Red Cell Ghosts Research Articles

Tangential flow filtration-facilitated purification of human red blood cell membrane fragments and its preferential use in removing unencapsulated material from resealed red blood cell ghosts compared to centrifugation.

The biodistribution of many therapeutics is controlled by the immune system. In addition, some molecules are cytotoxic when not encapsulated inside of larger cellular structures, such as hemoglobin (Hb) encapsulation inside of red blood cells (RBCs). To counter immune system recognition and cytotoxicity, drug delivery systems based on red blood cell membrane fragments (RBCMFs) have been proposed as a strategy for creating immunoprivileged therapeutics. However, the use of RBCMFs for drug delivery applications requires purification of RBCMFs at large scale from lysed RBCs free of their intracellular components. In this study, we were able to successfully use tangential flow filtration (TFF) to remove >99% of cell-free Hb from lysed RBCs at high concentrations (30%-40% v/v), producing RBCMFs that were 2.68 ± 0.17 μm in diameter. We were also able to characterize the RBCMFs more thoroughly than prior work, including measurement of particle zeta potential, along with individual TFF diacycle data on the cell-free Hb concentration in solution and time per diacycle, as well as concentration and size of the RBCMFs. In addition to purifying RBCMFs from lysed RBCs, we utilized a hypertonic solution to reseal purified RBCMFs encapsulating a model protein (Hb) to yield resealed Hb-encapsulated RBC ghosts (Hb-RBCGs). TFF was then compared against centrifugation as an alternative method for removing unencapsulated Hb from Hb-RBCGs, and the effects that each washing method on the resulting Hb-RBCG biophysical properties was assessed.

Energy Transfer on Cytoskeleton of Red Blood Cell Ghosts and their Efficiency Control by KCl Concentration-induced Cell Shrinkage.

Lipid bilayer membranes such as liposomes have been utilized as platforms for bioinspired artificial photosynthesis. Embedding functional compounds, including chromophores and catalysts, into two-dimensional lipid membranes allows their high local concentration and proximity, resulting in enhanced reactivity compared to that of homogeneous solutions. The control of photoreactions by the physical and chemical properties of membranes, such as fluidity and phase separation, has also been well studied in recent years. In contrast, it remains difficult to control chemical reactions via dynamic membrane deformation. Here, we report on the control of excitation energy transfer using red blood cell ghosts (RBCGs) as scaffolds, relying on their asymmetric lipid membranes and inherent and unique deformability. RBCGs, in which donor and acceptor molecules were chemically conjugated to a two-dimensional cytoskeleton located beneath the inner membrane, exhibited energy transfer, and their efficiency varied depending on the amount and ratio of donor and acceptor modifications, as confirmed by experimental and theoretical analysis. Furthermore, the KCl concentration-induced RBCG shrinkage enhanced the energy transfer efficiency. Our proposed method is expected to facilitate the construction of photoreaction systems that can be controlled via membrane deformation.

Erythrocyte Membrane Cloaked Cytokine Functionalized Gold Nanoparticles Create Localized Controlled Inflammation for Rapid In Vitro Wound Healing.

Due to impaired wound healing, millions of acute and chronic wound cases with increased morbidity have been recorded in the developed countries. The primary reason has been attributed to uncontrolled inflammation at the wound site, which makes healing impossible for years. The use of red blood cell (RBC) ghosts or erythrocyte membranes for different theranostic applications has gained significant attention in recent years due to their biocompatibility and biomimicking properties. Our study builds upon this concept by presenting a new approach for creating an improved and controlled inflammatory response by employing RBC ghost encapsulated tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) modified AuNPs (gold nanoparticles) for accelerating the wound healing at early postinjury stage (∼48 h). The results suggested that the developed GTNFα-IL6@AuNPs created a controlled and time dependent TNF-α response and showed increased reactive oxygen species generation at ∼12 h. Further, proper M1/M2 functional transition of macrophages was observed in macrophages at different time intervals. The expression results suggested that the levels of wound healing biomarkers like transforming growth factor-β (1.8-fold) and collagen (2.4-fold) increased while matrix metalloproteinase (3-8-fold) levels declined at later stages, which possibly increased the cell migration rate of NP treated cells to ∼90%. Hence, we are here reducing the timeline of the inflammatory phase of wound healing by actually creating a controlled inflammatory response at an early postinjury stage and further assisting in regaining the ability of cells for wound remodelation and repair. We intend that this new approach has the potential to improve the current treatment strategies for wound healing and skin repair under both in vitro and in vivo conditions.

Plasmalogens: Free Radical Reactivity and Identification of Trans Isomers Relevant to Biological Membranes.

Plasmalogens are membrane phospholipids with two fatty acid hydrocarbon chains linked to L-glycerol, one containing a characteristic cis-vinyl ether function and the other one being a polyunsaturated fatty acid (PUFA) residue linked through an acyl function. All double bonds in these structures display the cis geometrical configuration due to desaturase enzymatic activity and they are known to be involved in the peroxidation process, whereas the reactivity through cis-trans double bond isomerization has not yet been identified. Using 1-(1Z-octadecenyl)-2-arachidonoyl-sn-glycero-3-phosphocholine (C18 plasm-20:4 PC) as a representative molecule, we showed that the cis-trans isomerization can occur at both plasmalogen unsaturated moieties, and the product has characteristic analytical signatures useful for omics applications. Using plasmalogen-containing liposomes and red blood cell (RBC) ghosts under biomimetic Fenton-like conditions, in the presence or absence of thiols, peroxidation, and isomerization processes were found to occur with different reaction outcomes due to the particular liposome compositions. These results allow gaining a full scenario of plasmalogen reactivity under free radical conditions. Moreover, clarification of the plasmalogen reactivity under acidic and alkaline conditions was carried out, identifying the best protocol for RBC membrane fatty acid analysis due to their plasmalogen content of 15-20%. These results are important for lipidomic applications and for achieving a full scenario of radical stress in living organisms.

Effect of Mg-Gluconate on the Osmotic Fragility of Red Blood Cells, Lipid Peroxidation, and Ca2+-ATPase (PMCA) Activity of Placental Homogenates and Red Blood Cell Ghosts From Salt-Loaded Pregnant Rats.

Preeclampsia (PE) is a pregnancy-specific syndrome with multisystem involvement which leads to fetal, neonatal, and maternal morbidity and mortality. A model of salt-loaded pregnant rats has been previously studied, sharing several pathological characteristics of preeclamptic women. In this study, it was compared the effects of the treatment with an oral magnesium salt, magnesium gluconate (Mg-gluconate), on the osmotic fragility of red blood cells, lipid peroxidation, and PMCA activity of placental homogenates and red blood cell ghosts in salt-loaded pregnant rats. Mg-gluconate has a higher antioxidant capacity than MgSO4 due to the presence of several hydroxyl groups in the two anions of this salt. Salt-loaded pregnant rats received 1.8% NaCl solution ad libitum as a beverage during the last week of pregnancy. On day 22nd of pregnancy, the rats were euthanized and red blood cells and placenta were obtained. Salt-loaded pregnant rats showed an increased level of lipid peroxidation and a lowered PMCA activity in placental and red blood cell ghosts, as well as an increased osmotic fragility of their red blood cells. The treatment of the salt-loaded pregnant rats with Mg-gluconate avoids the rise in the level of lipid peroxidation and the concomitant lowering of the PMCA activity of their red blood cell membranes, reaching values similar to those from control pregnant rats. Also, this treatment prevents the increase of the osmotic fragility of their red blood cells, keeping values similar to those from control pregnant rats. Mg-gluconate seems to be an important candidate for the replacement of the MgSO4 treatment of preeclamptic women.

Peptide modification on the interior surface of red blood cell ghosts for construction of catalytic reactors.

We have succeeded in in situ synthesis of gold nanoparticles (Au NPs) on the interior surfaces of red blood cell ghosts (RBCGs) with a cytoskeleton chemically conjugated to a gold-binding peptide. The Au NP-deposited RBCG with the peptide exhibits enhanced catalytic reactivity for the reduction of 4-nitrophenol due to the uniform distribution of the Au NPs on the inner surface and the coverage of the Au NPs surface with the peptide.

Flow Cytometry Assay of Plasmodium Falciparum-Specific B-Cell Proportions.

Detection of P. falciparum-specific subpopulations of B-cells is important for studies of immunity in malaria. This protocol relies on the photostability and protein loading capacity of carboxylated quantum dots to detect a broad range of different P. falciparum-specific B-cells. Infected red blood cell ghosts, obtained by permeabilization of infected cells with Streptolysin O, are coupled with carboxyl quantum dots using N-ethyl-N-dimethylaminopropyl-carbodiimide condensation. Immunophenotyping of P. falciparum-specific B-cells is performed by flow cytometry using Fc-receptor block, quantum dot-infected red blood cell ghost conjugates, and fluorochrome-conjugated anti-human CD19 mouse monoclonal antibodies.

Broad lipid phase transitions in mammalian cell membranes measured by Laurdan fluorescence spectroscopy

Employing fluorescence spectroscopy and the membrane-embedded dye Laurdan we experimentally show that linear changes of cell membrane order in the physiological temperature regime are part of broad order-disorder-phase transitions which extend over a much broader temperature range. Even though these extreme temperatures are usually not object of live science research due to failure of cellular functions, our findings help to understand and predict cell membrane properties under physiological conditions as they explain the underlying physics of a broad order-disorder phase transition. Therefore, we analyzed the membranes of various cell lines, red blood cell ghosts and lipid vesicles by spectral decomposition in a custom-made setup in a temperature range from −40 °C to +90 °C. While the generalized polarization as a measure for membrane order of artificial lipid membranes like phosphatidylcholine show sharp transitions as known from calorimetry measurements, living cells in a physiological temperature range do only show linear changes. However, extending the temperature range shows the existence of broad transitions and their sensitivity to cholesterol content, pH and anaesthetic. Moreover, adaptation to culture conditions like decreased temperature and morphological changes like detachment of adherent cells or dendrite growth are accompanied by changes in membrane order as well. The observed changes of the generalized polarization are equivalent to temperature changes dT in the range of +12 K < dT < -6 K.

Estimation of Na\K - ATPase Enzyme Activity and Endogenous Digitalis in Patients with Diabetic Neuropathy

Background: Among the most common complications of diabetes is diabetic neuropathy (DN). Diabetic neuropathy is a heterogeneous group of disorders, which involves a different part of somatic and autonomic nervous systems, with a gradual loss of neural conductivity. Some studies have shown that they reduce the activity of the Na/K ATPase, however, elevated levels of endogenous sodium pump inhibitor in diabetic individuals, including those with neuropathy. Changes in this transfer enzyme are believed to be due to several diabetes complications. Objective: The study had designed to evaluate the Na/K ATPase enzymatic activity in the erythrocyte-membrane among three groups. The first group had represented the patients with type 2 diabetes mellitus (DM2) and neuropathy. The second group is diabetics without neuropathy. The third group was a healthy subject. As well, the study had estimated the inhibitory activity of endogenous digitalis among patient groups. Furthermore, the aim of this research was to see whether there was a connection between red blood cell membrane Na-K ATPase activity and the medical facts of the analysis subjects. Design and Methods: One-hundred fifty subjects had enrolled in this case-control study; 80 patients complained of diabetic neuropathy of both sexes, the mean age 59.3 years with an age range of 40-81, 40 DM2 without neuropathy (53.9 years), (35 – 70), and 30 healthy controls (30 years, 25 to 45). Patients in the first group were selected carefully according to their clinical manifestations and the nerve conduction study results. The evaluations of both inhibitory activities of endogenous digitalis and Na/K ATPase had completed using a spectrophotometer. Enzyme activity had expressed in micrograms of phosphate concentration per grams of red cell ghost total protein concentration. Results: The mean enzyme activity of Na/K ATPase was significantly lower (p&lt;0.001) in patients with diabetic neuropathy (381±17.9) compared with the diabetic group without neuropathy (498±22.9) and the normal controls (837±61.43). There was a significant inhibitory activity of endogenous digitals (17.87±2.15) in patients with DNP, compared with the diabetics without neuropathy (8.78±0.89) and healthy control (5.3±1.33). There was a significant association of enzyme activity with the following parameters: duration of diabetes, age, level of glycated hemoglobin and endogenous digitalis with the respective p-values (0.000, 0.000, 0.000 and 0.021). Gender showed no significant relationship with enzyme activity (p 0.43). Conclusions: In DM2 with neuropathy, hyperglycemia can much reduce the activity of erythrocyte Na/K ATPase. In addition, it may enhance the inhibitory activity of endogenous digitals. The timedependent increase in diabetic complications can be due to a strong association between diabetes duration and erythrocyte Na/K-ATPase activities.

The Kg‐antigen, RhAG with a Lys164Gln mutation, gives rise to haemolytic disease of the newborn

The Kg-antigen was first discovered in an investigation of a mother whose infant had haemolytic disease of the newborn (HDN). The antibody against the Kg-antigen is believed to be responsible for HDN. The Kg-antigen is provisionally registered under the number 700045, according to the Red Cell Immunogenetics and Blood Group Terminology. However, the molecular nature of the Kg-antigen has remained a mystery for over 30years. In this study, a monoclonal antibody against the Kg-antigen and the recombinant protein were developed that allowed for the immunoprecipitation analysis. Immunoprecipitants from the propositus' red blood cell ghosts were subjected to mass spectrometry analysis, and DNA sequence analysis of the genes was also performed. A candidate for the Kg-antigen was molecularly isolated and confirmed to be a determinant of the Kg-antigen by cell transfection and flow cytometry analyses. The Kg-antigen and the genetic mutation were then screened for in a Japanese population. The molecular nature of the Kg-antigen was shown to be RhAG with a Lys164Gln mutation. Kg phenotyping further clarified that 0.22% of the Japanese population studied was positive for the Kg-antigen. These findings provide important information on the Kg-antigen, which has been clinically presumed to give rise to HDN.

High-Speed Single-Cell Dielectric Spectroscopy.

Single-cell impedance cytometry is a label-free analysis technique that is now widely used to measure the electrical properties of a cell and to differentiate different subpopulations. Current techniques are limited to measuring the impedance of a single cell at one or two simultaneous frequencies. Also, there are no methods that extrapolate the intrinsic electrical properties of single cells. We demonstrate a new approach that uses multifrequency impedance measurements to determine the complete intrinsic electrical properties of thousands of single cells at high throughput. The applicability of the method is demonstrated by measuring the properties of red blood cells and red cell ghosts, deriving the unique values of conductivity and permittivity of the membrane and cytoplasm for each individual cell.

Two-step tumor-targeting therapy via integrating metabolic lipid-engineering with in situ click chemistry.

Highly efficient tumor-targeted therapy remains a great challenge due to the complexity and heterogeneity of tumor tissues. Herein, we developed an in vivo two-step tumor-targeting strategy by combining metabolic lipid-engineering with a stain-promoted azide-alkyne 1,3-dipolar cycloaddition (SPAAC) reaction, independent of the tumor microenvironment and cell phenotype. Firstly, exogenously-supplied azidoethyl-cholines (AECho) were metabolically incorporated into the cell membranes in tumor tissues through the intrinsic biosynthesis of phosphatidylcholine. The pre-inserted and accumulated azido groups (N3) could subsequently serve as 'artificial chemical receptors' for the specific anchoring of dibenzocyclooctyne (DBCO) modified biomimetic nanoparticles (DBCO-RBCG@ICG) via in situ click chemistry, resulting in significantly enhanced tumor-targeting and then an improved photothermal therapy effect. Such a two-step targeting strategy based on these cutting-edge techniques provided new insights into the universal and precise functionalization of living tissues for site-specific drug delivery in the diagnosis and treatment of various diseases.

Nanoerythrosomes tailoring: Lipid induced protein scaffolding in ghost membrane derived vesicles.

A peculiar polygonal protein scaffolding that resembles to spectrin-based skeleton of red blood cells can be reconstructed on the outer surface of vesicle-like nanoerythrosomes. The approximately 130 nm sized nanoerythrosomes are produced from red blood cell ghosts by addition of phospholipids (dipalmitoylphosphatidylcholine, DPPC). The scaffolding, constructed from the structural proteins of the cell membrane skeleton, covers the whole object resulting an enhanced stiffness. The protein pattern of the scaffolding is thermosensitive, reversible transformable in the biologically relevant temperature range. When the lipid additive is changed from DPPC to lysophospholipid (LPC), the protein network/scaffolding ceases to exist. By the variation of lipid type and ratio, a tailoring of the nanoerythrosomes can be achieved. During the tailoring process nanoerythrosomes or micelles, in a wide size range from 200 to 30 nm, are produced.

Anti tissue transglutaminase antibody in idiopathic autoimmune haemolytic anemia

BackgroundIn idiopathic autoimmune haemolytic anaemia (AIHA haemolytic antibodies are directed to every type of red cellsWestern blot studies have shown antibody positivity towards red cell anion channel complex which also includes band 4.2 a protein with similarities to tissue trans glutaminase. ObjectiveEvaluation of AIHA for anti tissue transglutaminase antibody (Anti tTG). Materials & methodsTwenty three AIHA patients were tested along with routine hamatogical work up, for a series of auto antibodies and red cell eluates and serum from the patents were tested against solubilised group O red cell ghosts on western blot. Other ancillary investigations were done to rule out complications and secondary causes of haemolysis. Results11/23 patients (48%) were positive for anti tTG, Four, 3 and 8,7 patients were positive for anti thyroid, anti b2 glycoprotein, lupus anticoagulant and ANA respectively. One patient with anti tTG had biopsy proven celiac disease. Three patient developed DVT and all of them were lupus anticoagulant as well as b2 gp-1 antibody positive.17 had become Coombs test negative on treatment while 21/23 had positive western blot test. Discussion & conclusionThere is strong association of anti tTG antibody with idiopathic AIHA. Aetiological association of this finding needs exploration.

Membrane order and ionic strength modulation of the inhibition of the membrane-bound acetylcholinesterase by epigallocatechin‑3‑gallate

In the present work, we analyzed how external factors can modulate the efficiency of epigallocatechin‑3‑O‑gallate (EGCG) inhibition of a membrane-bound isoform of the acetylcholinesterase. Increasing the ionic strength but not the osmolarity of the bulk medium proved to be an important factor. In addition, we verified a clear correlation between the inhibitory activity with the order degree of the membranes by using cholesterol-partially depleted red blood cell ghosts. These two factors i.e. high salt concentration in the bulk medium and less viscous membranes, allow a deeper insertion of the EGCG into the lipid bilayer, thus leading to a greater inhibition of AChE. As a corollary, we propose that any treatment or process that leads to a slight decrease in cholesterol content in the membranes can efficiently enhance the inhibitory activity of EGCG, which can have important consequences in all the pathologies where the inhibition of AChE is recommended.

Native Gel Electrophoresis Reveals Partial Physical Association of band 3 and aquaporins on the Erythrocyte Membrane

Band 3 is the most abundant membrane protein in human red cells. Band 3 primarily functions as a bi‐directional anion transporter for Cl− and HCO3− to support blood CO2 exchange. Because CO2 mainly exists in the form of HCO3− (aq) in the human body, the rate of CO2 blood respiration is dependent on the intraerythrocytic reaction CO2(g) + H2O □ HCO3− (aq) + H+ (aq). By FLIM‐FRET, we recently uncovered the existence of mobile interaction between band 3 and water channel AQP1. Their interaction could be disrupted when red cell vesicles were put in a hypotonic milieu, suggesting AQP1 bearing a functional role in the band 3‐central CO2 transport metabolon.ObjectiveAs there are at least two types of band 3 protein complexes on the erythrocyte membrane, the goal of this study was to probe the physical associations between aquaporins and different band 3 complexes.MethodWe utilized semi‐native and native gel electrophoresis to identify aquaporin complexes and to differentiate their associations with the different band 3 complexes.ResultsWater channel AQP1 and aquaglyceroporin AQP3 complexes were both identified in red cell ghosts by semi‐native gel electrophoresis. Some of the aquaporin complexes were found to be structurally linked to band 3 protein complexes.ConclusionThe physical association between AQP1 and band 3 and that between AQP3 and band 3 can be differentiated by semi‐native gel electrophoresis. Erythrocyte aquaporins also appear physically linked to other membrane‐associated proteins in the human red cells.Support or Funding InformationMOST 106‐2320‐B‐195‐002 from Taiwan Ministry of Science &amp; TechnologyThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impact of hydroxycarbamide and interferon-α on red cell adhesion and membrane protein expression in polycythemia vera.

Polycythemia vera is a chronic myeloproliferative neoplasm characterized by the JAK2V617F mutation, elevated blood cell counts and a high risk of thrombosis. Although the red cell lineage is primarily affected by JAK2V617F, the impact of mutated JAK2 on circulating red blood cells is poorly documented. Recently, we showed that in polycythemia vera, erythrocytes had abnormal expression of several proteins including Lu/BCAM adhesion molecule and proteins from the endoplasmic reticulum, mainly calreticulin and calnexin. Here we investigated the effects of hydroxycarbamide and interferon-α treatments on the expression of erythroid membrane proteins in a cohort of 53 patients. Surprisingly, while both drugs tended to normalize calreticulin expression, proteomics analysis showed that hydroxycarbamide deregulated the expression of 53 proteins in red cell ghosts, with overexpression and downregulation of 37 and 16 proteins, respectively. Within over-expressed proteins, hydroxycarbamide was found to enhance the expression of adhesion molecules such as Lu/BCAM and CD147, while interferon-α did not. In addition, we found that hydroxycarbamide increased Lu/BCAM phosphorylation and exacerbated red cell adhesion to its ligand laminin. Our study reveals unexpected adverse effects of hydroxycarbamide on red cell physiology in polycythemia vera and provides new insights into the effects of this molecule on gene regulation and protein recycling or maturation during erythroid differentiation. Furthermore, our study shows deregulation of Lu/BCAM and CD147 that are two ubiquitously expressed proteins linked to progression of solid tumors, paving the way for future studies to address the role of hydroxycarbamide in tissues other than blood cells in myeloproliferative neoplasms.

The Molecular Structure of Human Red Blood Cell Membranes From Highly Oriented, Solid Supported Multi-Lamellar Membranes

The preparation of Red Blood Cell (RBC) Ghosts is a well-known protocol in biological and medical research and describes the extraction of the membrane from RBCs. Another well-known protocol is the preparation of highly ordered stacks of artificial lipid bilayers on silicon wafers. There are various attempts to adapt this protocol to a native cell membrane. For the first time we were able to combine both described techniques and to prepare highly ordered stacks of RBC membranes on silicon wafers [1]. These systems can now be used as inexpensive and safe platforms for testing the effect of drugs and bacteria on RBC membranes in-vitro using biophysical techniques, such as X-ray and neutron diffraction, optical spectroscopy and atomic force microscopy. We present direct experimental evidence that these RBC membranes consist of nanometer sized domains of integral coiled-coil peptides, as well as liquid ordered (lo) and liquid disordered (ld) lipids. Lamellar spacings, membrane and hydration water layer thicknesses, areas per lipid tail and domain sizes were determined. The common drug aspirin was added to the RBC membranes and found to interact with RBC membranes and preferably partition in the head group region of the lo domain leading to a fluidification of the membranes, i.e., a thinning of the bilayers and an increase in lipid tail spacing. [1] Himbert S. et al., The Molecular Structure of Human Red Blood Cell Membranes from Highly Oriented, Solid Supported Multi-Lamellar Membranes, Scientific Reports 7, Article number: 39661 (2017).

Evidence that asymmetry of the membrane/cytoskeletal complex in human red blood cell ghosts is responsible for their biconcave shape

The main conclusion of the results reported in this article is that during centrifugation, sphered red blood cell ghosts become oriented in their attachment to a coverslip such that a dense band within the ghosts lies parallel to the centrifugal field. The result of the orientation of this dense band is that when the attached spherical ghosts are shrunken to become biconcave discs, they do so by directly collapsing on themselves without any lateral motion. This result is interpreted to suggest that a dense band, relative to the dimple, resides in the rim of the ghost and is responsible for its biconcave shape. These results confirm the conclusions reached in a previous publication in which there was the uncertainty that the shape change of the spherical ghosts to discs could not be directly imaged. The present work corrects this limitation by use of a chamber in which the tonicity of the solutions in the ghosts' surround could be altered by perfusion coupled with constant microscopic imaging. The identity of the components that are responsible for the differences in the density (mass) between the rim and the dimple regions of the cytoskeletal/membrane complex in the biconcave disk are unknown. It is also unknown what forces apply or what the explanation is for the unique orientation of the dense band during the ghosts' centrifugation, as described in this article. Nevertheless, the results reported in this article indicate the membrane's underlying cytoskeletal complex is asymmetrically distributed.

Time-lapse imaging of cell death in cell culture and whole living organisms using turn-on deep-red fluorescent probes.

Cell death is a central process in developmental biology and also an important indicator of disease status and treatment efficacy. Two related fluorescent probes are described that are molecular conjugates of one or two zinc dipicolylamine (ZnDPA) coordination complexes with an appended solvatochromic benzothiazolium squaraine dye. The probes were designed to target the anionic phospholipid, phosphatidylserine (PS), that is exposed on the surface of dead and dying cells. A series of spectrometric and microscopy studies using liposomes and red blood cell ghosts as models showed that the probe with two ZnDPA targeting units produced higher affinity, stronger fluorescence "turn-on" effect, and better image contrast than the probe with one ZnDPA. Both fluorescent probes enabled "no-wash" time-lapse microscopic imaging of mammalian cell death within a culture. The probe with two ZnDPA units was used for non-invasive time-lapse imaging of cell death during the development of Xenopus laevis (frog) embryos. In vivo fluorescence micrographs revealed probe accumulation within the embryo tail, head and spine regions that were undergoing regression and apoptosis during growth and maturation. These new fluorescent probes are likely to be useful for time-resolved, non-invasive in vivo imaging of cell death process in range of living organisms. From a broader perspective, it should be possible to utilize the negative solvatochromism exhibited by benzothiazolium squaraine dyes for development of various "turn-on" deep-red fluorescent probes and materials that target cell surface biomarkers for in vitro and in vivo imaging.