Drug uptake, lipid rafts, and vesicle trafficking modulate resistance to an anticancer lysophosphatidylcholine analogue in yeast.

The ether-phospholipid edelfosine, a prototype antitumor lipid (ATL), kills yeast cells and selectively kills several cancer cell types. To gain insight into its mechanism of action, we performed chemogenomic screens in the Saccharomyces cerevisiae gene-deletion strain collection, identifying edelfosine-resistant mutants. LEM3, AGP2, and DOC1 genes were required for drug uptake. Edelfosine displaced the essential proton pump Pma1p from rafts, inducing its internalization into the vacuole. Additional ATLs, including miltefosine and perifosine, also displaced Pma1p from rafts to the vacuole, suggesting that this process is a major hallmark of ATL cytotoxicity in yeast. Radioactive and synthetic fluorescent edelfosine analogues accumulated in yeast plasma membrane rafts and subsequently the endoplasmic reticulum. Although both edelfosine and Pma1p were initially located at membrane rafts, internalization of the drug toward endoplasmic reticulum and Pma1p to the vacuole followed different routes. Drug internalization was not dependent on endocytosis and was not critical for yeast cytotoxicity. However, mutants affecting endocytosis, vesicle sorting, or trafficking to the vacuole, including the retromer and ESCRT complexes, prevented Pma1p internalization and were edelfosine-resistant. Our data suggest that edelfosine-induced cytotoxicity involves raft reorganization and retromer- and ESCRT-mediated vesicular transport and degradation of essential raft proteins leading to cell death. Cytotoxicity of ATLs is mainly dependent on the changes they induce in plasma membrane raft-located proteins that lead to their internalization and subsequent degradation. Edelfosine toxicity can be circumvented by inactivating genes that then result in the recycling of internalized cell-surface proteins back to the plasma membrane.

A BODIPY-embedding miltefosine analog linked to cell-penetrating Tat(48-60) peptide favors intracellular delivery and visualization of the antiparasitic drug.

Therapeutic application of many drugs is often hampered by poor or denied access to intracellular targets. A case in point is miltefosine (MT), an orally active antiparasitic drug, which becomes ineffective when parasites develop dysfunctional uptake systems. We report here the synthesis of a fluorescent BODIPY-embedding MT analogue with appropriate thiol functionalization allowing linkage to the cell-penetrating Tat(48-60) peptide through disulfide or thioether linkages. The resulting constructs are efficiently internalized into the otherwise MT-invulnerable R40 Leishmania strain, resulting in fast parasite killing, and hence successful avoidance of the resistance. In the disulfide-linked conjugate, an additional fluoro tag on the Tat moiety allows to monitor its reductive cleavage within the cytoplasm. Terminally differentiated cells such as peritoneal macrophages, impervious to MT unless infected by Leishmania, can uptake the drug in its Tat-conjugated form. The results afford proof-of-principle for using CPP vectors to avert drug resistance in parasites, and/or for tackling leishmaniasis by modulating macrophage uptake.

Synthesis and photophysics of novel biocompatible fluorescent oxocines and azocines in aqueous solution.

The spectroscopic properties in water solution of the different prototropic forms of the strongly fluorescent hemiacetal 4,9-dihydroxy-1,2-dihydro-4,11a-methanooxocino[4,5-b]benzofuran-5(4H)-one (1a, monardine), the aza analogue 4,9-dihydroxy-3,4-dihydro-1H-4,11a-methanobenzofuro[2,3-d]azocin-5(2H)-one (2a, azamonardine) and the respective 2-carboxyl derivatives (1b, 2b) have been studied by experimental and quantum-chemical methods. Monardine and carboxymonardine are the major products of new fluorogenic, room-temperature reactions of hydroxytyrosol or salvianic acid in aqueous solution, respectively, and present unique photophysical properties. Near neutral pH (pKa = 7.2) monardine switches from a weakly emitting, UV-absorbing (382 nm) neutral species to a VIS-absorbing (426 nm), blue emitting (464 nm) anion form, with a fluorescence quantum yield ϕF = 1 and single-exponential decay τF = 2.74 ns. This binary-like spectroscopic change from the neutral to the anionic form was interpreted based on time-dependent density functional theory (TDDFT) calculations as due to (i) the reversal of (n,π*) and (π,π*) lowest-lying singlet excited states, and (ii) a change in the triplet-state distribution accompanying monardine ionization which may abolish de-excitation via intersystem crossing. A similar fluorogenic reaction takes place with catecholamines such as dopamine and DOPA, to yield fluorescent azocines 2a and 2b which, depending on pH, may be present as cationic, neutral or anionic species. TDDFT computations of these forms were also carried out to assign the corresponding excitation transitions and emission properties. Besides the analytical interest of the fluorogenic reactions, the photochemical stability and biocompatibility of the bright-dark pH-controlled molecular switches 1a and 1b may facilitate novel labels and probes to be developed for superresolution fluorescence microscopy.

Intracellular triggering of Fas aggregation and recruitment of apoptotic molecules into Fas-enriched rafts in selective tumor cell apoptosis.

We have discovered a new and specific cell-killing mechanism mediated by the selective uptake of the antitumor drug 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH(3), Edelfosine) into lipid rafts of tumor cells, followed by its coaggregation with Fas death receptor (also known as APO-1 or CD95) and recruitment of apoptotic molecules into Fas-enriched rafts. Drug sensitivity was dependent on drug uptake and Fas expression, regardless of the presence of other major death receptors, such as tumor necrosis factor (TNF) receptor 1 or TNF-related apoptosis-inducing ligand R2/DR5 in the target cell. Drug microinjection experiments in Fas-deficient and Fas-transfected cells unable to incorporate exogenous ET-18-OCH(3) demonstrated that Fas was intracellularly activated. Partial deletion of the Fas intracellular domain prevented apoptosis. Unlike normal lymphocytes, leukemic T cells incorporated ET-18-OCH(3) into rafts coaggregating with Fas and underwent apoptosis. Fas-associated death domain protein, procaspase-8, procaspase-10, c-Jun amino-terminal kinase, and Bid were recruited into rafts, linking Fas and mitochondrial signaling routes. Clustering of rafts was necessary but not sufficient for ET-18-OCH(3)-mediated cell death, with Fas being required as the apoptosis trigger. ET-18-OCH(3)-mediated apoptosis did not require sphingomyelinase activation. Normal cells, including human and rat hepatocytes, did not incorporate ET-18-OCH(3) and were spared. This mechanism represents the first selective activation of Fas in tumor cells. Our data set a framework for the development of more targeted therapies leading to intracellular Fas activation and recruitment of downstream signaling molecules into Fas-enriched rafts.

The phenyltetraene lysophospholipid analog PTE-ET-18-OMe as a fluorescent anisotropy probe of liquid ordered membrane domains (lipid rafts) and ceramide-rich membrane domains.

The conjugated phenyltetraene PTE-ET-18-OMe (all-(E)-1-O-(15'-phenylpentadeca-8',10',12',14'-tetraenyl)-2-O-methyl-rac-glycero-3-phosphocholine) is a recently developed fluorescent lysophospholipid analog of edelfosine, (Quesada et al. (2004) J. Med. Chem. 47, 5333-5335). We investigated the use of this analog as a probe of membrane structure. PTE-ET-18-OMe was found to have several properties that are favorable for fluorescence anisotropy (polarization) experiments in membranes, including low fluorescence in water and moderately strong association with lipid bilayers. PTE-ET-18-OMe has absorbance and fluorescence properties similar to those of diphenylhexatriene (DPH) probes, with about as large a difference between its fluorescence anisotropy in liquid disordered (Ld) and ordered states (gel and Lo) as observed for DPH. Also like DPH, PTE-ET-18-OMe has a moderate affinity for both gel state ordered domains and Lo state ordered domains (rafts). However, unlike fluorescent sterols or DPH (Megha and London (2004) J. Biol. Chem. 279, 9997-10004), PTE-ET-18-OMe is not displaced from ordered domains by ceramide. Also unlike DPH, PTE-ET-18-OMe shows only slow exchange between the inner and outer leaflets of membrane bilayers, and can thus be used to examine anisotropy of an individual leaflet of a lipid bilayer. Since PTE-ET-18-OMe is a zwitterionic molecule, it should not be as influenced by electrostatic interactions as are other probes that do not cross the lipid bilayer but have a net charge. We conclude that PTE-ET-18-OMe has some unique properties that should make it a useful fluorescence probe of membrane structure.

Synthesis of BODIPY-labeled alkylphosphocholines with leishmanicidal activity, as fluorescent analogues of miltefosine.

Two general synthetic methods are described, by which the highly fluorescent and photostable BODIPY group can be inserted in and aligned with the alkyl backbone of linear lipids. These methods have been used to prepare strongly emitting analogues of the leishmanicidal drug miltefosine, in which the antiparasite activity in vitro of the original drug is preserved.

Photodegradation of bisphenol A and related compounds under natural-like conditions in the presence of riboflavin: kinetics, mechanism and photoproducts.

The aerobic riboflavin (Rf)-sensitized photodegradation of the endocrine disruptor 4,4'-isopropylidenebisphenol (bisphenol A, BPA), and of the related compounds 4,4'-isopropylidenebis(2,6-dibromophenol) and 4,4'-isopropylidenebis(2,6-dimethylphenol) has been studied in water and water-methanol mixtures through visible-light continuous photolysis, polarographic detection of oxygen uptake, stationary and time-resolved fluorescence spectroscopy, time-resolved near-IR phosphorescence detection and laser flash photolysis techniques. Bisphenols (BPs) quench excited singlet and triplet states of Rf, with rate constants close to the diffusion limit. BPs and dissolved molecular oxygen, employed in similar concentrations, competitively quench triplet excited Rf. As a consequence, superoxide radical anion and singlet molecular oxygen (O2(1Deltag)) are produced by electron- and energy-transfer processes, respectively, as demonstrated by auxiliary experiments employing selective quenchers of both oxidative species and the exclusive O2(1Deltag) generator Rose Bengal. As a global result, the photodegradation of Rf is retarded, whereas BPs are degraded, mainly by an O2(1Deltag)-mediated mechanism, which constitutes a relatively efficient process in the case of BPA. Oxidation, dimerization and fragmentation products have been identified in the photooxidation of BPA. Results indicate that BPs in natural waters can undergo spontaneous photodegradation under environmental conditions in the presence of adequate photosensitizers.

Synthesis and biological evaluation of fluorescent leishmanicidal analogues of hexadecylphosphocholine (miltefosine) as probes of antiparasite mechanisms.

The leishmanicidal mechanism of miltefosine (hexadecylphosphocholine, MT) is not clearly understood. Valuable insights into its mode of action could be obtained by fluorescence techniques, given suitably emitting analogues. In this regard, the synthesis and biological characterization of two fully competent MT fluorescent analogues is reported here: all-(E)-13-phenyltrideca-6,8,10,12-tetraenylphosphocholine (PTE-MT) and all-(E)-13-phenyltrideca-8,10,12-trien-6-ynylphosphocholine (PTRI-MT). Both compounds show large absorption coefficients and a modest, but usable, fluorescence yield. Their activities were very similar to that of MT and were recognized by the MT uptake system of Leishmania. Their localization in living L. donovani promastigotes by confocal microscopy show a homogeneous intracellular distribution of the fluorescence. The concentration of PTRI-MT within the parasites (ca. 1.7 mM) showed a 100-fold enrichment relative to its external concentration. These results are consistent with a multiple target leishmanicidal mechanism for MT and validate the application of these analogues for pharmacokinetic and diagnostic studies concerning the chemotherapy of leishmaniasis.

Synthesis, photophysical properties, and laser behavior of 3-amino and 3-acetamido BODIPY dyes.

The asymmetrically substituted BODIPY dyes 9a and 9b have been synthesized through a key redox step involving the alpha-nitroso derivative of the starting pyrrol. Both dyes emit fluorescence with quantum yields of ca. 0.7, but only 8b behaves as a good laser dye, with an efficiency of 48% in ethanol solution.

Kinetics and mechanism of the sensitized photodegradation of uracil--modeling the fate of related herbicides in aqueous environments.

The dye-sensitized photodegradation of uracil (UR), the parent compound of several profusely employed herbicides, has been studied as a model of their environmental fate. In order to mimic conditions frequently found in nature, aqueous solutions of UR have been irradiated with visible light in the presence of the natural sensitizer riboflavin (Rf). The results indicate that UR is photostable in acid media, but is quickly degraded in pH 7 or pH 9 solutions, where singlet molecular oxygen [O2(1Delta(g))] and, to a lesser extent, superoxide radical anion (O2*-)-both species photogenerated from triplet excited Rf, 3Rf*-participate in the photodegradation. At pH 7, UR is slowly degraded through an O2*- -mediated mechanism, whereas Rf disappears through its reaction with O2(1Delta(g)) and, in the form of 3Rf*, with UR. On the contrary, at pH 9 Rf is photoprotected through two processes: its regeneration from the formed Rf radical species-a back electron transfer that also produces O2*- -and the elimination from the medium of O2(1Delta(g)) by its reaction with UR. The overall result of the preservation of ground state Rf is the continuity of the photosensitized process and, hence, of the UR degradation. Media with higher pH values could not be employed due to the fast photodegradation of Rf. With rose bengal (RB) as photosensitizer, the rate constants found for the overall interaction between UR and the photogenerated O2(1Delta(g)) were in the range 5 x 10(5) M(-1) s(-1) (at pH 7) to 1.3 x 10(8) M(-1) s(-1) (in 1 M NaOH aqueous solution, mainly physical quenching). The maximum O2(1Delta(g)0-mediated photooxidation efficiencies with RB were reached at pH 11, where only the O2(1Delta(g)0-reactive quenching with UR was observed.

Dynamics of bolaamphiphilic fluorescent polyenes in lipid bilayers from polarization emission spectroscopy.

The rotational motions of the biamphiphilic polyenes (bolapolyenes) dimethyl all-(E)-octacosa-10,12,14,16,18-pentaenedioate (DE28:5) and dimethyl all-(E)-tetratriaconta-13,15,17,19,21-pentaenedioate (DE34:5), with head-to-head distances of 34 and 42A, respectively, have been examined by fluorescence anisotropy methods. The membrane-spanning bolapolyenes, which contain a central emitting pentaene group tethered to two methoxycarbonyl opposite polar heads by symmetric C(8) (DE28:5) and C(11) (DE34:5) polymethylene chains, were dispersed in lipid bilayers of DPPC or DMPC, and the stationary and picosecond-resolved emission was recorded as a function of temperature. In fluid-phase DMPC bilayers, three relaxation times could be determined, assigned to fast (0.2 and 2ns) single-bond isomerization processes localized on the alkyl chains, and to whole-molecule oscillations ( approximately 11ns), respectively. The anisotropy decay parameters were further analyzed in terms of a diffusive model for wobbling in a Gaussian ordering potential, to assess the anchoring effect of the symmetric polar heads. In this way, the average rotational diffusion constant of the bolapolyenes, D( perpendicular), could be estimated as 0.022-0.026rad(2) ns(-1) (DMPC bilayers, 35 degrees Celsius), a value that is only 1/3 of that corresponding to the related pentaene fatty acid spanning a single membrane monolayer. In contrast, the amplitude of the equilibrium orientational distribution (theta(half-cone) approximately 50 degrees ) is very similar for both the transmembrane and the single-headed polyenes. The reorientational oscillations of the central emitting group in the bolapolyenes necessarily would produce large-amplitude (2-5A) and very fast (ns) translational motions of the polar heads.

Dye-sensitized photodegradation of the fungicide carbendazim and related benzimidazoles.

The present work studies the visible-light-promoted photodegradation of the colorless fungicide carbendazim (methyl 2-benzimidazolecarbamate) and several 2-substituted benzimidazoles (SBZ's), in water or water-methanol solution, in the presence of air and, as a photosensitizer, the synthetic xanthene dye Rose Bengal (RB) or the natural pigment riboflavin (Rf). The results indicate that the degradation of each particular SBZ depends on its chemical structure and on the sensitizer employed. In the presence of RB, the degradation always operates via a singlet molecular oxygen (O(2)((1)Delta(g)))-mediated mechanism, through a highly efficient process, as deduced from the comparison of the rate constants for physical and chemical quenching of O(2)((1)Delta(g)). In the presence of Rf, the visible-light irradiation of any of the studied SBZ's produces a series of competitive processes that depend on the relative concentrations of Rf and SBZ. These processes include the quenching of excited singlet and triplet Rf states by the SBZ and the generation of both O(2)((1)Delta(g)) and superoxide radical anion (O(2)(-)), the latter generated by electron transfer from excited Rf species to the dissolved oxygen. The overall result is the photodegradation of the SBZ and the photoprotection of the sensitizer.

Photodegradation of hydroxylated N-heteroaromatic derivatives in natural-like aquatic environments. A review of kinetic data of pesticide model compounds.

In the present review, the results published by our group and others related with the study of the kinetic behavior and, in some cases, the mechanism of the dye-promoted photooxygenation of several hydroxypyridines, hydroxyquinolines and hydroxypyrimidines--some of them with the basic molecular structures of known pesticides, and of a few non-hydroxylated model compounds, are compiled and discussed. The main aim was to examine the experimental conditions that maximize the photodegradation efficiencies of all these compounds, under dye-sensitized photooxidation conditions similar to those frequently found in nature, with a natural dye sensitizer such as riboflavin (vitamin B2), a pigment habitually present in natural waters. The usual mechanism of action of this compound is rather complex, in many cases with the concurrent involvement of the oxidative species singlet molecular oxygen (O2(1Deltag)) and superoxide radical anion. In order to simplify the study of the processes, the results found using the synthetic dye Rose Bengal (RB), a sensitizer that generates O2(1Deltag) with high efficiency, are also discussed. RB and similar O2(1Deltag)-generators could be used for the efficient non-natural photodegradation of related pesticides in aqueous solutions under controlled conditions.

The solid state, solution and tubulin-bound conformations of agents that promote microtubule stabilization.

Taxol (paclitaxel), a complex diterpene obtained from Taxus brevifolia and its semisynthetic analogue Taxotere are two of the most important new drugs for cancer chemotherapy. Their mechanism of cytotoxic action involves stabilization of microtubules leading to mitotic arrest. A similar mechanism has been proposed for an expanding set of other natural products, for instance, the epothilones, eleutherobin, the sarcodictyins, discodermolide, laulimalide, Rhazinilam, WS9885B, certain steroids and a group of polyisoprenyl benzophenones. In this review, we focus on the conformations of small molecule microtubule (MT) stabilizing compounds which have been isolated or synthesized and subjected to structural analysis. NMR and fluorescense spectroscopies, X-ray crystallography, high resolution microscopy (electron crystallography) and theoretical calculations comprise the most common methods used in this context. In particular, we describe how the structures were determined and with what accuracy. We also discuss the conformational diversity apparent from the three dimensional structures and compare the various proposals for bioactive conformations at the target MT binding sites. Of critical importance are the recently disclosed models for Taxol and its biomimetics binding to beta-tubulin. Several different conformational schemes derived from both pharmacophore construction and modeled protein ligand complexes are compared and critically evaluated. Although full consensus has yet to be reached, emphasis is placed on pharmacophore models for the various anti-MT agents that are internally consistent and encompass more than one structural class.

Fluorescent phenylpolyene analogues of the ether phospholipid edelfosine for the selective labeling of cancer cells.

Edelfosine (ET-18-OCH3), a synthetic antitumor ether lipid, is taken up by malignant but not by normal cells, triggering apoptosis in a large variety of human tumor cells. The synthesis of the first fluorescent edelfosine analogue (6), with apoptotic activity comparable to that of the parent drug, is described. Fluorescence microscopy experiments show that 6 selectively labels human cancer cells, accumulating into specific domains of the plasma membrane.

Methacrylate-tethered analogs of the laser dye PM567--synthesis, copolymerization with methyl methacrylate and photostability of the copolymers.

The synthesis and characterization of new analogs of the laser dye PM567 (4,4-difluoro-1,3,5,7,8-pentamethyl-2,6-diethyl-4-bora-3a,4a-diaza-s-indacene) with the 8-position substituted by a linear chain with n methylenes (n = 1, 3, 5, 10 or 15) tethered with an acetoxy or methacryloyloxy group (PnAc and PnMA, respectively) is described. The monomeric dyes PnMA have been successfully copolymerized with methyl methacrylate (MMA), yielding linear copolymers of high optical quality where the covalently bonded chromophore is separated from the polymeric main chain by a spacer of variable length. The photostability of the solid polymeric materials under UV (310 nm) irradiation (method ASTM G 53-77) has been compared with those of the model dyes PnAc and PM567 as solid solutions in poly-MMA (PnAc-PMMA and PM567-PMMA, respectively). In all the cases, the chromophore bound to the polymer is more photostable than that simply dissolved in the same polymer, with photodegradation quantum yields in the range 2.3 x 10(-5) to 4.8 x 10(-5), which was interpreted as due to additional modes for the dissipation of the absorbed energy along the polymeric chain. In both polymer solutions and copolymers, the length of the polymethylene chain has low or null influence on the photostability. In ethanol solution, PnAc model dyes with polymethylene chains with three or more methylene groups show about the same photostability; this is of an order of magnitude higher than that of the parent dyes P1Ac and PM567 in the same solvent.

Rose Bengal-sensitized photooxidation of the dipeptides L-tryptophyl-L-phenylalanine, L-tryptophyl-L-tyrosine and L-tryptophyl-L-tryptophan: kinetics, mechanism and photoproducts.

The Rose Bengal-sensitized photooxidations of the dipeptides l-tryptophyl-l-phenylalanine (Trp-Phe), l-tryptophyl-l-tyrosine (Trp-Tyr) and l-tryptophyl-l-tryptophan (Trp-Trp) have been studied in pH 7 water solution using static photolysis and time-resolved methods. Kinetic results indicate that the tryptophan (Trp) moiety interacts with singlet molecular oxygen (O(2)((1)Delta(g))) both through chemical reaction and through physical quenching, and that the photooxidations can be compared with those of equimolecular mixtures of the corresponding free amino acids, with minimum, if any, influence of the peptide bond on the chemical reaction. This is not a common behavior in other di- and polypeptides of photooxidizable amino acids. The ratio between chemical (k(r)) and overall (k(t)) rate constants for the interaction O(2)((1)Delta(g))-dipeptide indicates that Trp-Phe and Trp-Trp are good candidates to suffer photodynamic action, with k(r)/k(t) values of 0.72 and 0.60, respectively (0.65 for free Trp). In the case of Trp-Tyr, a lower k(r)/k(t) value (0.18) has been found, likely as a result of the high component of physical deactivation of O(2)((1)Delta(g)) by the tyrosine moiety. The analysis of the photooxidation products shows that the main target for O(2)((1)Delta(g)) attack is the Trp group and suggests a much lower accumulation of kynurenine-type products, as compared with free Trp. This is possibly because of the occurrence of another accepted alternative pathway of oxidation that gives rise to 3a-oxidized hydrogenated pyrrolo[2,3-b]indoles.

New Transmembrane Polyene Bolaamphiphiles as Fluorescent Probes in Lipid Bilayers.

Inspired by Archaebacterial lipids, transmembrane probes anchor a sensing fluorescent polyene with Ångström resolution deep within a lipid layer. These new bolaamphiphiles are obtained in good yields from a double cross-coupling between esters with a terminal acetylene group and conjugated 1,ω-dihalopolyenes, followed by partial reduction of the triple bond.

Defeating Leishmania resistance to miltefosine (hexadecylphosphocholine) by peptide-mediated drug smuggling: a proof of mechanism for trypanosomatid chemotherapy.

Miltefosine (hexadecylphosphocholine, HePC), the first orally active drug successful against leishmaniasis, is especially active on the visceral form of the disease. Resistance mechanisms are almost exclusively associated to dysfunction in HePC uptake systems. In order to evade the requirements of its cognate receptor/translocator, HePC-resistant Leishmania donovani parasites (R40 strain) were challenged with constructs consisting of an ω-thiol-functionalized HePC analogue conjugated to the cell-penetrating peptide (CPP) Tat(48-60), either through a disulfide or a thioether bond. The conjugates enter and kill both promastigote and intracellular amastigote forms of the R40 strain. Intracellular release of HePC by reduction of the disulfide-based conjugate was confirmed by means of double tagging at both the CPP (Quasar 670) and HePC (BODIPY) moieties. Scission of the conjugate, however, is not mandatory, as the metabolically more stable thioether conjugate retained substantial activity. The disulfide conjugate is highly active on the bloodstream form of Trypanosoma b. brucei, naturally resistant to HePC. Our results provide proof-of-mechanism for the use of CPP conjugates to avert drug resistance by faulty drug accumulation in parasites, as well as the possibility to extend chemotherapy into other parasites intrinsically devoid of membrane translocation systems.

Fluorescent labeling of Acanthamoeba assessed in situ from corneal sectioned microscopy.

Acanthamoeba keratitis is a serious pathogenic corneal disease, with challenging diagnosis. Standard diagnostic methods include corneal biopsy (involving cell culture) and in vivo reflection corneal microscopy (in which the visualization of the pathogen is challenged by the presence of multiple reflectance corneal structures). We present a new imaging method based on fluorescence sectioned microscopy for visualization of Acanthamoeba. A fluorescent marker (MT-11-BDP), composed by a fluorescent group (BODIPY) inserted in miltefosine (a therapeutic agent against Acanthamoeba), was developed. A custom-developed fluorescent structured illumination sectioned corneal microscope (excitation wavelength: 488 nm; axial/lateral resolution: 2.6 µm/0.4-0.6 µm) was used to image intact enucleated rabbit eyes, injected with a solution of stained Acanthamoeba in the stroma. Fluorescent sectioned microscopic images of intact enucleated rabbit eyes revealed stained Acanthamoeba trophozoites within the stroma, easily identified by the contrasted fluorescent emission, size and shape. Control experiments show that the fluorescent maker is not internalized by corneal cells, making the developed marker specific to the pathogen. Fluorescent sectioned microscopy shows potential for specific diagnosis of Acanthamoeba keratitis. Corneal confocal microscopy, provided with a fluorescent channel, could be largely improved in specificity and sensitivity in combination with specific fluorescent marking.