29 research outputs found
Intracellular Delivery Using Fluorophore-CPP Conjugates and Light: Mechanisms and Implications
Cell-penetrating peptides (CPPs) can induce translocation of conjugated
macromolecules across the plasma membrane of live cells. The major route of uptake of
these CPPs by cells is through endocytosis. However, intracellular cytosolic delivery
efficiency of these reagents is inefficient because CPP-cargo conjugates typically remain
trapped inside endosomes. As a result, macromolecules are unable to reach their
cytosolic targets and exert biological function. The fluorophore-CPP conjugate
(Fl-CPPs) where the prototypical CPP TAT is conjugated to the fluorophore TMR gets
entrapped inside endosomes of live cells upon incubation. Interestingly, irradiation of the
endosomally contained TMR-TAT with moderate doses of light induces release of
Fl-CPPs into the cytosol. However, the mechanism of this phenomenon is not clear.
Also, the endosomal release of TMR-TAT is accompanied by loss of plasma membrane
integrity, membrane blebbing, and cell-death. I investigated the molecular basis of the
photo-induced endosomolytic activity of Fl-CPPs and the mechanisms behind Fl-CPP
mediated cell death.
I reported that Fl-CPPs act as photosensitizer molecules that can destroy
membranes such as endosomal membranes, membranes of simpler model systems RBCs
and liposomes. I showed that the CPP moiety of Fl-CPPs binds to negatively charged
phospholipids of target membranes and brings the attached fluorophore into close
proximity of the membrane. Upon irradiation of Fl-CPPs, reactive oxygen species (ROS)
such as singlet oxygen and superoxide are produced that cause oxidation of membrane
lipids. In addition, CPPs have a latent ability to cause damage of photo-oxidized lipid
membranes. Thus, CPPs and singlet oxygen generators act in synergy to cause
photolysis of membranes. I further establish structure activity relationship of Fl-CPPs to
understand how structure of Fl-CPPs impact synergistic activity on membranes. These
factors should therefore be considered for the development of effective delivery agents.
I also explored mechanisms behind cell death that accompanied TMR-TAT mediated PCI. I showed that the lysis of endocytic organelles by TMR-TAT caused a rapid increase in the concentration of calcium in the cytosol followed by accumulation of calcium in the mitochondria. Ruthenium red and cyclosporin A, inhibitors of calcium import in mitochondria and of the mitochondria permeability transition pore were able to inhibit cell death
Conjugation to the Cell-Penetrating Peptide TAT Potentiates the Photodynamic Effect of Carboxytetramethylrhodamine
Cell-penetrating peptides (CPPs) can transport macromolecular cargos into live cells. However, the cellular delivery efficiency of these reagents is often suboptimal because CPP-cargo conjugates typically remain trapped inside endosomes. Interestingly, irradiation of fluorescently labeled CPPs with light increases the release of the peptide and its cargos into the cytosol. However, the mechanism of this phenomenon is not clear. Here we investigate the molecular basis of the photo-induced endosomolytic activity of the prototypical CPPs TAT labeled to the fluorophore 5(6)-carboxytetramethylrhodamine (TMR).We report that TMR-TAT acts as a photosensitizer that can destroy membranes. TMR-TAT escapes from endosomes after exposure to moderate light doses. However, this is also accompanied by loss of plasma membrane integrity, membrane blebbing, and cell-death. In addition, the peptide causes the destruction of cells when applied extracellularly and also triggers the photohemolysis of red blood cells. These photolytic and photocytotoxic effects were inhibited by hydrophobic singlet oxygen quenchers but not by hydrophilic quenchers.Together, these results suggest that TAT can convert an innocuous fluorophore such as TMR into a potent photolytic agent. This effect involves the targeting of the fluorophore to cellular membranes and the production of singlet oxygen within the hydrophobic environment of the membranes. Our findings may be relevant for the design of reagents with photo-induced endosomolytic activity. The photocytotoxicity exhibited by TMR-TAT also suggests that CPP-chromophore conjugates could aid the development of novel Photodynamic Therapy agents
Photoinactivation of Gram Positive and Gram Negative Bacteria with the Antimicrobial Peptide (KLAKLAK)<sub>2</sub> Conjugated to the Hydrophilic Photosensitizer Eosin Y
We test the hypothesis that the antimicrobial peptide
(KLAKLAK)<sub>2</sub> enhances the photodynamic activity of the photosensitizer
eosin Y upon conjugation. The conjugate eosin-(KLAKLAK)<sub>2</sub> was obtained by solid-phase peptide synthesis. Photoinactivation
assays were performed against the Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa, and multidrug resistant Acinetobacter baumannii AYE, as well as the Gram-positive bacteria Staphylococcus
aureus, and Staphylococcus epidermidis. Partitioning assays were performed with E. coli and S. aureus. Photohemolysis and
photokilling assays were also performed to assess the photodynamic
activity of the conjugate toward mammalian cells. Eosin-(KLAKLAK)<sub>2</sub> photoinactivates 99.999% of 10<sup>8</sup> CFU/mL of most
bacteria tested at a concentration of 1 μM or below. In contrast,
neither eosin Y nor (KLAKLAK)<sub>2</sub> cause any significant photoinactivation
under similar conditions. The increase in photodynamic activity of
the photosensitizer conferred by the antimicrobial peptide is in part
due to the fact that (KLAKLAK)<sub>2</sub> promotes the association
of eosin Y to bacteria. Eosin-(KLAKLAK)<sub>2</sub> does not significantly
associate with red blood cells or the cultured mammalian cell lines
HaCaT, COS-7, and COLO 316. Consequently, little photodamage or photokilling
is observed with these cells under conditions for which bacterial
photoinactivation is achieved. The peptide (KLAKLAK)<sub>2</sub> therefore
significantly enhances the photodynamic activity of eosin Y toward
both Gram-positive and Gram-negative bacteria while interacting minimally
with human cells. Overall, our results suggest that antimicrobial
peptides such as (KLAKLAK)<sub>2</sub> might serve as attractive agents
that can target photosensitizers to bacteria specifically
Improving the Endosomal Escape of Cell-Penetrating Peptides and Their Cargos: Strategies and Challenges
Cell penetrating peptides (CPPs) can deliver cell-impermeable therapeutic cargos into cells. In particular, CPP-cargo conjugates tend to accumulate inside cells by endocytosis. However, they often remain trapped inside endocytic organelles and fail to reach the cytosolic space of cells efficiently. In this review, the evidence for CPP-mediated endosomal escape is discussed. In addition, several strategies that have been utilized to enhance the endosomal escape of CPP-cargos are described. The recent development of branched systems that display multiple copies of a CPP is presented. The use of viral or synthetic peptides that can disrupt the endosomal membrane upon activation by the low pH of endosomes is also discussed. Finally, we survey how CPPs labeled with chromophores can be used in combination with light to stimulate endosomal lysis. The mechanisms and challenges associated with these intracellular delivery methodologies are discussed
Photoinduced Membrane Damage of <i>E. coli</i> and <i>S. aureus</i> by the Photosensitizer-Antimicrobial Peptide Conjugate Eosin-(KLAKLAK)<sub>2</sub>
<div><p>Background/Objectives</p><p>Upon irradiation with visible light, the photosensitizer-peptide conjugate eosin-(KLAKLAK)<sub>2</sub> kills a broad spectrum of bacteria without damaging human cells. Eosin-(KLAKLAK)<sub>2</sub> therefore represents an interesting lead compound for the treatment of local infection by photodynamic bacterial inactivation. The mechanisms of cellular killing by eosin-(KLAKLAK)<sub>2</sub>, however, remain unclear and this lack of knowledge hampers the development of optimized therapeutic agents. Herein, we investigate the localization of eosin-(KLAKLAK)<sub>2</sub> in bacteria prior to light treatment and examine the molecular basis for the photodynamic activity of this conjugate.</p><p>Methodology/Principal Findings</p><p>By employing photooxidation of 3,3-diaminobenzidine (DAB), (scanning) transmission electron microscopy ((S)TEM), and energy dispersive X-ray spectroscopy (EDS) methodologies, eosin-(KLAKLAK)<sub>2</sub> is visualized at the surface of <i>E. coli</i> and <i>S. aureus</i> prior to photodynamic irradiation. Subsequent irradiation leads to severe membrane damage. Consistent with these observations, eosin-(KLAKLAK)<sub>2</sub> binds to liposomes of bacterial lipid composition and causes liposomal leakage upon irradiation. The eosin moiety of the conjugate mediates bacterial killing and lipid bilayer leakage by generating the reactive oxygen species singlet oxygen and superoxide. In contrast, the (KLAKLAK)<sub>2</sub> moiety targets the photosensitizer to bacterial lipid bilayers. In addition, while (KLAKLAK)<sub>2</sub> does not disrupt intact liposomes, the peptide accelerates the leakage of photo-oxidized liposomes.</p><p>Conclusions/Significance</p><p>Together, our results suggest that (KLAKLAK)<sub>2</sub> promotes the binding of eosin Y to bacteria cell walls and lipid bilayers. Subsequent light irradiation results in membrane damage from the production of both Type I & II photodynamic products. Membrane damage by oxidation is then further aggravated by the (KLAKLAK)<sub>2</sub> moiety and membrane lysis is accelerated by the peptide. These results therefore establish how photosensitizer and peptide act in synergy to achieve bacterial photo-inactivation. Learning how to exploit and optimize this synergy should lead to the development of future bacterial photoinactivation agents that are effective at low concentrations and at low light doses.</p></div
Eosin-(KLAKLAK)<sub>2</sub> lyses LUVs of bacterial lipid composition, but not of mammalian composition.
<p>Eosin-(KLAKLAK)<sub>2</sub> (10 µM) was mixed with LUVs (200 µM) of (A) “Human” (Hum) or (B) “Bacterial” (Bac) lipid composition, each containing a self-quenching concentration of calcein (60 mM). Samples were irradiated for the times indicated and leakage was detected as an increase in fluorescence intensity after release of calcein and subsequent unquenching. Average values are shown for triplicate experiments with error bars representing the standard deviation.</p
Experimental design of DAB photo-oxidation and visualization by TEM.
<p>(A) Light excitation of eosin-(KLAKLAK)<sub>2</sub> results in production of singlet oxygen and superoxide, which can polymerize DAB to provide an enhanced staining of osmium at the location of eosin-(KLAKLAK)<sub>2</sub>. (B) Light irradiation has two purposes in this experiment, 1) to excite eosin-(KLAKLAK)<sub>2</sub> for photodynamic activity (step 1), then following fixation of samples, 2) to polymerize DAB at the location of the PS-AMP conjugate (step 4).</p
Ce6 and (KLAKLAK)<sub>2</sub> display synergistic leakage activity towards Bac LUVs.
<p>(A) Bac LUVs in the presence or absence of Ce6 were kept in the dark for 10 min before addition of 0, 1, or 10 µM (KLAKLAK)<sub>2</sub>. (B) Same as in (A), but samples were irradiated with light for 10 min before addition of (KLAKLAK)<sub>2</sub> (two-tailed <i>t</i> test, * = p<0.05, ** = <0.01, *** = p<0.001). (C) Synergy of (KLAKLAK)<sub>2</sub> and Ce6 leakage determined for light and dark conditions using values from (A) and (B).</p
Bromine atoms from eosin-(KLAKLAK)<sub>2</sub> serve as a marker for detection by STEM-EDS in bacteria samples.
<p>(A) STEM dark field image of <i>S. aureus</i> treated with eosin-(KLAKLAK)<sub>2</sub> and light for 2 min. (B) Elemental analysis by EDS for the square area indicated in (A), showing the distinct presence of Br from eosin-(KLAKLAK)<sub>2</sub> (the transitions of C, O, P, and other elements are predominant at lower energy levels and thus not seen here). (C) EDS element profiles of the line scan depicted in (A), showing the coincident intensities of Os, Br, and P elements at the interior, cell wall, and extracellular material, with more than 250 counts for Br at the membrane and extracellular regions.</p