Interaction of molecular probes with living cells and tissues. Part 2. A structure-activity analysis of mitochondrial staining by cationic probes, and a discussion of the synergistic nature of image-based and biochemical approaches.

Cultured rat fibroblasts were exposed to 41 cationic fluorescent probes of very varied hydrophilicity/lipophilicity. Outcome of probe-cell interaction fell into one of the following categories: probe failed to enter the cells; probe accumulated on cell surfaces; probe accumulated in mitochondria, and/or in other intracellular regions. The observations were analysed using a Simplistic Chinese Box (SCB) approach, and the following conclusions were reached. It was the hydrophilic probes which failed to enter cells, whilst extremely lipophilic probes were retained on the cell surfaces. Only the slightly lipophilic cationic probes were permeant, and accumulated in mitochondria. Using the probes log P values to model hydrophilicity/lipophilicity, effective cationic mitochondrial stains can be specified numerically so: 0 less than log P probe less than +5. This SCB model was used to rationalise a variety of earlier observations on the action of mitochondrial probes. The applicability of the SCB approach to integrate image-based and biochemical investigations was demonstrated by using the action of chlorpromazine on mitochondrial action as a case example.     

Interactions of molecular probes with living cells and tissues. Part 1. Some general mechanistic proposals,making use of a simplistic Chinese box model

Summary A simple and generalised model — termed the simplistic Chinese box [SCB] model — for the interaction of molecular probes with living systems is described. The SCB model includes the following assumptions. That living systems may be considered as built from biologically defined boxes, e.g. whole cell nucleus nucleoli. That movement of molecular probes into and through these boxes is strongly infuenced by box wall permeability, which in turn is largely dependent on the simple physicochemical properties of probe and wall. That retention of probes in boxes is influenced by permeability of the walls and by trapping of probes by boxes and walls, the latter effect also being strongly dependent on simple physicochemical, properties. That important physicochemical properties include electric charge, hydrophilicity/lipophilicity, non-specific protein binding, and molecular size. That, since all these factors can be expressed or modelled numerically, SAR methodology is an appropriate technique for analysing molecular probe investigations. This paper is dedicated to the memory of Prof. A.W. Rogers     

lmmunocytochemical Detection of Bromodeoxyuridine in Tissues of the Toad Bufo arenarum Hensel

Abstract

The concept of mitochondrial targeting for chemo- and photochemotherapy of neoplastic diseases has its origin in the observation that enhanced mitochondrial transmembrane potential is a common tumor cell phenotype. As a result of this enhanced transmembrane potential, a number of cationic dyes accumulate in larger amounts and are retained for longer periods in the mitochondria of tumor cells than in normal cells. Only a relatively small number of (photo)toxic dyes known to localize in energized cell mitochondria are capable of inducing the destruction of tumor cells with desirable degrees of selectivity, however. We investigated how lipophilic character may affect the degree of specificity with which cationic dyes localize in energized cell mitochondria and how mitochondrial specificity may affect tumor cell selectivity. To this end, we used fluorescence microscopy to characterize the subcellular localization of ethyl violet and seven analogs of the prototypical mitochondria-specific dye, rhodamine 123. All cationic rhodamines studied here (?0.62 < log D_ow < 1.60, where D_ow represents the n-octanol/water distribution coefficient) were found to show considerable mitochondrial specificity, while the more lipophilic ethyl violet (log D_ow = 2.37) did not. Ethyl violet was found to localize not only in mitochondria, but also in lysosomes. We also compared the degree of selective tumor cell killing induced by ethyl violet and two phototoxic rhodamines, i.e., the dibromo derivatives of rhodamine 123 and its n-octyl ester analog. While ethyl violet induces the destruction of human uterine sarcoma (MES-SA) cells and normal green monkey kidney cells (CV-1) with comparable efficiency, the mitochondria-specific dibromorhodamines were found to induce the destruction of MES-SA cells with considerable selectivity. Our findings are consistent with the premise that mitochondrial localization per se does not provide successful selective tumor cell killing using mitochondrial targeting. Our results reinforce the hypothesis that while most cationic dyes can be expected to localize at least to some extent in energized cell mitochondria, only those showing virtually absolute mitochondrial specificity can actually mediate the destruction of tumor cells with desirable selectivity. These findings also support the hypothesis that the probability of success of mitochondrial targeting in photochemotherapy of neoplastic diseases is bound to be higher when the D_ow associated with the drug candidate falls within approximately two orders of magnitude of that of rhodamine 123.     

Mitochondrial accumulation of a lipophilic cation conjugated to an ionisable group depends on membrane potential, pH gradient and pK a: implications for the design of mitochondrial probes and therapies

Mitochondria play key roles in a broad range of biomedical situations, consequently there is a need to direct bioactive compounds to mitochondria as both therapies and probes. A successful approach has been to target compounds to mitochondria by conjugation to lipophilic cations, such as triphenylphosphonium (TPP), which utilize the large mitochondrial membrane potential (Δψ_m, negative inside) to drive accumulation. This has proven effective both in vitro and in vivo for a range of bioactive compounds and probes. However so far only neutral appendages have been targeted to mitochondria in this way. Many bioactive functional moieties that we would like to send to mitochondria contain ionisable groups with pK _a in the range that creates an assortment of charged species under physiological conditions. To see if such ionisable compounds can also be taken up by mitochondria, we determined the general requirements for the accumulation within mitochondria of a TPP cation conjugated to a carboxylic acid or an amine. Both were taken up by energised mitochondria in response to the protonmotive force. A lipophilic TPP cation attached to a carboxylic acid was accumulated to a greater extent than a simple TPP cation due to the interaction of the weakly acidic group with the pH gradient (ΔpH). In contrast, a lipophilic TPP cation attached to an amine was accumulated less than the simple cation due to exclusion of the weakly basic group by the ΔpH. From these data we derived a simple equation that describes the uptake of lipophilic cations containing ionisable groups as a function of Δψ_m, ΔpH and pK _a. These findings may facilitate the rational design of additional mitochondrial targeted probes and therapies.     

Influence of trans-membrane potential and of hydrophobic interactions on dye accumulation in mitochondria of living cells

The lipophilic cationic fluorescent dye azopentylmethylindocarbocyanine (APMC) specifically stains the mitochondria in living cells. The dye contains a photosensitive diazirine ring and is suitable for photoaffinity labelling of mitochondrial proteins. By a combination of photoaffinity labelling of cell cultures of mouse fibroblasts (LM) with APMC, lysis of the labelled cells, subsequent micro-gel electrophoresis and detection of the fluorescence of the labelled proteins in the gel lanes with a sensitive microfluorimeter, we determined the number, apparent molecular masses, and relative intensity of the labelled proteins. In LM cells, three proteins with apparent molecular masses of 31, 40, and 74 kDa were labelled with high intensity, and proteins of 28, 29, 44, 48, 49, 66, and 105 kDa with low intensity. Two effects mainly determine the binding of lipophilic dye cations to mitochondrial proteins in living cells: (1) interaction of the trans-membrane potential of the inner mitochondrial membrane with the dye cations; and (2) hydrophobic interactions between the strongly lipophilic proteins of the inner membrane and the lipophilic dye molecules. Preincubation of the cell cultures with drugs that dissipate the trans-membrane potential, such as valinomycin, 2,4-dinitrophenol (DNP) and 3-chlorcarbonylcyanidephenylhydrazon (CCCP), strongly reduces or even prevents APMC labelling of mitochondrial proteins. The influence of hydrophobic interactions was investigated by competitive staining experiments using dyes with very different lipophilic properties. The lipophilicity of the dyes was characterized by their R _m values in reversed phase thin-layer chromatography. Prestaining with an excess of strongly lipophilic cationic acridine and phenanthridine dyes, such as pentyl acridinium orange chloride (PAO), nonyl acridinium orange chloride (NAO) and tetramethylpropidium chloride (MP), respectively, completely prevents protein labelling with APMC. Obviously, the dyes occupy the same mitochondrial binding sites as APMC. At equal concentrations the intensity of the 40-kDa signal is strongly reduced, whereas the 31-kDa and 74-kDa signals are unaffected. Using phenanthridine dyes with lower lipophilicity, namely propidium chloride (P), M, and N reduces the peak of the 40-kDa protein in APMC labelling, indicating that the 40-kDa protein preferentially binds lipophilic dye cations.     

Monitoring of relative mitochondrial membrane potential in living cells by fluorescence microscopy

Permeant cationic fluorescent probes are shown to be selectively accumulated by the mitochondria of living cells. Mitochondria-specific interaction of such molecules is apparently dependent on the high trans- membrane potential (inside negative) maintained by functional mitochondria. Dissipation of the mitochondrial trans-membrane and potential by ionophores or inhibitors of electron transport eliminates the selective mitochondrial association of these compounds. The application of such potential-dependent probes in conjunction with fluorescence microscopy allows the monitoring of mitochondrial membrane potential in individual living cells. Marked elevations in mitochondria- associated probe fluorescence have been observed in cells engaged in active movement. This approach to the analysis of mitochondrial membrane potential should be of value in future investigations of the control of energy metabolism and energy requirements of specific biological functions at the cellular level.     

A predictive model for the selective accumulation of chemicals in tumor cells

Cationic lipophilic dyes can accumulate in mitochondria, and especially in mitochondria of tumor cells. We investigated the chemical properties and the processes allowing selective uptake into tumor cells using the Fick–Nernst–Planck equation. The model simulates uptake into cytoplasm and mitochondria and is valid for neutral molecules and ions, and thus also for weak electrolytes. The differential equation system was analytically solved for the steady-state and the dynamic case. The parameterization was for a generic human cell, with a 60 mV more negative potential at the inner mitochondrial membrane of generic tumor cells. The chemical input data were the lipophilicity (logK_OW), the acid/base dissociation constant (pK_a) and the electric charge (z). Accumulation in mitochondria occurred for polar acids with pK_a between 5 and 9 owing to the ion trap, and for lipophilic bases with pK_a>11 or permanent cations owing to electrical attraction. Selective accumulation in tumor cells was found for monovalent cations or strong bases with logK_OW of the cation between –2 and 2, with the optimum near 0. The results are in agreement with experimental results for rhodamine 123, a series of cationic triarylmethane dyes, F16 and MKT-077, an anticancer drug targeting tumor mitochondria.     

Rapid uptake of lipophilic triphenylphosphonium cations by mitochondria in vivo following intravenous injection: Implications for mitochondria-specific therapies and probes

Background

Mitochondrial dysfunction contributes to a range of pathologies, consequently there is a need to monitor mitochondrial function and to intervene pharmacologically to prevent mitochondrial damage. One approach to this is to deliver antioxidants, probes and pharmacophores to mitochondria by conjugation to the lipophilic triphenylphosphonium (TPP) cation that is taken up selectively by mitochondria driven by the membrane potential.

Conclusions

Oral administration of TPP-conjugated antioxidants protects against mitochondrial damage in vivo. However, there is also a need to deliver molecules rapidly to mitochondria to respond quickly to pathologies and for the real-time assessment of mitochondrial function.

Methods

To see if this was possible we investigated how rapidly TPP cations were taken up by mitochondria in vivo following intravenous (iv) administration.

Results

AlkylTPP cations were accumulated selectively by mitochondria within mice within 5 min of iv injection. The extent of uptake was enhanced 10–30-fold relative to simple alkylTPP cations by attaching functional groups to the TPP cation via long, hydrophobic alkyl chains. Conclusions: Mitochondria-targeted antioxidants, probes and pharmacophores can be delivered into mitochondria within minutes of iv administration.

General significance

These findings greatly extend the utility of mitochondria-targeted lipophilic cations as therapies and probes.     

Fluorescent cationic probes for nuclei of living cells: why are they selective? A quantitative structure–activity relations analysis

Selectivity of nuclear probes is controlled by competitive accumulation of the probe by cellular organelles as well as the high affinity for nucleic acids. Physicochemical features of probes which favor nucleic acid binding include cationic character and a planar aromatic system above a minimum size. Features of probes which permit entry into cells are low protein and lipid binding. Features which reduce accumulation in non-nuclear sites include high base strength and hydrophilicity of the cation. The overall quantitative structure–activity (QSAR) model specifying nuclear accumulation may be expressed as follows: CBN<40; 8>log P _{neutral species}>0; AI<8; Z>0; -5<log P _cation<0; pK _a >10; LCF>17; LCF/CBN>0.70 (where CBN is the conjugated bond number, log P _x the logarithm of the water–octanol partition coefficient of species x, AI the amphilicity index, Z the electric charge, pK _a the negative logarithm of the equilibrium constant for the free base–protonated base reaction, and LCF the largest conjugated fragment). Preliminary applications of the QSAR model—to the selection of anticancer drugs, minimization of dye and drug toxicity and the designed synthesis of fluorescent probes—are outlined.     

Respiratory control depression by tetraalkylammonium bromides in rat liver mitochondria.

Six different lipophilic (hydrophobic) organic cations, tetraethyl-, tetrapropyl, tetrabutyl-, tetrapentyl-, tetrahexyl-, and tetraheptylammonium bromide, depressed respiratory control in rat liver mitochondria. Evaluation of mitochondrial responses in terms of a quadratic equation in log P (an index of lipophilicity) indicated that the NADH dehydrogenase receptor site for inhibitor (diminution of control of glutamate, alpha-ketoglutarate, and beta-hydroxybutyrate respiration) was more lipophilic than receptor sites for flavin-linked substrates (reduction of control of succinate, choline and alpha-glycerophosphate respiration). The succinate dehydrogenase receptor site for inhibition by the tetraalkylammonium bromides was more hydrophillic (less lipophilic) than the choline or alpha-glycerophosphate dehydrogenase receptor sites. Depression of respiratory control may be a function of charge density and of lipophilicity at specific inner membranal sites and the susceptible site may differ for different respiratory substrates.     

Interaction of cationic <Emphasis Type="Italic">meso</Emphasis>-porphyrins with liposomes,mitochondria and erythrocytes

Two series of cationic porphyrins meso-(3N-methylpyridinium)phenylporphyrin (3P1, 3P2c, 3P2t, 3P3 and 3P4) and meso-(4N-methylpyridinium)phenylporphyrin (4P1, 4P2c, 4P2t, 4P3 and 4P4) were studied to obtain a comprehensive understanding of factors that influence the binding of cationic porphyrins to liposomes and mitochondria, as well as their photodynamic efficiencies in erythrocytes. Binding and photodynamic efficiency were found to be inversely proportional to the number of positively charged groups and directly proportional to n-octanol/water partition coefficients (log P_OW), except for the cis molecules 3P2c and 4P2c. In the cis molecules, binding and photodynamic efficiency were much higher than expected, indicating that specific interactions not accounted by log P_OW enhance photodynamic efficiency. The effect of mitochondrial transmembrane electrochemical potentials on cationic porphyrin binding constants was estimated to be as large as 15%, and may be useful to selectively target this organelle when promoting photodynamic therapy to induce apoptosis.     

Mitochondrial functional changes during hepatic hyperplasia and azo dye carcinogenesis.

Resting and active-state respiratory velocities, respiratory control, high amplitude volume changes, and latent ATPase activities were examined in hepatic mitochondria from rats fed 3'-methyl-4-dimethylaminoazobenzene (3'MeDAB) for production of liver tumors and from rats in three phases of liver regeneration subsequent to subtotal hepatectomies. Tetrabutylammonium bromide, a lipophilic probe capable of selectively inhibiting phosphorylating oxidation or uncoupling oxidation from phosphorylation, was used to detect subtle alterations in lipophilicity characteristics of the organelles and it was concluded that mitochondria from pre-hyperplastic, hyperplastic, and neoplastic tissues had a higher than normal degree of membrane lipophilicity at specific functional sites. Control of respiration by ADP was markedly augmented in all experimental groups; this behavior, plus depressed sensitivity to swelling agents and energized contraction, were similar in mitochondria from hepatomas and from 3-day regenerating livers. These mitochondrial functions were even more pronounced, however, in cells in pre-hyperplastic states (6 and 16 h subsequent to partial hepatectomy). Many forms of liver damage result in mitochondrial alterations which elevate the capacity for oxidative phosphorylation. Such changes associated with induction of azo dye oncogenesis are mimicked by the degree of hyperplasia in the tissue following the first mitotic wave of regeneration; implications relevant to hepatocarcinogenesis are discussed.     

Inhibition of mitochondrial respiration by neutral, monocationic, and dicationic bis-pyridines related to the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium cation (MPP+)

The cytotoxic effect of the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+) is believed to be associated with a compromise in cellular energy arising as a consequence of its persistent inhibition of mitochondrial respiration. MPP+ is a rather weak inhibitor of electron transport, but it undergoes passive accumulation inside actively respiring mitochondria in response to the transmembrane electrochemical potential gradient. In order to test the prediction that dicationic analogs of MPP+ might be concentrated to a much greater extent and thereby exert especially potent inhibition of respiration on the intact organelle, we synthesized four differently spaced bis-pyridines, each in neutral, monocationic, and dicationic forms, and evaluated their inhibitory activities in intact mitochondria and in electron transport particles (ETP). Compared to the neutrals, the monocations and especially the dications exhibit reduced inhibition in ETP, but the inhibition in mitochondria is enhanced selectively for the cationic inhibitors presumably on account of their accumulation in the mitochondrial matrix. This enhancement is limited by the relatively poor ability of the cationic bis-pyridines to enter mitochondria, as judged from experiments which evaluated the rate of onset of inhibition (without preincubation), in the absence and presence of tetraphenylborate (TPB-). The dications appear to be transported less well than the monocations, and only the most lipophilic dication exhibited a substantially greater accumulation-dependent enhancement of inhibitory activity on mitochondria than did the corresponding monocation. The compounds studied here constitute a novel class of respiratory chain probes which may be useful for a variety of studies on mitochondria.     

Targeting lipophilic cations to mitochondria

Mitochondrial function and dysfunction contributes to a range of important aspects of biomedical research. Consequently there is considerable interest in developing approaches to modify and report on mitochondria in cells and in vivo. One approach has been to target bioactive molecules to mitochondria by conjugating them to lipophilic cations. Due to the large mitochondrial membrane potential, the cations are accumulated within mitochondria inside cells. This approach had been used to develop mitochondria-targeted antioxidants that selectively block mitochondrial oxidative damage and prevent some types of cell death and also to develop probes of mitochondrial function. Here we outline some of the background to the development of these compounds.     

Photoaffinity labelling with fluorescence detection

A micromethod was developed for investigating the interactions between fluorescent dyes and cellular proteins. The lipophilic cationic dye APMC (azopentylmethylcarbocyanine) contains a photosensitive diazirine ring and is suitable for photoaffinity labelling. By combining photoaffinity labelling of cultured cells, micro-gel electrophoresis and detection of the fluorescence with a microfluorimeter, we established a highly sensitive and rapid procedure to identify APMC labelled proteins. Cells which had been incubated for 10 min with 10^–8 M APMC could be analysed for APMC binding without difficulty. Under our experimental conditions this corresponds to about 0.2 nmol APMC per mg protein. The lipophilic APMC specifically stains the mitochondria in living HeLa and LM cells. The fluorescing mitochondria can be easily detected under a fluorescence microscope. By photoaffinity labelling we were able to show that at low dye concentrations APMC preferentially marks four proteins with apparent molecular masses of 31, 40, 66, and 74 kDa. In order to establish that these are mitochondrial proteins, we isolated and analysed the mitochondria from incubated HeLa and LM cells; again, the same four proteins were detected. They are most probably proteins of the inner mitochondrial membranes, which accumulate the lipophilic APMC cations.     

Interactions of molecular probes with living cells and tissues. Part 1. Some general mechanistic proposals, making use of a simplistic Chinese box model

A simple and generalised model-termed the simplistic Chinese box [SCB] model-for the interaction of molecular probes with living systems is described. The SCB model includes the following assumptions. That living systems may be considered as built from biologically defined boxes, e.g. whole cell, nucleus, nucleoli. That movement of molecular probes into and through these boxes is strongly influenced by box wall permeability, which in turn is largely dependent on the simple physicochemical properties of probe and wall. That retention of probes in boxes is influenced by permeability of the walls and by trapping of probes by boxes and walls, the latter effect also being strongly dependent on simple physicochemical properties. That important physicochemical properties include electric charge, hydrophilicity/lipophilicity, non-specific protein binding, and molecular size. That, since all these factors can be expressed or modelled numerically, SAR methodology is an appropriate technique for analysing molecular probe investigations.     

Effects of mitochondria-selective fluorescent probes on mitochondrial movement in Arabidopsis mesophyll cells evaluated by using the quantification

Mitochondria-selective fluorescent probes such as MitoTracker are often used for mitochondria imaging in various plants. Although some of the probes are reported to induce mitochondria dysfunction in animal cells, the effect on plant cells remains to be determined. In the present study, we applied quantitative methods to analyze mitochondrial movement, speed frequency, and speed-angle changes, based on trajectory analysis of mitochondria in mesophyll protoplast cells of Arabidopsis thaliana expressing the mitochondria-localized fluorescent protein. Using the quantitative method, we assessed whether MitoTracker Red (FM and CMXRos) induce mitochondria dysfunction in A. thaliana. Although both the fluorescent probes well-stained mitochondria, the CMXRos probe, not the FM probe, gave a severe effect on mitochondrial movement at the low concentration (10 nM), indicating a MitoTracker-induced mitochondria dysfunction in A. thaliana. These results revealed that our quantitative method based on mitochondrial movement can be used to determine the appropriate concentrations of mitochondria-selective fluorescent probes in plants.     

Passive Membrane Penetration of the Serotonin Precursor 5‐Hydroxytryptophan is Controlled by Its Zwitterion

Species‐specific partition coefficients in the octanol/water system were determined for the neurotransmitter serotonin (5‐HT) and its precursor 5‐hydroxytryptophan (5‐HTP). The pH‐independent partition coefficients (p) of the individual microspecies were determined by combination of experimentally measured distribution constants and a custom‐tailored evaluation method, using highly similar auxiliary compounds. Experimental microscopic partition coefficients for triprotic molecules have only been reported before for thyroxine and its derivatives. The parabolic pH‐distribution profile of 5‐HT shows the dominance of the lipophilic non‐charged microspecies, with a log p of 0.66. However, the most lipophilic non‐charged form of 5‐HTP, with a log p of 0.31, has no significant contribution to the distribution coefficient at any pH value. Instead, the less lipophilic zwitterionic protonation isomer dominates the distribution in the pH range 2.10 – 11.11. Although the non‐charged microspecies of 5‐HTP is 151 times more lipophilic than its zwitterionic protonation isomer, the overwhelming dominance of the zwitterionic form ensures that its contribution to the overall lipophilicity exceeds 1320 times that of the non‐charged one. This fact is another counter‐example of the widespread belief that passive diffusion into lipophilic media is predominated by the non‐charged species. The lipophilicity profile of 5‐HT and 5‐HTP is depicted in terms of species‐specific lipophilicities.