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1.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 51(1): 81-86, 2020 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-31950794

RESUMO

Objective: To preliminarily investigate the differences of protein composition between immature dendritic cells (DC2.4) and their derived exosomes (DC-Exo) using a relatively rapid and sample-saving method based on nano-flow liquid chromatography tandem mass spectrometry (nanoLC-MS/MS). Methods: The supernatant of DC2.4 cells culture medium was collected and gradient centrifugation was applied to primarily extract and isolate DC-Exo; then sucrose density gradient ultracentrifugation was adopted to purify the DC-Exo. Bradford protein assay was used to determine the total protein content of the purified DC-Exo, and dynamic light scattering and transmission electron microscope were conducted to characterize the morphology and size distribution of the DC-Exo. Afterwards, protein samples including DC2.4 cells and DC-Exo were prepared by FASP enzymolysis method. Samples were performed nanoLC-MS/MS assay. The µLPickUp sample loading mode was used and only 1 µg of protein sample was required for each assay. The phase of Transport liquid and Micro A were both 0.05% trifluoroacetic acid-2% acetonitrile (ACN) aq. ( V/ V). Acclaim ® PepMap RSLC column was used to separate sample compositions and the gradient elute was adopted where the mobile phase consisted of (A) 0.1% formic acid (FA) and (B) 0.08% FA-80% ACN aq. ( V/ V) with flow rate of 0.3 µL/min. Positive APCI nanospray interface was used and "one-drive-ten" schema was set to collect primary information. The collected data was then searched and matched based on Uniport Mouse Fasta file as protein database in this case, and the re-annotated data was further sorted out and analyzed. Results: In the current study, relatively high yield of DC-Exo samples with sizes of 40-200 nm were obtained. The lyophilized protein samples prepared by FASP method could be loaded directly after redissolution, and only 1 µg of protein sample is required. The annotated results showed that DC2.4 cells contained 998 kinds of proteins, among which 227 were highly expressed and 535 were unique; while DC-Exo contained only 348 types of proteins, among which 18 were uniquely and highly expressed. There were 306 kinds of consensus proteins in both DC2.4 cells and DC-Exo, among them 7 kinds were highly expressed. Conclusion: The nanoLC-MS/MS method developed in this study only requires very small amount of protein samples, and it could primarily differentiate the protein compositions between DC2.4 cells and their derived exosomes rapidly.


Assuntos
Cromatografia Líquida , Células Dendríticas , Exossomos , Proteínas , Espectrometria de Massas em Tandem , Animais , Células Dendríticas/química , Exossomos/química , Camundongos , Proteínas/química
2.
Anal Bioanal Chem ; 412(3): 601-609, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31897558

RESUMO

Numerous studies have shown that exosomes are closely related to the pathogenesis of various diseases, especially cancers. Therefore, a rapid and sensitive method for exosome detection will be of great importance for the diagnosis and prognosis of diseases. We report here a method for exosome detection based on the CD63 aptamer and clustered regular interspaced short palindromic repeats (CRISPR)/Cas12a system. This method consists mainly of exosomal membrane protein recognition based on the CD63 aptamer and signal amplification based on CRISPR/Cas12a. The CD63 aptamer, as an easily adaptable nucleic acid strand, is responsible for the conversion of the amounts of exosomes into nucleic acid detection, whereas CRISPR/Cas12a is responsible for highly specific nucleic acid signal amplification. The detection range of the method was determined as 3 × 103-6 × 107 particles per microliter. Additionally, we successfully applied this method to detect exosomes in clinical samples from both healthy individuals and patients with lung cancer, and the results were highly consistent with those obtained by nanoparticle tracking analysis. In general, this method provides a highly sensitive and specific method for the detection of exosomes and offers an avenue toward future exosome-based diagnosis of diseases.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Exossomos/química , Tetraspanina 30/análise , Células A549 , Sistemas CRISPR-Cas , Exossomos/patologia , Humanos , Neoplasias Pulmonares/patologia , Técnicas de Amplificação de Ácido Nucleico/métodos
4.
Chem Commun (Camb) ; 55(95): 14339-14342, 2019 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-31720594

RESUMO

Exosomes are emerging as a promising source of disease biomarkers. However, glycans from exosomes have been less studied. Here, for the first time, the N-glycome of human serum exosomes is reported and the potential of N-glycans from exosomes as a source for biomarker discovery is revealed.


Assuntos
Biomarcadores/sangue , Biomarcadores/química , Exossomos/química , Polissacarídeos/sangue , Polissacarídeos/química , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/química , Humanos , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/química
5.
Parasit Vectors ; 12(1): 467, 2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31597577

RESUMO

BACKGROUND: Pathogenic protozoans use extracellular vesicles (EVs) for intercellular communication and host manipulation. Acanthamoeba castellanii is a free-living protozoan that may cause severe keratitis and fatal granulomatous encephalitis. Although several secreted molecules have been shown to play crucial roles in the pathogenesis of Acanthamoeba, the functions and components of parasite-derived EVs are far from understood. METHODS: Purified EVs from A. castellanii were confirmed by electron microscopy and nanoparticle tracking analysis. The functional roles of parasite-derived EVs in the cytotoxicity to and immune response of host cells were examined. The protein composition in EVs from A. castellanii was identified and quantified by LC-MS/MS analysis. RESULTS: EVs from A. castellanii fused with rat glioma C6 cells. The parasite-derived EVs induced an immune response from human THP-1 cells and a cytotoxic effect in C6 cells. Quantitative proteomic analysis identified a total of 130 proteins in EVs. Among the identified proteins, hydrolases (50.2%) and oxidoreductases (31.7%) were the largest protein families in EVs. Furthermore, aminopeptidase activities were confirmed in EVs from A. castellanii. CONCLUSIONS: The proteomic profiling and functional characterization of EVs from A. castellanii provide an in-depth understanding of the molecules packaged into EVs and their potential mechanisms mediating the pathogenesis of this parasite.


Assuntos
Acanthamoeba castellanii/fisiologia , Exossomos/química , Exossomos/fisiologia , Proteômica , Ceratite por Acanthamoeba/parasitologia , Acanthamoeba castellanii/patogenicidade , Acanthamoeba castellanii/ultraestrutura , Aminopeptidases/análise , Animais , Infecções Protozoárias do Sistema Nervoso Central/parasitologia , Meios de Cultura , DNA Complementar/biossíntese , Exossomos/imunologia , Exossomos/ultraestrutura , Humanos , Microscopia Eletrônica de Transmissão , Neuroglia/parasitologia , RNA de Protozoário/genética , RNA de Protozoário/isolamento & purificação , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células THP-1/imunologia , Células THP-1/parasitologia
6.
Nanoscale ; 11(33): 15589-15595, 2019 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-31403149

RESUMO

Exosomes have been recognized as promising sources of biomarkers for early cancer diagnosis due to their important role in the occurrence and metastasis of cancer, and so the development of a sensitive low-cost detection method for exosomes is highly desirable. In this paper, we report a fluorescence method for the competitive detection of exosomes based on an aptamer specific to CD63 (an exosome transmembrane protein). Aptamer-modified magnetic beads were hybridized with a Cy3-labeled short sequence complementary to a region of the aptamer. In the presence of exosomes, the CD63 on the exosomes bound to the aptamer, resulting in the shedding of the short sequence into the supernatant. The quantity of the exosomes could be estimated by detecting the fluorescence intensity in the supernatant. This method could detect exosomes at a concentration as low as 1.0 × 105 particles per µL under optimal conditions, and the feasibility of the method for exosome detection in complex clinical samples was also proved using simulated serum samples. The detection cost and difficulty are significantly reduced compared to conventional methods, while ensuring sensitivity, and so this method provides a basis for subsequent exosome detection in specific cancer cells.


Assuntos
Aptâmeros de Nucleotídeos/química , Exossomos/química , Corantes Fluorescentes/química , Imagem Óptica/métodos , Células A549 , Humanos , Nanopartículas de Magnetita/química , Temperatura Ambiente
7.
Anal Bioanal Chem ; 411(25): 6591-6601, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31372698

RESUMO

Exosomes are vesicles secreted by cells having a size range from 30 to 150 nm and carrying genetic materials that are important for intercellular functions, including cancer progression. Mounting evidence shows that tumor cells secrete more exosomes than normal cells. Thus, it is important to be able to efficiently isolate and quantify exosomes for potential use in clinical diagnostics, as well as to develop a deeper understanding of their role in intercellular processes. Current methods for exosome isolation and quantification are time-consuming and expensive. Few of these methods are able to combine exosome isolation and quantification into a singular operation scheme. However, a new efficient, rapid, and low-cost isolation and quantification method for exosomes in human urine samples using polyester (PET) capillary-channeled polymer (C-CP) fibers in a hydrophobic interaction chromatography (HIC) protocol has been developed. The process has been verified via scanning electron microscopy (SEM) before and after the capture of exosomes on the fiber surfaces. Sample load and elution rates were optimized to affect high resolution and throughput. Isolated exosomes were quantified based on a UV absorbance response curve created using a commercial human urine-derived exosome standard with an exosome concentration of 7.32 × 1011 mL-1. The loading capacity of a 30-cm C-CP PET column was ~ 7 × 1011 exosomes. An inter-injection washing method with PBS was developed to improve the reproducibility with a 2.9% RSD achieved for 7 complete isolation cycles. Graphical abstract.


Assuntos
Cromatografia Líquida de Alta Pressão/instrumentação , Exossomos/química , Poliésteres/química , Urina/química , Desenho de Equipamento , Humanos , Interações Hidrofóbicas e Hidrofílicas , Urinálise/instrumentação
8.
Molecules ; 24(16)2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31416240

RESUMO

Exosomes contain different functional bimolecular characteristics related to physiological or pathological processes and are now recognized as new biomarkers in different human cancers. Rapid detection and classification of cancer-related exosomes might be helpful in the rapid screening of patients that may have cancer. Here, we report a surface enhanced Raman scattering technology for rapid and label-free exosomal detection (Exo-SERS) to aid in the discrimination of different cancer cells based on specific Raman phenotypes and multivariate statistical analysis. The results demonstrated that exosomes derived from both tumor cells and normal cells exhibit special, unique Raman phenotypes. Using the Exo-SERS method, the cancer cells were accurately discriminated from normal cells, and subtle molecular changes between the different cell types could be detected with high sensitive. This research provides a rapid, label-free and non-destructive manner for detecting and discriminating between cancer types.


Assuntos
Biomarcadores Tumorais , Exossomos/química , Neoplasias/diagnóstico , Análise Espectral Raman , Linhagem Celular Tumoral , Exossomos/classificação , Exossomos/ultraestrutura , Ouro/química , Humanos , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Análise Multivariada , Fenótipo , Análise Espectral Raman/métodos
9.
Nat Commun ; 10(1): 3838, 2019 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-31444335

RESUMO

Developing biomimetic nanoparticles without loss of the integrity of proteins remains a major challenge in cancer chemotherapy. Here, we develop a biocompatible tumor-cell-exocytosed exosome-biomimetic porous silicon nanoparticles (PSiNPs) as drug carrier for targeted cancer chemotherapy. Exosome-sheathed doxorubicin-loaded PSiNPs (DOX@E-PSiNPs), generated by exocytosis of the endocytosed DOX-loaded PSiNPs from tumor cells, exhibit enhanced tumor accumulation, extravasation from blood vessels and penetration into deep tumor parenchyma following intravenous administration. In addition, DOX@E-PSiNPs, regardless of their origin, possess significant cellular uptake and cytotoxicity in both bulk cancer cells and cancer stem cells (CSCs). These properties endow DOX@E-PSiNPs with great in vivo enrichment in total tumor cells and side population cells with features of CSCs, resulting in anticancer activity and CSCs reduction in subcutaneous, orthotopic and metastatic tumor models. These results provide a proof-of-concept for the use of exosome-biomimetic nanoparticles exocytosed from tumor cells as a promising drug carrier for efficient cancer chemotherapy.


Assuntos
Doxorrubicina/administração & dosagem , Portadores de Fármacos/química , Composição de Medicamentos/métodos , Exossomos/química , Neoplasias/tratamento farmacológico , Animais , Linhagem Celular Tumoral/citologia , Linhagem Celular Tumoral/metabolismo , Linhagem Celular Tumoral/transplante , Modelos Animais de Doenças , Exocitose , Feminino , Humanos , Masculino , Camundongos , Nanopartículas/química , Neoplasias/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Porosidade , Estudo de Prova de Conceito , Silício/química , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Esferoides Celulares/transplante , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Cancer Sci ; 110(10): 3173-3182, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31464035

RESUMO

Ferroptosis is an iron-dependent, lipid peroxide-driven cell death caused by inhibition of the cystine/glutamate transporter, which is of importance for the survival of triple-negative breast cancer (TNBC) cells. Erastin is a low molecular weight chemotherapy drug that induces ferroptosis; however, poor water solubility and renal toxicity have limited its application. Exosomes, as drug delivery vehicles with low immunogenicity, high biocompatibility and high efficiency, have attracted increasing attention in recent years. Herein, we developed a formulation of erastin-loaded exosomes labeled with folate (FA) to form FA-vectorized exosomes loaded with erastin (erastin@FA-exo) to target TNBC cells with overexpression of FA receptors. The characterization, drug release, internalization and anti-tumor effect in vitro of erastin@FA-exo were determined. Erastin@FA-exo could increase the uptake efficiency of erastin into MDA-MB-231 cells; compared with erastin@exo and free erastin, erastin@FA-exo has a better inhibitory effect on the proliferation and migration of MDA-MB-231 cells. Furthermore, erastin@FA-exo promoted ferroptosis with intracellular depletion of glutathione and reactive oxygen species overgeneration. Western blot analyses revealed that erastin@FA-exo suppressed expression of glutathione peroxidase 4 (GPX4) and upregulated expression of cysteine dioxygenase (CDO1). We conclude that targeting and biocompatibility of exosome-based erastin preparations provide an innovative and powerful delivery platform for anti-cancer therapy.


Assuntos
Exossomos/química , Ácido Fólico/química , Piperazinas/farmacologia , Neoplasias de Mama Triplo Negativas/metabolismo , Morte Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisteína Dioxigenase/metabolismo , Sistemas de Liberação de Medicamentos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glutationa Peroxidase/metabolismo , Humanos , Piperazinas/química , Espécies Reativas de Oxigênio/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico
11.
Biosens Bioelectron ; 142: 111503, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31376716

RESUMO

Exosomes, lipid bilayer membrane vesicles, can guide various pathological and physiological processes. However, reliable, convenient and sensitive methods for exosome determination for early cancer diagnosis are still technically challenging. Herein, an electrochemical aptasensor based on click chemistry and the DNA hybridization chain reaction (HCR) for signal amplification has been developed for the ultrasensitive detection of tumor exosomes. CD63 aptamer was first immobilized on a glassy carbon electrode for capturing exosomes, and 4-oxo-2-nonenal alkyne (alkynyl-4-ONE) molecules, functionalized lipid electrophiles, were conjugated to the exosomes via the reaction of amino and aldehyde groups. Azide-labeled DNA probe as an anchor was then connected to the exosomes by copper (I)-catalyzed click chemistry. Signal amplification was achieved by HCR, and the numerous linked horseradish peroxidase (HRP) molecules could catalyze the reaction of o-phenylenediamine (OPD) and H2O2. The concentration of exosomes could be quantified by monitoring the electrochemical reduction current of 2,3-diaminophenazine (DAP). Under the optimal conditions, this method allowed the sensitive detection of exosomes in the range of 1.12 × 102 to 1.12 × 108 particles/µL with a limit of detection (LOD) of 96 particles/µL. Furthermore, the present assay enabled sensitive and accurate quantification of exosomes in human serum, and it has high potential for exosome analysis in clinical samples.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Neoplasias da Mama/patologia , Exossomos/patologia , Alquinos/química , Azidas/química , Neoplasias da Mama/sangue , Neoplasias da Mama/química , Química Click/métodos , Sondas de DNA/química , Técnicas Eletroquímicas/métodos , Exossomos/química , Feminino , Humanos , Células MCF-7 , Hibridização de Ácido Nucleico , Tetraspanina 30/análise
12.
J Nanobiotechnology ; 17(1): 85, 2019 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-31319859

RESUMO

BACKGROUND: Lack of effective tumor-specific delivery systems remains an unmet clinical challenge for successful translation of innovative therapies, such as, therapeutic oligonucleotides. In the past decade, exosomes have been suggested to be ideal drug delivery systems with application in a broad range of pathologies including cancer, due to their organotropic properties. Tumor-derived exosomes, having tumor-homing properties, can efficiently reach cancer cells and therefore behave as carriers for improved drug delivery to the primary tumor and metastases. However, due to their complex composition, and still undefined biological functions, safety concerns arise hampering their translation to the clinics. RESULTS: We propose here the development of exosome-mimetic nanosystems (EMNs) that simulate natural tumor-derived exosomes with respect to their structure and functionality, but with a controlled composition, for the targeted delivery of therapeutic oligonucleotides to lung adenocarcinoma cells (microRNA-145 mimics). Making use of the well-known liposome technology, EMNs can be engineered, loaded with the therapeutic compounds, and tailored with specific proteins (integrin α6ß4) providing them organotropic properties. EMNs show great similarities to natural exosomes with respect to their physicochemical properties, drug loading capacity, and ability to interact with the cancer target cells in vitro and in vivo, but are easier to manufacture, can be produced at high yields, and are safer by definition. CONCLUSIONS: We have designed a multifunctional nanoplatform mimicking exosomes, EMNs, and proved their potential to reach cancer cells with a similar efficient that tumor-derived exosomes but providing important advantages in terms of production methodology and regulations. Additionally, EMNs are highly versatile systems that can be tunable for a broader range of applications.


Assuntos
Antineoplásicos/química , Materiais Biomiméticos/química , Exossomos/química , MicroRNAs/química , Nanocápsulas/química , Animais , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Técnicas de Transferência de Genes , Humanos , Integrinas/química , Integrinas/metabolismo , Camundongos , MicroRNAs/administração & dosagem , Propriedades de Superfície
14.
J Dairy Sci ; 102(8): 6726-6737, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31155266

RESUMO

Previous studies have demonstrated that bovine milk contains mRNA and microRNA that are largely encapsulated in milk-derived exosomes. However, little information is available about long noncoding RNAs (lncRNA) in bovine milk. Increasing evidence suggests that lncRNA are of particular interest given their key role in gene expression and development. We performed a comprehensive analysis of lncRNA in bovine milk exosomes by RNA sequencing. We used a validated human in vitro digestion model to investigate the stability of lncRNA encapsulated in bovine milk exosomes during the digestion process. We identified 3,475 novel lncRNA and 6 annotated lncRNA. The lncRNA shared characteristics with those of other mammals in terms of length, exon number, and open reading frames. However, lncRNA showed higher expression than mRNAs. We selected 12 lncRNA of high-expression abundance and identified them by PCR. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that lncRNA regulate immune function, osteoblastogenesis, neurodevelopment, reproduction, cell proliferation, and cell-cell communication. We also investigated the 12 lncRNA using quantitative real-time PCR to reveal their expression profiles in milk exosomes during different stages of lactation (colostrum 2 d, 30 d, 150 d, and 270 d); their resulting expression levels in milk exosomes showed variations across the stages. A digestion experiment showed that bovine milk exosome lncRNA was resistant to in vitro digestion with different digestive juices, including saliva, gastric juice, pancreatic juice, and bile juice. Taken together, these results show for the first time that cow milk contains lncRNA, and that their abundance varied at different stages of lactation. As expected, bovine milk exosomal lncRNA were stable during in vitro digestion. These findings provide a basis for further understanding of the physiological role of milk lncRNA.


Assuntos
Leite/química , RNA Longo não Codificante/análise , Animais , Bovinos , Colostro/metabolismo , Digestão , Estabilidade de Medicamentos , Exossomos/química , Exossomos/metabolismo , Feminino , Expressão Gênica , Genoma , Humanos , Lactação/fisiologia , MicroRNAs/genética , Gravidez , RNA Longo não Codificante/genética , RNA Longo não Codificante/fisiologia , RNA Mensageiro/genética , Análise de Sequência de RNA/veterinária
15.
Anal Chim Acta ; 1073: 79-89, 2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31146839

RESUMO

We investigated the effect of oxidative stress (OS) on lipidomic perturbations in the subcellular fractions and exosomes of human embryonic kidney (HEK) 293 cells using asymmetrical flow field-flow fractionation (AF4) and nanoflow ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry (nUHPLC-ESI-MS/MS). We treated HEK 293 cells with hydrogen peroxide (H2O2) and fractionated the cell lysates using AF4 to determine the change in size and population of the subcellular fractions and exosomes, and to obtain narrow size fractions for lipid analysis. A total of 438 lipids from 642 identified species-including oxidized lipids-were quantified. The relative amount of secreted exosomes increased by 28% during OS, whereas the amount of subcellular species decreased by 35%. There was a significant increase in the level of oxidized phospholipids in the mitochondrion-enriched subcellular fractions, but not in the exosomes. Most high-abundance triacylglycerol (TG) species increased in the stressed cells, whereas they decreased in the exosomes. During OS, ceramides involved in the apoptotic mitochondrial pathway were accumulated in the subcellular fractions, whereas their levels were unaffected in the exosomes. The present study demonstrated that AF4 and nUHPLC-ESI-MS/MS can be used to investigate lipid alterations in subcellular and extracellular species during OS, and the pathological relationships in diseases caused by reactive oxygen species.


Assuntos
Exossomos/metabolismo , Fracionamento por Campo e Fluxo , Lipídeos/análise , Organelas/metabolismo , Estresse Oxidativo , Cromatografia Líquida de Alta Pressão , Exossomos/química , Células HEK293 , Humanos , Organelas/química , Espectrometria de Massas em Tandem
16.
J Biosci ; 44(2)2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31180050

RESUMO

To investigate the source of serum exosomal HOTAIR, to uncover the diagnostic and prognostic values of serum exosomal HOTAIR, and to discern the expression of serum exosomal HOTAIR between neoadjuvant chemotherapy and response to tamoxifen therapy. Samples were collected from the Third Affiliated Hospital of Kunming Medical University, Tumor Hospital of Yunnan. Exosomes were isolated from serum, cell culture medium and tumor tissues. We used transmission electron microscopy and western immunoblotting assay to characterize exosomes, and real-time PCR (qPCR) to assess HOTAIR expression. Neoadjuvant chemotherapy and tamoxifen therapy were carried out according to established guidelines. Breast cancer patients expressed higher serum exosomal HOTAIR than did healthy individuals (P\0.001). Serum exosomal HOTAIR levels 3 months after surgery were markedly decreased compared with levels before surgery (P\0.001), and the expression level of exosomal HOTAIR in cell culture medium increased with time in both breast cancer cell lines (72 h greater than 48 h greater than 24 h, 48 h vs 24 h [ [P less than 0.05]; 72 h vs 24 h [P less than 0.01]. Expression of serum exosomal HOTAIR in nude mice was notably greater than in the mock control group (P less than 0.001). The results of the ROC analysis revealed an AUC for serum exosomal HOTAIR of 0.9178 with a 95% CI of 0.8407-1.017 (P less than 0.01). The AUC for the CA15-3 cell line was 0.7378 (95% CI, 0.5585-0.9170; P = 0.03). High expression of exosomal HOTAIR led to a worse disease-free survival (P = 0.0481) and overall survival (P = 0.0463). In the high-expression chemotherapy group, six patients achieved a partial response (PR) and eight demonstrated stable disease (SD) and nine patients achieved PR and two SD in the low-expression group (P = 0.048). In the low-expression tamoxifen group, one patient had a recurrence of breast cancer and another 10 patients exhibited no recurrence, while six showed recurrence, and seven had none in the highexpression group (P = 0.035). We isolated exosomes successfully, and demonstrated that serum exosomal HOTAIR originated from primary breast cancer tissue. We conclude that serum exosomal HOTAIR exhibits the potential to be a diagnostic and prognostic biomarker. High expression of serum exosomal HOTAIR was also correlated with poor neoadjuvant chemotherapy and response to tamoxifen therapy.


Assuntos
Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Carcinoma Lobular/genética , Exossomos/química , Regulação Neoplásica da Expressão Gênica , RNA Longo não Codificante/genética , Adulto , Idoso , Animais , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/sangue , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/mortalidade , Carcinoma Ductal de Mama/sangue , Carcinoma Ductal de Mama/tratamento farmacológico , Carcinoma Ductal de Mama/mortalidade , Carcinoma Lobular/sangue , Carcinoma Lobular/tratamento farmacológico , Carcinoma Lobular/mortalidade , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Exossomos/metabolismo , Feminino , Humanos , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , RNA Longo não Codificante/metabolismo , Transdução de Sinais , Análise de Sobrevida , Tamoxifeno/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Biosens Bioelectron ; 141: 111397, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31200334

RESUMO

Nowadays, exosomes that carry abundant information have attracted increasing attention as potent biomarkers of liquid biopsy and ideal candidates for early diagnosis and treatment of cancers. In this work, we propose a "principle-of-proof" biosensing method for amplified electrochemical detection of exosomes by using HepG2-derived exosomes as models. Specifically, target exosomes are enriched on anti-CD63-functionalized immunobeads and then recognized by a DNA chain containing CD63 aptamer region, which subsequently initiates a catalytic molecule machine that relies on cascade toehold-mediated strand displacement reaction. Benefiting from high efficiency of the molecule machine, the method shows a linear range from 1 × 105 to 5 × 107 particles/mL and a detection limit of 1.72 × 104 particles/mL toward target exosomes, better than most existing detection methods. Moreover, the method demonstrates a high specificity even in serum samples and suggests a potential use in clinic, which may provide sufficient information for disease diagnosis, especially early detection and prognosis monitoring of tumors.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Exossomos/química , Tetraspanina 30/análise , Anticorpos Imobilizados/química , Química Click , Técnicas Eletroquímicas/métodos , Células Hep G2 , Humanos
18.
Mol Cell Biochem ; 459(1-2): 1-6, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31073888

RESUMO

Exosomes are 40- to 100- nm cell-originated vesicles derived from endocytic compartments that are released into almost all biological fluids. Exosomes are cell-created vesicles that inherit identical phospholipid membrane, explaining a wide application of electroporation as a technique for exosomes loading with exogenous cargoes. Another way of loading exosomes with therapeutic cargo is to overexpress a certain gene in exosome-donor cells or treat cell line with drug of interest that later will be gently enveloped into vesicles based on the process of EV biogenesis. Similarly, to visualize siRNA loading into exosomes as well as the exosomal product delivery to recipient cells, we have conducted an experiment where chemical-based exosome transfection was used. In this review, we discuss different ways of extracellular vesicle loading with exogenous cargoes and their advantages/limitations as well as novel alternative techniques of substance incorporation into nanoparticles.


Assuntos
Portadores de Fármacos/química , Portadores de Fármacos/uso terapêutico , Exossomos/química , Nanopartículas/química , Nanopartículas/uso terapêutico , Animais , Humanos
19.
Lab Chip ; 19(10): 1877-1886, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31044204

RESUMO

Extracellular vesicles (EVs), particularly exosomes (30-150 nm), are an emerging delivery system in mediating cellular communications, which have been observed for priming immune responses by presenting parent cell signaling proteins or tumor antigens to immune cells. Therefore, preparation of antigenic exosomes that can play therapeutic roles, particularly in cancer immunotherapy, is emerging. However, standard benchtop methods (e.g., ultracentrifugation and filtration) lack the ability to purify antigenic exosomes specifically among other microvesicle subtypes, due to the non-selective and time-consuming (>10 h) isolation protocols. Exosome engineering approaches, such as the transfection of parent cells, also suffer from poor yields, low purity, and time-consuming operations. In this paper, we introduce a streamlined microfluidic cell culture platform for integration of harvesting, antigenic modification, and photo-release of surface engineered exosomes in one workflow, which enables the production of intact, MHC peptide surface engineered exosomes for cytolysis activation. A PDMS microfluidic cell culture chip is simply cast from a 3D-printed mold. This proof-of-concept study demonstrated the enhanced ability of harvested exosomes in antigen presentation and T cell activation, by decorating melanoma tumor peptides on the exosome surface (e.g., gp-100, MART-1, and MAGE-A3). Such surface engineered antigenic exosomes were harvested in real-time from the on-chip culture of leukocytes isolated from human blood, leading to much faster cellular uptake. The activation of gp100-specific CD8 T cells which were purified from the spleen of 2 Pmel1 transgenic mice was evaluated using surface engineered exosomes prepared from murine antigen presenting cells. Antigen-specific CD8 T cell proliferation was significantly induced by the engineered exosomes compared to that by native, non-engineered exosomes. This microfluidic platform serves as an automated and highly integrated cell culture device for rapid and real-time production of therapeutic exosomes that could advance cancer immunotherapy.


Assuntos
Engenharia Celular , Exossomos/química , Imunoterapia , Técnicas Analíticas Microfluídicas , Neoplasias/terapia , Animais , Antígenos de Neoplasias/química , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Exossomos/imunologia , Humanos , Camundongos , Neoplasias/imunologia , Impressão Tridimensional
20.
Int J Nanomedicine ; 14: 2349-2369, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31040661

RESUMO

Background: In view of the growing importance of nanotechnologies, the detection/identification of nanoparticles type has been considered of utmost importance. Although the characterization of synthetic/organic nanoparticles is currently considered a priority (eg, drug delivery devices, nanotextiles, theranostic nanoparticles), there are many examples of "naturally" generated nanostructures - for example, extracellular vesicles (EVs), lipoproteins, and virus - that provide useful information about human physiology or clinical conditions. For example, the detection of tumor-related exosomes, a specific type of EVs, in circulating fluids has been contributing to the diagnosis of cancer in an early stage. However, scientists have struggled to find a simple, fast, and low-cost method to accurately detect/identify these nanoparticles, since the majority of them have diameters between 100 and 150 nm, thus being far below the diffraction limit. Methods: This study investigated if, by projecting the information provided from short-term portions of the back-scattered laser light signal collected by a polymeric lensed optical fiber tip dipped into a solution of synthetic nanoparticles into a lower features dimensional space, a discriminant function is able to correctly detect the presence of 100 nm synthetic nanoparticles in distilled water, in different concentration values. Results and discussion: This technique ensured an optimal performance (100% accuracy) in detecting nanoparticles for a concentration above or equal to 3.89 µg/mL (8.74E+10 particles/mL), and a performance of 90% for concentrations below this value and higher than 1.22E-03 µg/mL (2.74E+07 particles/mL), values that are compatible with human plasmatic levels of tumor-derived and other types of EVs, as well as lipoproteins currently used as potential biomarkers of cardiovascular diseases. Conclusion: The proposed technique is able to detect synthetic nanoparticles whose dimensions are similar to EVs and other "clinically" relevant nanostructures, and in concentrations equivalent to the majority of cell-derived, platelet-derived EVs and lipoproteins physiological levels. This study can, therefore, provide valuable insights towards the future development of a device for EVs and other biological nanoparticles detection with innovative characteristics.


Assuntos
Tecnologia Biomédica/métodos , Técnicas Biossensoriais/métodos , Nanopartículas/química , Fibras Ópticas , Análise Discriminante , Exossomos/química , Vesículas Extracelulares , Análise de Fourier , Humanos , Polímeros/química , Soluções
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