Proteogenomic characterization of difficult-to-treat breast cancer with tumor cells enriched through laser microdissection.

BACKGROUND: Breast cancer (BC) is the most commonly diagnosed cancer and the leading cause of cancer death among women globally. Despite advances, there is considerable variation in clinical outcomes for patients with non-luminal A tumors, classified as difficult-to-treat breast cancers (DTBC). This study aims to delineate the proteogenomic landscape of DTBC tumors compared to luminal A (LumA) tumors. METHODS: We retrospectively collected a total of 117 untreated primary breast tumor specimens, focusing on DTBC subtypes. Breast tumors were processed by laser microdissection (LMD) to enrich tumor cells. DNA, RNA, and protein were simultaneously extracted from each tumor preparation, followed by whole genome sequencing, paired-end RNA sequencing, global proteomics and phosphoproteomics. Differential feature analysis, pathway analysis and survival analysis were performed to better understand DTBC and investigate biomarkers. RESULTS: We observed distinct variations in gene mutations, structural variations, and chromosomal alterations between DTBC and LumA breast tumors. DTBC tumors predominantly had more mutations in TP53, PLXNB3, Zinc finger genes, and fewer mutations in SDC2, CDH1, PIK3CA, SVIL, and PTEN. Notably, Cytoband 1q21, which contains numerous cell proliferation-related genes, was significantly amplified in the DTBC tumors. LMD successfully minimized stromal components and increased RNA-protein concordance, as evidenced by stromal score comparisons and proteomic analysis. Distinct DTBC and LumA-enriched clusters were observed by proteomic and phosphoproteomic clustering analysis, some with survival differences. Phosphoproteomics identified two distinct phosphoproteomic profiles for high relapse-risk and low relapse-risk basal-like tumors, involving several genes known to be associated with breast cancer oncogenesis and progression, including KIAA1522, DCK, FOXO3, MYO9B, ARID1A, EPRS, ZC3HAV1, and RBM14. Lastly, an integrated pathway analysis of multi-omics data highlighted a robust enrichment of proliferation pathways in DTBC tumors. CONCLUSIONS: This study provides an integrated proteogenomic characterization of DTBC vs LumA with tumor cells enriched through laser microdissection. We identified many common features of DTBC tumors and the phosphopeptides that could serve as potential biomarkers for high/low relapse-risk basal-like BC and possibly guide treatment selections.

Early Middle Palaeolithic culture in India around 385-172 ka reframes Out of Africa models.

Luminescence dating at the stratified prehistoric site of Attirampakkam, India, has shown that processes signifying the end of the Acheulian culture and the emergence of a Middle Palaeolithic culture occurred at 385 ± 64 thousand years ago (ka), much earlier than conventionally presumed for South Asia. The Middle Palaeolithic continued at Attirampakkam until 172 ± 41 ka. Chronologies of Middle Palaeolithic technologies in regions distant from Africa and Europe are crucial for testing theories about the origins and early evolution of these cultures, and for understanding their association with modern humans or archaic hominins, their links with preceding Acheulian cultures and the spread of Levallois lithic technologies. The geographic location of India and its rich Middle Palaeolithic record are ideally suited to addressing these issues, but progress has been limited by the paucity of excavated sites and hominin fossils as well as by geochronological constraints. At Attirampakkam, the gradual disuse of bifaces, the predominance of small tools, the appearance of distinctive and diverse Levallois flake and point strategies, and the blade component all highlight a notable shift away from the preceding Acheulian large-flake technologies. These findings document a process of substantial behavioural change that occurred in India at 385 ± 64 ka and establish its contemporaneity with similar processes recorded in Africa and Europe. This suggests complex interactions between local developments and ongoing global transformations. Together, these observations call for a re-evaluation of models that restrict the origins of Indian Middle Palaeolithic culture to the incidence of modern human dispersals after approximately 125 ka.

Author Correction: Informed-Proteomics: open-source software package for top-down proteomics.

In the version of this article initially published, the authors erroneously reported the search mode that was used for ProSightPC 3.0 in the Online Methods and in Supplementary Table 3.

Exposure to an Environmental Mixture of Polycyclic Aromatic Hydrocarbons Induces Hepatic Cytochrome P450 Enzymes in Mice.

Cytochrome P450 enzymes (CYPs) play an important role in bioactivating or detoxifying polycyclic aromatic hydrocarbons (PAHs), common environmental contaminants. While it is widely accepted that exposure to PAHs induces CYPs, effectively increasing rates of xenobiotic metabolism, dose- and time-response patterns of CYP induction are not well-known. In order to better understand dose- and time-response relationships of individual CYPs following induction, we exposed B6129SF1/J mice to single or repeated doses (2-180 µmol/kg/d) of benzo[a]pyrene (BaP) or Supermix-10, a mixture of the top 10 most abundant PAHs found at the Portland Harbor Superfund Site. In hepatic microsomes from exposed mice, we measured amounts of active CYPs using activity-based protein profiling and total CYP expression using global proteomics. We observed rapid Cyp1a1 induction after 6 h at the lowest PAH exposures and broad induction of many CYPs after 3 daily PAH doses at 72 h following the first dose. Using samples displaying Cyp1a1 induction, we observed significantly higher metabolic affinity for BaP metabolism (Km reduced 3-fold), 3-fold higher intrinsic clearance, but no changes to the Vmax. Mice dosed with the highest PAH exposures exhibited 1.7-5-fold higher intrinsic clearance rates for BaP compared to controls and higher Vmax values indicating greater amounts of enzymes capable of metabolizing BaP. This study demonstrates exposure to PAHs found at superfund sites induces enzymes in dose- and time-dependent patterns in mice. Accounting for specific changes in enzyme profiles, relative rates of PAH bioactivation and detoxification, and resulting risk will help translate internal dosimetry of animal models to humans and improve risk assessments of PAHs at superfund sites.

Phosphoproteome Analysis Reveals Estrogen-ER Pathway as a Modulator of mTOR Activity Via DEPTOR.

ER-positive breast tumors represent ∼70% of all breast cancer cases. Although their treatment with endocrine therapies is effective in the adjuvant or recurrent settings, the development of resistance compromises their effectiveness. The binding of estrogen to ERα, a transcription factor, triggers the regulation of the target genes (genomic pathway). Additionally, a cytoplasmic fraction of estrogen-bound ERα activates oncogenic signaling pathways such as PI3K/AKT/mTOR (nongenomic pathway). The upregulation of the estrogenic and the PI3K/AKT/mTOR signaling pathways are frequently associated with a poor outcome. To better characterize the connection between these two pathways, we performed a phosphoproteome analysis of ER-positive MCF7 breast cancer cells treated with estrogen or estrogen and the mTORC1 inhibitor rapamycin. Many proteins were identified as estrogen-regulated mTORC1 targets and among them, DEPTOR was selected for further characterization. DEPTOR binds to mTOR and inhibits the kinase activity of both mTOR complexes mTORC1 and mTORC2, but mitogen-activated mTOR promotes phosphorylation-mediated DEPTOR degradation. Although estrogen enhances the phosphorylation of DEPTOR by mTORC1, DEPTOR levels increase in estrogen-stimulated cells. We demonstrated that DEPTOR accumulation is the result of estrogen-ERα-mediated transcriptional upregulation of DEPTOR expression. Consequently, the elevated levels of DEPTOR partially counterbalance the estrogen-induced activation of mTORC1 and mTORC2. These results underscore the critical role of estrogen-ERα as a modulator of the PI3K/AKT/mTOR signaling pathway in ER-positive breast cancer cells. Additionally, these studies provide evidence supporting the use of dual PI3K/mTOR or dual mTORC1/2 inhibitors in combination with endocrine therapies as a first-line treatment option for the patients with ER-positive advanced breast cancer.

Informed-Proteomics: open-source software package for top-down proteomics.

Top-down proteomics, the analysis of intact proteins in their endogenous form, preserves valuable information about post-translation modifications, isoforms and proteolytic processing. The quality of top-down liquid chromatography-tandem MS (LC-MS/MS) data sets is rapidly increasing on account of advances in instrumentation and sample-processing protocols. However, top-down mass spectra are substantially more complex than conventional bottom-up data. New algorithms and software tools for confident proteoform identification and quantification are needed. Here we present Informed-Proteomics, an open-source software suite for top-down proteomics analysis that consists of an LC-MS feature-finding algorithm, a database search algorithm, and an interactive results viewer. We compare our tool with several other popular tools using human-in-mouse xenograft luminal and basal breast tumor samples that are known to have significant differences in protein abundance based on bottom-up analysis.

Structure Dependent Determination of Organophosphate Targets in Mammalian Tissues Using Activity-Based Protein Profiling.

Acute and chronic exposures to organophosphates (OPs), including agricultural pesticides, industrial chemicals, and chemical warfare agents, remain a significant worldwide health risk. The mechanisms by which OPs alter development and cognition in exposed individuals remain poorly understood, in part due to the large number of structurally diverse OPs and the wide range of affected proteins and signaling pathways. To investigate the influence of structure on OP targets in mammalian systems, we have developed a series of probes for activity-based protein profiling (ABPP) featuring two distinct reactive groups that mimic OP chemical reactivity. FOP features a fluorophosphonate moiety, and PODA and CODA utilize a dialkynyl phosphate ester; both reactive group types target serine hydrolase activity. As the oxon represents the highly reactive and toxic functional group of many OPs, the new probes described herein enhance our understanding of tissue-specific reactivity of OPs. Chemoproteomic analysis of mouse tissues treated with the probes revealed divergent protein profiles, demonstrating the influence of probe structure on protein targeting. These targets also vary in sensitivity toward different OPs. The simultaneous use of multiple probes in ABPP experiments may therefore offer more comprehensive coverage of OP targets; FOP consistently labeled more targets in both brain and liver than PODA or CODA, suggesting the dialkyne warhead is more selective for enzymes in major signaling pathways than the more reactive fluorophosphonate warhead. Additionally, the probes can be used to assess reactivation of OP-inhibited enzymes by N-oximes and may serve as diagnostic tools for screening of therapeutic candidates in a panel of protein targets. These applications will help clarify the short- and long-term effects of OP toxicity beyond acetylcholinesterase inhibition, investigate potential points of convergence for broad spectrum therapeutic development, and support future efforts to screen candidate molecules for efficacy in various model systems.

The Effect of a Rapid Assessment Zone on Emergency Department Operations and Throughput.

STUDY OBJECTIVE: We examine the effects of a front-end flow model designated the rapid assessment zone on multiple emergency department (ED) operational metrics. METHODS: This was a retrospective, before-after study of consecutive patient visits at an urban community ED. Six-month periods were compared before and after an intervention in 2017 that changed patient flow and the intake process. A lead nurse role splits patient flow immediately on patient arrival according to only age and chief complaint, allowing direct bedding without the bottlenecks of vital sign measurement, full triage assessment, or Emergency Severity Index assignment. A new patient care area (designated rapid assessment zone) preferentially expedites treatment of patients likely to remain ambulatory and serves as flexible acute care space when needed by individual cases and the ED. The outcomes measured were ED length of stay, arrival-to-provider time, the rate of leaving before treatment completion, and the rate of leaving before being seen. Data were analyzed with nonparametric testing, χ2 analysis, and multiple linear regression, controlling for patient visit characteristics, ED daily census volumes, and measurements of boarding patients. RESULTS: We analyzed 43,847 visits in the preintervention and 44,792 visits in the postintervention periods. The intervention was associated with the following changes: median ED length of stay from 203 to 171 minutes (-15.8%), median arrival-to-provider time from 28 to 13 minutes (-53.6%), leaving before treatment completion from 1.0% to 0.8% (-20%), and leaving before being seen from 3.1% to 0.5% (-84%). Regression analysis accounting for multiple confounders demonstrated that the reduced length of stay after rapid assessment zone implementation persisted across Emergency Severity Index levels 2 to 5 and all ED daily census levels. CONCLUSION: The rapid assessment zone model aims to decrease front-end bottlenecks and minimize serial intake assessments at a high-volume, urban ED. It was associated with improved patient throughput and decreased early patient departure. It may represent a useful model for similar centers.

Spatially Resolved Proteome Mapping of Laser Capture Microdissected Tissue with Automated Sample Transfer to Nanodroplets.

Current mass spectrometry (MS)-based proteomics approaches are ineffective for mapping protein expression in tissue sections with high spatial resolution because of the limited overall sensitivity of conventional workflows. Here we report an integrated and automated method to advance spatially resolved proteomics by seamlessly coupling laser capture microdissection (LCM) with a recently developed nanoliter-scale sample preparation system termed nanoPOTS (Nanodroplet Processing in One pot for Trace Samples). The workflow is enabled by prepopulating nanowells with DMSO, which serves as a sacrificial capture liquid for microdissected tissues. The DMSO droplets efficiently collect laser-pressure catapulted LCM tissues as small as 20 µm in diameter with success rates >87%. We also demonstrate that tissue treatment with DMSO can significantly improve proteome coverage, likely due to its ability to dissolve lipids from tissue and enhance protein extraction efficiency. The LCM-nanoPOTS platform was able to identify 180, 695, and 1827 protein groups on average from 12-µm-thick rat brain cortex tissue sections having diameters of 50, 100, and 200 µm, respectively. We also analyzed 100-µm-diameter sections corresponding to 10-18 cells from three different regions of rat brain and comparatively quantified ∼1000 proteins, demonstrating the potential utility for high-resolution spatially resolved mapping of protein expression in tissues.

A Probe-Enabled Approach for the Selective Isolation and Characterization of Functionally Active Subpopulations in the Gut Microbiome.

Commensal microorganisms in the mammalian gut play important roles in host health and physiology, but a central challenge remains in achieving a detailed mechanistic understanding of specific microbial contributions to host biochemistry. New function-based approaches are needed that analyze gut microbial function at the molecular level by coupling detection and measurements of in situ biochemical activity with identification of the responsible microbes and enzymes. We developed a platform employing ß-glucuronidase selective activity-based probes to detect, isolate, and identify microbial subpopulations in the gut responsible for this xenobiotic metabolism. We find that metabolic activity of gut microbiota can be plastic and that between individuals and during perturbation, phylogenetically disparate populations can provide ß-glucuronidase activity. Our work links biochemical activity with molecular-scale resolution without relying on genomic inference.

Multifunctional Activity-Based Protein Profiling of the Developing Lung.

Lung diseases and disorders are a leading cause of death among infants. Many of these diseases and disorders are caused by premature birth and underdeveloped lungs. In addition to developmentally related disorders, the lungs are exposed to a variety of environmental contaminants and xenobiotics upon birth that can cause breathing issues and are progenitors of cancer. In order to gain a deeper understanding of the developing lung, we applied an activity-based chemoproteomics approach for the functional characterization of the xenometabolizing cytochrome P450 enzymes, active ATP and nucleotide binding enzymes, and serine hydrolases using a suite of activity-based probes (ABPs). We detected P450 activity primarily in the postnatal lung; using our ATP-ABP, we characterized a wide range of ATPases and other active nucleotide- and nucleic acid-binding enzymes involved in multiple facets of cellular metabolism throughout development. ATP-ABP targets include kinases, phosphatases, NAD- and FAD-dependent enzymes, RNA/DNA helicases, and others. The serine hydrolase-targeting probe detected changes in the activities of several proteases during the course of lung development, yielding insights into protein turnover at different stages of development. Select activity-based probe targets were then correlated with RNA in situ hybridization analyses of lung tissue sections.

Time-resolved proteome profiling of normal lung development.

Biochemical networks mediating normal lung morphogenesis and function have important implications for ameliorating morbidity and mortality in premature infants. Although several transcript-level studies have examined normal lung development, corresponding protein-level analyses are lacking. Here we performed proteomics analysis of murine lungs from embryonic to early adult ages to identify the molecular networks mediating normal lung development. We identified 8,932 proteins, providing a deep and comprehensive view of the lung proteome. Analysis of the proteomics data revealed discrete modules and the underlying regulatory and signaling network modulating their expression during development. Our data support the cell proliferation that characterizes early lung development and highlight responses of the lung to exposure to a nonsterile oxygen-rich ambient environment and the important role of lipid (surfactant) metabolism in lung development. Comparison of dynamic regulation of proteomic and recent transcriptomic analyses identified biological processes under posttranscriptional control. Our study provides a unique proteomic resource for understanding normal lung formation and function and can be freely accessed at Lungmap.net.

BRCA2 minor transcript lacking exons 4-7 supports viability in mice and may account for survival of humans with a pathogenic biallelic mutation.

The breast cancer gene, BRCA2, is essential for viability, yet patients with Fanconi anemia-D1 subtype are born alive with biallelic mutations in this gene. The hypomorphic nature of the mutations is believed to support viability, but this is not always apparent. One such mutation is IVS7+2T>G, which causes premature protein truncation due to skipping of exon 7. We previously identified a transcript lacking exons 4-7, which restores the open-reading frame, encodes a DNA repair proficient protein and is expressed in IVS7+2T>G carriers. However, because the exons 4-7 encoded region contains several residues required for normal cell-cycle regulation and cytokinesis, this transcript's ability to support viability can be argued. To address this, we generated a Brca2 knock-in mouse model lacking exons 4-7 and demonstrated that these exons are dispensable for viability as well as tumor-free survival. This study provides the first in vivo evidence of the functional significance of a minor transcript of BRCA2 that can play a major role in the survival of humans who are homozygous for a clearly pathogenic mutation. Our results highlight the importance of assessing protein function restoration by premature truncating codon bypass by alternative splicing when evaluating the functional significance of variants such as nonsense and frame-shift mutations that are assumed to be clearly pathogenic. Our findings will impact not only the assessment of variants that map to this region, but also influence counseling paradigms and treatment options for such mutation carriers.

Proteome Profiling of 1 to 5 Spiked Circulating Tumor Cells Isolated from Whole Blood Using Immunodensity Enrichment, Laser Capture Microdissection, Nanodroplet Sample Processing, and Ultrasensitive nanoLC-MS.

Proteome profiling of circulating tumor cells (CTCs) can provide crucial insight into disease progression and the role of CTCs in tumor metastasis. We describe an integrated workflow to measure global protein expression in 1-5 spiked CTCs enriched from whole blood by immunodensity gradient centrifugation. Enriched CTCs were purified and collected by laser capture microdissection, prepared using a recently developed nanodroplet-based processing platform (nanoPOTS), and finally analyzed by ultrasensitive nanoLC-MS/MS. The workflow was capable of identifying an average of 164 and 607 protein groups from samples comprising 1 and 5 LNCaP cells, respectively, that were isolated from human whole blood. A panel of prostate cancer-specific proteins were identified and quantified, which was used to differentiate between spiked CTCs and white blood cells.

Quantitative proteomics identifies altered O-GlcNAcylation of structural, synaptic and memory-associated proteins in Alzheimer's disease.

Protein modification by O-linked ß-N-acetylglucosamine (O-GlcNAc) is emerging as an important factor in the pathogenesis of sporadic Alzheimer's disease (AD); however, detailed molecular characterization of this important protein post-translational modification at the proteome level has been highly challenging, owing to its low stoichiometry and labile nature. Herein, we report the most comprehensive, quantitative proteomics analysis for protein O-GlcNAcylation in postmortem human brain tissues with and without AD by the use of isobaric tandem mass tag labelling, chemoenzymatic photocleavage enrichment, and liquid chromatography coupled to mass spectrometry. A total of 1850 O-GlcNAc peptides covering 1094 O-GlcNAcylation sites were identified from 530 proteins in the human brain. One hundred and thirty-one O-GlcNAc peptides covering 81 proteins were altered in AD brains as compared with controls (q < 0.05). Moreover, alteration of O-GlcNAc peptide abundance could be attributed more to O-GlcNAcylation level than to protein level changes. The altered O-GlcNAcylated proteins belong to several structural and functional categories, including synaptic proteins, cytoskeleton proteins, and memory-associated proteins. These findings suggest that dysregulation of O-GlcNAcylation of multiple brain proteins may be involved in the development of sporadic AD. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Simultaneous Proteomic Discovery and Targeted Monitoring using Liquid Chromatography, Ion Mobility Spectrometry, and Mass Spectrometry.

Current proteomic approaches include both broad discovery measurements and quantitative targeted analyses. In many cases, discovery measurements are initially used to identify potentially important proteins (e.g. candidate biomarkers) and then targeted studies are employed to quantify a limited number of selected proteins. Both approaches, however, suffer from limitations. Discovery measurements aim to sample the whole proteome but have lower sensitivity, accuracy, and quantitation precision than targeted approaches, whereas targeted measurements are significantly more sensitive but only sample a limited portion of the proteome. Herein, we describe a new approach that performs both discovery and targeted monitoring (DTM) in a single analysis by combining liquid chromatography, ion mobility spectrometry and mass spectrometry (LC-IMS-MS). In DTM, heavy labeled target peptides are spiked into tryptic digests and both the labeled and unlabeled peptides are detected using LC-IMS-MS instrumentation. Compared with the broad LC-MS discovery measurements, DTM yields greater peptide/protein coverage and detects lower abundance species. DTM also achieved detection limits similar to selected reaction monitoring (SRM) indicating its potential for combined high quality discovery and targeted analyses, which is a significant step toward the convergence of discovery and targeted approaches.

Proteomic Analysis of Single Mammalian Cells Enabled by Microfluidic Nanodroplet Sample Preparation and Ultrasensitive NanoLC-MS.

We report on the quantitative proteomic analysis of single mammalian cells. Fluorescence-activated cell sorting was employed to deposit cells into a newly developed nanodroplet sample processing chip, after which samples were analyzed by ultrasensitive nanoLC-MS. An average of circa 670 protein groups were confidently identified from single HeLa cells, which is a far greater level of proteome coverage for single cells than has been previously reported. We demonstrate that the single-cell proteomics platform can be used to differentiate cell types from enzyme-dissociated human lung primary cells and identify specific protein markers for epithelial and mesenchymal cells.

Activity-Based Probes for Isoenzyme- and Site-Specific Functional Characterization of Glutathione S-Transferases.

Glutathione S-transferases (GSTs) comprise a diverse family of phase II drug metabolizing enzymes whose shared function is the conjugation of reduced glutathione (GSH) to endo- and xenobiotics. Although the conglomerate activity of these enzymes can be measured, the isoform-specific contribution to the metabolism of xenobiotics in complex biological samples has not been possible. We have developed two activity-based probes (ABPs) that characterize active GSTs in mammalian tissues. The GST active site is composed of a GSH binding "G site" and a substrate binding "H site". Therefore, we developed (1) a GSH-based photoaffinity probe (GSTABP-G) to target the "G site", and (2) an ABP designed to mimic a substrate molecule and have "H site" activity (GSTABP-H). The GSTABP-G features a photoreactive moiety for UV-induced covalent binding to GSTs and GSH-binding enzymes. The GSTABP-H is a derivative of a known mechanism-based GST inhibitor that binds within the active site and inhibits GST activity. Validation of probe targets and "G" and "H" site specificity was carried out using a series of competition experiments in the liver. Herein, we present robust tools for the characterization of enzyme- and active site-specific GST activity in mammalian model systems.

Plasma Protein Turnover Rates in Rats Using Stable Isotope Labeling, Global Proteomics, and Activity-Based Protein Profiling.

Protein turnover is important for general health on cellular and organism scales providing a strategy to replace old, damaged, or dysfunctional proteins. Protein turnover also informs of biomarker kinetics, as a better understanding of synthesis and degradation of proteins increases the clinical utility of biomarkers. Here, turnover rates of plasma proteins in rats were measured in vivo using a pulse-chase stable isotope labeling experiment. During the pulse, rats (n = 5) were fed 13C6-labeled lysine ("heavy") feed for 23 days to label proteins. During the chase, feed was changed to an unlabeled equivalent feed ("light"), and blood was repeatedly sampled from rats over 10 time points for 28 days. Plasma samples were digested with trypsin and analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). MaxQuant was used to identify peptides and proteins and quantify heavy/light lysine ratios. A system of ordinary differential equations was used to calculate protein turnover rates. Using this approach, 273 proteins were identified, and turnover rates were quantified for 157 plasma proteins with half-lives ranging 0.3-103 days. For the ∼70 most abundant proteins, variability in turnover rates among rats was low (median coefficient of variation: 0.09). Activity-based protein profiling was applied to pooled plasma samples to enrich serine hydrolases using a fluorophosphonate (FP2) activity-based probe. This enrichment resulted in turnover rates for an additional 17 proteins. This study is the first to measure global plasma protein turnover rates in rats in vivo, measure variability of protein turnover rates in any animal model, and utilize activity-based protein profiling for enhancing turnover measurements of targeted, low-abundant proteins, such as those commonly used as biomarkers. Measured protein turnover rates will be important for understanding of the role of protein turnover in cellular and organism health as well as increasing the utility of protein biomarkers through better understanding of processes governing biomarker kinetics.

Surface-Modified Liposomal Formulation of Amphotericin B: In vitro Evaluation of Potential Against Visceral Leishmaniasis.

Surface modification of liposomes with targeting ligands is known to improve the efficacy with reduced untoward effects in treating infective diseases like visceral leishmaniasis (VL). In the present study, modified ligand (ML), designed by modifying polysaccharide with a long chain lipid was incorporated in liposomes with the objective to target amphotericin B (Amp B) to reticuloendothelial system and macrophages. Conventional liposomes (CL) and surface modified liposomes (SML) were characterized for size, shape, and entrapment efficiency (E.E.). Amp B SML with 3% w/w of ML retained the vesicular nature with particle size of ∼205 nm, E.E. of ∼95% and good stability. SML showed increased cellular uptake in RAW 264.7 cells which could be attributed to receptor-mediated endocytosis. Compared to Amp B solution, Amp B liposomes exhibited tenfold increased safety in vitro in RAW 264.7 and J774A.1 cell lines. Pharmacokinetics and biodistribution studies revealed high t 1/2, area under the curve (AUC)0-24, reduced clearance and prolonged retention in liver and spleen with Amp B SML compared to other formulations. In promastigote and amastigote models, Amp B SML showed enhanced performance with low 50% inhibitory concentration (IC50) compared to Amp B solution and Amp B CL. Thus, due to the targeting ability of ML, SML has the potential to achieve enhanced efficacy in treating VL.