Papers by Yana Reshetnyak
Frontiers in Oncology
Despite significant progress in the development of novel STING agonists (STINGa), applications ap... more Despite significant progress in the development of novel STING agonists (STINGa), applications appear to be challenged by the low efficiency and poor selectivity of these agents. A pH Low Insertion Peptide (pHLIP) extends the lifetime of a STINGa in the blood and targets it to acidic cancer-associated fibroblasts (CAFs), tumor-associated macrophages (TAMs), myeloid derived suppressor cells (mMDSCs) and dendritic cells (DCs). CAFs constitute 25% of all live cells within CT26 tumors, and M2-type TAMs and mMDSCs are the most abundant among the immune cells. The resulting activation of cytokines within the tumor microenvironment (TME) triggers the eradication of small (100 mm3) and large (400-700 mm3) CT26 tumors in mice after a single dose of pHLIP-STINGa. The tumor stroma was destroyed (the number of CAFs was reduced by 98%), intratumoral hemorrhage developed, and the level of acidity within the TME was reduced. Further, no tumors developed in 20 out of 25 tumor-free mice re-challenge...
pHLIP Peptides Target Acidity in Activated Macrophages
Molecular Imaging and Biology
PD53-10 IDENTIFYING Bladder Urothelial Carcinomas Using Fluorescent Signal Intensity of PH Low Inserting Peptides
Journal of Urology
hypomethylated regions and enrichment of genes associated with oxidative phosphorylation, cilium ... more hypomethylated regions and enrichment of genes associated with oxidative phosphorylation, cilium assembly, and ciliopathies in hypermethylated loci. CONCLUSIONS: Significant changes of DNA methylation were observed in mice exposed to ECS vapor vs. vehicle control. EC metabolites in urine may result in aberrant methylation patterns in the bladder urothelium. Results are suggestive of a pre-malignant phenotype driven by EC exposure via stimulation of MYC targets and silencing of genes responsible for establishing primary cilia. Loss of primary cilia in epithelial cells has been associated with loss of cell polarity and increased cell migration.
Hypoxia and tissue acidification occur in the macrophage-rich regions of advanced atherosclerotic... more Hypoxia and tissue acidification occur in the macrophage-rich regions of advanced atherosclerotic lesions due to the higher oxygen demand of activated immune cells and insufficient oxygen supply. Our group and others originally identified microRNA-33 (miR-33) as critical regulator of cellular lipid homeostasis and lipoprotein metabolism controlling the development of atherosclerosis. Our prior work has demonstrated that pH Low-Insertion Peptides (pHLIP) can be used to direct miR-33 inhibitors to acidic microenvironments and protect against kidney fibrosis. Here we utilize anti-miR-33 conjugated pHLIP constructs to target macrophages located in atherosclerosis plaques. The inhibition of miR-33 using pHLIP-directed targeting increased collagen content and decreased lesional lipid accumulation within atherosclerotic plaques in a murine model of atherosclerosis regression. Single cell RNA sequencing analysis revealed higher expression of fibrotic genes (Col2a1, Col3a1, Fn1, Dcn, etc) an...
Additional file 1: of Decoration of Nanovesicles with pH (Low) Insertion Peptide (pHLIP) for Targeted Delivery
Table S1. Parameters for the preparation of several samples with and without pHLIP. Figure S1. SA... more Table S1. Parameters for the preparation of several samples with and without pHLIP. Figure S1. SAXS intensity spectra of TWEEN20 based vesicles without (black dots) and with (red dots) pHLIP, together with the fitting curves obtained with a multishell model. The core has the same electron density of the solvent (water) and the three shells are constituted by the inner headgroups layer, the hydrophobic layer, the outer headgroups layer. Parameters, thickness and electron density are reported in the Table S2. Table S2. Parameters, thickness and electron density obtained by SAXS analysis. Figure S2. Samples stability in terms of size and Îś-potential variations over time. Figure S3. Z-Average variations for all samples in contact with human serum. The Îś-potential values are around -7 mV for all samples. (DOCX 635 kb)
Proceedings of MOL2NET 2017, International Conference on Multidisciplinary Sciences, 3rd edition, 2017
Biophysical Journal, 2018
Proceedings of the National Academy of Sciences, 2020
Significance The process of peptide insertion across a membrane is of fundamental interest. Obtai... more Significance The process of peptide insertion across a membrane is of fundamental interest. Obtained in this study’s results, combined with our recent constant-pH molecular dynamics simulations and kinetics experiments with liposomes of different bilayer thicknesses, allowed us to complete a generalized model of the insertion/exit and folding/unfolding of polypeptides of the pHLIP family and expand the general view of peptide conformations and dynamic excursions of the bilayer that can accompany interactions with peptides. In addition to fundamental significance, pH-dependent pHLIP insertion into membranes of acidic cells within diseased tissues finds wide application in medicine for targeted delivery of imaging and therapeutic agents.
Biophysical Journal, 2018
The physical properties of lipid bilayers, such as curvature and fluidity, can affect the interac... more The physical properties of lipid bilayers, such as curvature and fluidity, can affect the interactions of polypeptides with membranes, influencing biological events. Additionally, given the growing interest in peptide-based therapeutics, understanding the influence of membrane properties on membrane-associated peptides has potential utility. pH low insertion peptides (pHLIPs) are a family of water-soluble peptides that can insert across cell membranes in a pH-dependent manner, enabling the use of pH to follow peptide-lipid interactions. Here we study pHLIP interactions with liposomes varying in size and composition, to determine the influence of several key membrane physical properties. We find that pHLIP binding to bilayer surfaces at neutral pH is governed by the ease of access to the membrane's hydrophobic core, which can be facilitated by membrane curvature, thickness, and the cholesterol content of the membrane. After surface binding, if the pH is lowered, the kinetics of pHLIP folding to form a helix and subsequent insertion across the membrane depends on the fluidity and energetic dynamics of the membrane. We showed that pHLIP is capable of forming a helix across lipid bilayers of different thicknesses at low pH. However, the kinetics of the slow phase of insertion corresponding to the translocation of C-terminal end of the peptide across lipid bilayer, vary approximately twofold, and correlate with bilayer thickness and fluidity. Although these influences are not large, local curvature variations in membranes of different fluidity could selectively influence surface binding in mixed cell populations.
Nanoscale Research Letters, 2018
Acidity at surface of cancer cells is a hallmark of tumor microenvironments, which does not depen... more Acidity at surface of cancer cells is a hallmark of tumor microenvironments, which does not depend on tumor perfusion, thus it may serve as a general biomarker for targeting tumor cells. We used the pH (low) insertion peptide (pHLIP) for decoration of liposomes and niosomes. pHLIP senses pH at the surface of cancer cells and inserts into the membrane of targeted cells, and brings nanomaterial to close proximity of cellular membrane. DMPC liposomes and Tween 20 or Span 20 niosomes with and without pHLIP in their coating were fully characterized in order to obtain fundamental understanding on nanocarrier features and facilitate the rational design of acidity sensitive nanovectors. The samples stability over time and in presence of serum was demonstrated. The size, ζ-potential, and morphology of nanovectors, as well as their ability to entrap a hydrophilic probe and modulate its release were investigated. pHLIP decorated vesicles could be useful to obtain a prolonged (modified) release of biological active substances for targeting tumors and other acidic diseased tissues.
Molecular imaging and biology : MIB : the official publication of the Academy of Molecular Imaging, Oct 13, 2016
Acidification of extracellular space promotes tumor development, progression, and invasiveness. p... more Acidification of extracellular space promotes tumor development, progression, and invasiveness. pH (low) insertion peptides (pHLIP(®) peptides) belong to the class of pH-sensitive membrane peptides, which target acidic tumors and deliver imaging and/or therapeutic agents to cancer cells within tumors. Ex vivo fluorescent imaging of tissue and organs collected at various time points after administration of different pHLIP(®) variants conjugated with fluorescent dyes of various polarity was performed. Methods of multivariate statistical analyses were employed to establish classification between fluorescently labeled pHLIP(®) variants in multidimensional space of spectral parameters. The fluorescently labeled pHLIP(®) variants were classified based on their biodistribution profile and ability of targeting of primary tumors. Also, submillimeter-sized metastatic lesions in lungs were identified by ex vivo imaging after intravenous administration of fluorescent pHLIP(®) peptide. Different...
Clinical Cancer Research, 2015
Optoacoustic tomography has been used for the detection of pancreatic ductal adenocarcinoma targe... more Optoacoustic tomography has been used for the detection of pancreatic ductal adenocarcinoma targeted by pH-low insertion peptide (pHLIP) conjugated to near-infrared fluorescent dye. It was proved that tumor targeting is pH dependent. The approach could have major implication for detection and monitoring of pancreatic and other cancers. Clin Cancer Res; 21(20); 4502–4. ©2015 AACR. See related article by Kimbrough et al., p. 4576
Proceedings of the National Academy of Sciences, 2014
Significance Solid tumors adapt a glycolytic phenotype for their energetic requirements, leading ... more Significance Solid tumors adapt a glycolytic phenotype for their energetic requirements, leading to acidification of the extracellular environment. Targeting this global event is important to gauge the pace of tumor growth and invasiveness, as well as to provide a basis for predicting disease response to pH-dependent chemotherapies. To realize this goal, a noninvasive method is necessary to measure tumor extracellular acidification to meet clinical needs. This study explores the utility of pH (low) insertion peptide, an acidosis-targeting peptide, as a PET-based imaging probe to provide a method for quantifying extracellular pH and its correlation to known acidity markers, such as hypoxia, carbonic anhydrase IX, and lactate dehydrogenase A, within the prostate tumor tissue.
Biophysical Journal, 2010
Antimicrobial Peptides (AMPs) are fast microbe-killing molecules found in virtually-all living or... more Antimicrobial Peptides (AMPs) are fast microbe-killing molecules found in virtually-all living organisms. Membrane-active AMPs are of particular interest, since they do not easily induce bacterial resistance. Accordingly, these peptides offer promising design principles for developing potent peptide antibiotics, especially for fighting conventional antibiotic-resistant bacteria. Here, we present a physical basis for optimizing the selective membrane-disrupting activity of cationic AMPs. Our approach explains the vital feats of the peptide, shedding quantitative insights into their design principles: Threshold peptide coverage on the membrane surface required for disruption can easily be reached for microbes, but not for the host cell-large peptide charge (> 4) is shown to be the key ingredient for determining the optimal activity-selectivity of AMPs (in an ambientsalt dependent way). Our results also illustrate how reduced fluidity of the host cell membrane by cholesterol enhances the selectivity.
Tumor treatment by pHLIP-targeted antigen delivery
Frontiers in Bioengineering and Biotechnology
Targeted antigen delivery allows activation of the immune system to kill cancer cells. Here we re... more Targeted antigen delivery allows activation of the immune system to kill cancer cells. Here we report the targeted delivery of various epitopes, including a peptide, a small molecule, and a sugar, to tumors by pH Low Insertion Peptides (pHLIPs), which respond to surface acidity and insert to span the membranes of metabolically activated cancer and immune cells within tumors. Epitopes linked to the extracellular ends of pH Low Insertion Peptide peptides were positioned at the surfaces of tumor cells and were recognized by corresponding anti-epitope antibodies. Special attention was devoted to the targeted delivery of the nine residue HA peptide epitope from the Flu virus hemagglutinin. The HA sequence is not present in the human genome, and immunity is readily developed during viral infection or immunization with KLH-HA supplemented with adjuvants. We tested and refined a series of double-headed HA-pHLIP agents, where two HA epitopes were linked to a single pH Low Insertion Peptide p...
Targeting bladder urothelial carcinoma with pHLIP-ICG and inhibition of urothelial cancer cell proliferation by pHLIP-amanitin
Frontiers in Urology
Acidity is a useful biomarker for the targeting of metabolically active cells in tumors. pH Low I... more Acidity is a useful biomarker for the targeting of metabolically active cells in tumors. pH Low Insertion Peptides (pHLIPs) sense the pH at the surfaces of tumor cells and can facilitate intracellular delivery of cell-permeable and cell-impermeable cargo molecules. In this study we have shown the targeting of malignant lesions in human bladders by fluorescent pHLIP agents, intracellular delivery of amanitin toxin by pHLIP for the inhibition of urothelial cancer cell proliferation, and enhanced potency of pHLIP-amanitin for cancer cells with 17p loss, a mutation frequently present in urothelial cancers. Twenty-eight ex-vivo bladder specimens, from patients undergoing robotic assisted laparoscopic radical cystectomy for bladder cancer, were treated via intravesical incubation for 15-60 minutes with pHLIP conjugated to indocyanine green (ICG) or IR-800 near infrared fluorescent (NIRF) dyes at concentrations of 4-8 μM. White light cystoscopy identified 47/58 (81%) and NIRF pHLIP cystosc...
Algorithms, 2009
The fluorescence properties of tryptophan residues are sensitive to the microenvironment of fluor... more The fluorescence properties of tryptophan residues are sensitive to the microenvironment of fluorophores in proteins. Therefore, fluorescence characteristics are widely used to study structural transitions in proteins. However, the decoding of the structural information from spectroscopic data is challenging. Here we present a review of approaches developed for the decomposition of multi-component protein tryptophan fluorescence spectra and correlation of these spectral parameters with protein structural properties.
The physical causes for wide variation of Stokes shift values in emission spectra of tryptophan f... more The physical causes for wide variation of Stokes shift values in emission spectra of tryptophan fluorophores in proteins have been proposed in the model of discrete states (
Scientific Reports
Fluorescence imaging has seen enduring use in blood flow visualization and is now finding a new r... more Fluorescence imaging has seen enduring use in blood flow visualization and is now finding a new range of applications in image-guided surgery. In this paper, we report a translational study of a new fluorescent agent for use in surgery, pHLIP ICG, where ICG (indocyanine green) is a surgical fluorescent dye used widely for imaging blood flow. We studied pHLIP ICG interaction with the cell membrane lipid bilayer, the pharmacology and toxicology in vitro and in vivo (mice and dogs), and the biodistribution and clearance of pHLIP ICG in mice. The pHLIP ICG tumor targeting and imaging efficacy studies were carried out in several murine and human mouse tumor models. Blood vessels were imaged in mice and pigs. Clinical Stryker imaging instruments for endoscopy and open surgery were used in the study. Intravenously administered pHLIP ICG exhibits a multi-hour circulation half-life, offering protracted delineation of vasculature. As it clears from the blood, pHLIP ICG targets tumors and tumo...
Targeting Acidic Diseased Tissues by pH-Triggered Membrane-Associated Peptide Folding
Frontiers in Bioengineering and Biotechnology
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Papers by Yana Reshetnyak