates of these two dyes with oligoarginine moieties can also be prepared.

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by the higher loading efficiency and/or elimination of internal quenching upon their internalization. Compared to the previously reported PEPP1,29 the new conjugates show better and more diverse intracellular distribution, ranging from endosomal to whole cell localization. The cytoplasmic accumulation was demonstrated for the linear oligoarginine conjugate with the PEG linker (PEPP2), while whole cell location with nuclear accumulation at higher concentrations was observed for branched (PEPP3, T1, T3, and T4) conjugates. Surprisingly, the targeting sequence in PEPP4 and PEPP5 did not lead to their mitochondrial accumulation. This can be explained by (i) special requirements for mitochondrial targeting of small molecules such as PtCP; (ii) limitations imposed by the endosomal cell entry (e.g., no escape from endosomes for PEPP4, Figure S5, Supporting Information); and (iii) functional inactivation of the targeting sequence within the supra-molecular structure (sterical factors). Comparative analysis of branched (PEPP3) and linear (PEPP4) conjugates shows that both types use temperatureand ATP-dependent endocytosis mechanisms of cell entry. The latter is unaffected by MBCD and partially inhibited by EIPA and CPZ. This data points to the involvement of mixed uptake mechanisms. For the two different cell lines, MEF and HCT116, no significant differences were observed in cellular uptake, suggesting low cell-specificity. Cell penetration of the conjugates appears to be different from the unconjugated oligoarginine peptide, for which the uptake can vary in different cell lines.2,43 The structure−activity relationships established for the panel of PtCP conjugates can be exploited to design new cellpenetrating conjugates of porphyrin dyes with predetermined properties. This was illustrated by investigating icO2 sensing properties of one chosen conjugate, PEPP3, and preparing its homologues from the PdCP and CPK dyes (PEPP3B and PEPP3C conjugates, respectively). In the icO2 sensing experiments, PEPP3 showed good analytical performance. It can be used at lower concentrations than the existing peptide-based probes,28,29 with equal or shorter loading time (6−16 h) and without any significant effect on cell viability (Figure6). Furthermore, cell staining with PEPP3 did not interfere with the other cellular markers (LysoTracker Red, LysoTracker Green, Transferrin, MitoTracker Red, and MitoTracker Green; data not shown), thus indirectly confirming the minimal impact of the former on the function of these organelles. This probe was calibrated over physiological O2 concentrations and tested in time-lapse respiration experiments with live cells on a TR-F reader, both under normoxic and hypoxic macro-environments. The profiles of cell oxygenation and responses to model drugs and metabolic stimulations measured with this conjugate were consistent with results obtained with the other probes. 28,34,49 PEPP3B and PEPP3C conjugates based on the analogues of PtCP also demonstrated efficient accumulation in the cells and gave localization patterns similar to those of PEPP3. The PdCP based PEPP3B conjugate showed suitability for monitoring low O2 under a hypoxic environment, while PEPP3C showed pH sensitivity, which was shifted toward the acidic range probably due to the multiple charges on R-residues. The latter should be kept in mind when designing the intracellular probes with pHdependent properties. This study can also be used to develop new cell-permeable porphyrin based probes and drugs (e.g., PDT photosensitizers) targeted to different subcellular locations.

DISCUSSION

A number of PtCP conjugates with long linear and short branched oligoarginine peptides, an additional mitochondriatargeting sequence, different side groups, and linkers were prepared and characterized. Testing these conjugates with several common cell lines allowed us to establish structure− activity relationships, which can aid in further rational design and application of such structures. For the monosubstituted conjugates with CFR9 peptides, the hydrophilic PEG850 linker did not improve their cell loading and water solubility; however, this was achieved with tetra-substituted PtCP conjugates with the R2-R3 peptides. We also showed that the overall structure can affect the photophysical properties of PtCP moiety. Analyzing the uptake of the conjugates by mammalian cells, we observed a large variation in its efficiency and subsequent intracellular distribution. The presence of hydroxyl groups (PEPP6 and T2) and the PEG850 linker (Figures 2−3 and S3 (Supporting Information)) slightly reduced the efficacy of cellular uptake. For the tetra-substituted conjugates, we found that a minimum 8 Arg residues is required for efficient internalization. Branched PtCP conjugates showed lower brightness (emission quantum yields) in aqueous solutions; however, TR-F signals in stained cells were comparable to the signals produced by the linear conjugates. This can be explained 2516



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ASSOCIATED CONTENT S Supporting Information * Experimental procedures and the synthesis of initial dyes. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION Corresponding Author *Phone: +353-21-4901699. Fax: +353-21-4901698. E-mail: d. [email protected]. Notes The authors declare no conflict of interest.

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ACKNOWLEDGMENTS

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ABBREVIATIONS

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REFERENCES

This work was supported by the Science Foundation of Ireland, grant 07/IN.1/B1804, and by the Higher Education Authority (HEA) of Ireland.

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