RESUMO
Herb-based extracellular vesicles (EV), inherently replete with bioactive proteins, RNA, lipids, and other medicinal compounds, are noncytotoxic and uniquely capable of cellular delivery to meet the ever-stringent challenges of ongoing clinical applications. EVs are abundant in nature, affordable, and scalable, but they are also incredibly fragile and stuffed with many biomolecules. To address the low drug binding abilities and poor stability of EVs, we demonstrated herb-based EVs (isolated from neem, mint, and curry leaves) conjugated with chitosan (CS) and PEGylated graphene oxide (GP) that led to their transformation into robust and efficient vectors. The designed conjugates successfully delivered estrogen receptor α (ERα1)-targeting siRNA to breast cancer MCF7 cells. Our data revealed that neem-based EV-CS-GP conjugates were most efficient in cellular siRNA delivery, which could be attributed to hyaluronic acid-mediated recognition of neem EVs by MCF7 cells via CD44 receptors. Our approach shows a futuristic direction in designing clinically viable, sustainable, nontoxic EV-based vehicles that can deliver a variety of functional siRNA cargos.
Assuntos
Neoplasias da Mama , Quitosana , Receptor alfa de Estrogênio , Vesículas Extracelulares , Grafite , Polietilenoglicóis , RNA Interferente Pequeno , Humanos , Quitosana/química , Grafite/química , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Receptor alfa de Estrogênio/metabolismo , Células MCF-7 , Polietilenoglicóis/química , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Teste de Materiais , Tamanho da Partícula , Feminino , Sobrevivência Celular/efeitos dos fármacosRESUMO
Aberrantly glycosylated mucin 1 is a critical prognostic biomarker in breast epithelial cancers. Hypoglycosylated mucin 1 coats the surface of the cancer cells, where O-glycans are predominantly linked via an N-acetylgalactosamine moiety (GalNAc). Cancer cell-derived extracellular vesicles (EVs) carry biomarkers from parent cancer cells to the recipient cells and, therefore, could potentially be leveraged for diagnostics and noninvasive disease monitoring. We devised a label-free approach for identifying glycoprotein mucin 1 overexpression on breast cancer EVs. While exploring a plethora of biochemical (enzyme-linked immunosorbent assay, flow cytometry, and SDS-PAGE) and label-free biophysical techniques (circular dichroism and infrared spectroscopy (IR)) along with multivariate analysis, we discovered that mucin 1 is significantly overexpressed in breast cancer EVs and aberrant glycosylation in mucin 1 could be critically addressed using IR and multivariate analysis targeting the GalNAc sugar. This approach emerges as a convenient and comprehensive method of distinguishing cancer EVs from normal samples and holds potential for nonintrusive breast cancer liquid biopsy screening.
Assuntos
Vesículas Extracelulares , Neoplasias , Mucina-1 , GlicosilaçãoRESUMO
Epigenetic dysregulation including DNA methylation and histone modifications is being increasingly recognized as a promising biomarker for the diagnosis and prognosis of cancer. Herein, we devised a label-free analytical toolbox comprising IR, UV-vis, CD spectroscopy, and cyclic voltammetry, which is capable to differentiate significantly hyper-methylated breast cancer chromosomes from the normal breast epithelial counterparts.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/genética , Epigênese Genética , Metilação de DNA , Biomarcadores , CromossomosRESUMO
Cancer cells secrete extracellular vesicles (EVs) covered with a carbohydrate polymer, hyaluronan (HA), linked to tumor malignancy. Herein, we have unravelled the contour lengths of HA on a single cancer cell-derived EV surface using single-molecule force spectroscopy (SMFS), which divulges the presence of low molecular weight HA (LMW-HA < 200 kDa). We also discovered that these LMW-HA-EVs are significantly more elastic than the normal cell-derived EVs. This intrinsic elasticity of cancer EVs could be directly allied to the LMW-HA abundance and associated labile water network on EV surface as revealed by correlative SMFS, hydration dynamics with fluorescence spectroscopy, and molecular dynamics simulations. This method emerges as a molecular biosensor of the cancer microenvironment.
Assuntos
Vesículas Extracelulares , Neoplasias , Humanos , Ácido Hialurônico/química , Peso Molecular , Microambiente TumoralRESUMO
Colorectal cancer typically begins from a nonmalignant polyp formation in the large intestine that, over time, develops into colorectal cancer. The growth of benign polyps can be checked if detected in the early stages of the disease. Doctors usually recommend colonoscopy to average and high-risk individuals for colorectal cancer screening. Elevated carcinoembryonic antigen (CEA) is a broadly used biomarker for colorectal cancer. The genetic and epigenetic alteration of genes such as p53, BRAF, APC, and PIK3CA is also correlated with colorectal cancer in various clinical studies. In general, tissue biopsy is most frequently used for colorectal cancer diagnosis, but the whole tumor heterogeneity cannot be accessed by this technique. Furthermore, such a highly invasive technique is not suitable for repeated testing. Recently, extracellular vesicles (EVs), lipid bilayer enclosed sacs secreted from colorectal cancer cells, are emerging as a diagnostic tool for colon cancer detection. The major advantages of using EVs for colon cancer diagnosis are (i) EVs can be isolated in a noninvasive manner from the body fluid and (ii) EV incorporated cargoes (mostly RNAs) reveal various aspects of colorectal cancer. EV-RNAs are also implicated in tumor invasion and influence the immune system for the further spread of tumors. However, due to the lack of standardized EV detection strategies, diagnostic applicability is limited. Herein, we review the recent literature on the pathobiological dependence of colorectal cancer on EV-RNAs. Further, we present the advantages of identification and characterization of EV-RNAs to explore the connection between differential expression of extracellular vesicle incorporated RNAs and colorectal cancer. How this approach may potentially translate into point of care colorectal cancer diagnostics is also discussed.
RESUMO
DNA origami has emerged as a versatile platform for diverse applications, namely, photonics, electronics, (bio) sensing, smart actuator, and drug delivery. In the last decade, DNA origami has been extensively pursued for efficient anticancer therapy. However, challenges remain to develop strategies that improve the targeting efficiency and drug delivery capability of the DNA origami nanostructures. In this direction, we developed folate-functionalized DNA origami that effectively targets and delivers doxorubicin (DOX), a well-known anticancer drug to the folate receptor alpha (FOLR1) expressing triple-negative breast cancer (TNBC) cells in vitro. We show that folate-functionalized DNA origami structure targets and kills FOLR1 overexpressing cells with better efficacy than nontargeted origami. We envision that this study will open up the possibility of target specific delivery of anticancer drug combinations using the versatile DNA origami nanostructures to the drug resistant cancer cells.
RESUMO
To realize a customizable biogenic delivery platform, herein we propose combining cell-derived extracellular vesicles (EVs) derived from breast cancer cell line MCF-7 with synthetic cationic liposomes using a fusogenic agent, polyethylene glycol (PEG). We performed a fluorescence resonance energy transfer (FRET)-based lipid-mixing assay with varying PEG 1000 concentrations (0%, 15%, and 30%) correlated with flow cytometry-based analysis and supported by dimensional analysis by dynamic light scattering (DLS), transmission electron microscopy (TEM), and atomic force microscopy (AFM) to validate our fusion strategy. Our data revealed that these hybrid vesicles at a particular concentration of PEG (â¼15%) improved the cellular delivery efficiency of a model siRNA molecule to the EV parental breast cancer cells, MCF-7, by factors of 2 and 4 compared to the loaded liposome and EV precursors, respectively. The critical rigidity/pliability balance of the hybrid systems fused by PEG seems to be playing a pivotal role in improving their delivery capability. This approach can provide clinically viable delivery solutions using EVs.
Assuntos
Neoplasias da Mama , Vesículas Extracelulares , Cátions , Feminino , Humanos , Lipossomos , PolietilenoglicóisRESUMO
The significant role of a vesicle is well recognized; however, only lately has the advancement in biomedical applications started to uncover their usefulness. Although the concept of vesicles originates from cell biology, it later transferred to chemistry and material science to develop nanoscale artificial vesicles for biomedical applications. Herein, we examine different synthetic and biological vesicles and their applications in the biomedical field in general. As our understanding of biological vesicles increases, more suitable biomimicking synthetic vesicles will be developed. The comparative discussion between synthetic and natural vesicles for biomedical applications is a relevant topic, and we envision this could enable the development of a proper approach to realize the next-generation treatment goals.
Assuntos
Materiais Biocompatíveis/química , Pesquisa Biomédica , Vesículas Extracelulares/química , Humanos , Teste de Materiais , Tamanho da PartículaRESUMO
Extracellular vesicles (EVs), naturally occurring nanosized vesicles secreted from cells, are essential for intercellular communication. They carry unique biomolecules on the surface or interior that are of great interest as biomarkers for various pathological conditions such as cancer. In this work, we use high-resolution atomic force microscopy (AFM) and spectroscopy (AFS) techniques to demonstrate differences between EVs derived from colon cancer cells and colon epithelial cells at the single-vesicle level. We observe that EV populations are significantly increased in the cancer cell media compared to the normal cell EVs. We show that both EVs display an EV marker, CD9, while EVs derived from the cancer cells are slightly higher in density. Hyaluronan (HA) is a nonsulfated glycosaminoglycan linked to malignant tumor growth according to recent reports. Interestingly, at the single-vesicle level, colon cancer EVs exhibit significantly increased HA surface densities compared to the normal EVs. Spectroscopic measurements such as Fourier transform infrared (FT-IR), circular dichroism (CD), and Raman spectroscopy unequivocally support the AFM and AFS measurements. To our knowledge, it represents the first report of detecting HA-coated EVs as a potential colon cancer biomarker. Taken together, this sensitive approach will be useful in identifying biomarkers in the early stages of detection and evaluation of cancer.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias do Colo/metabolismo , Vesículas Extracelulares/metabolismo , Ácido Hialurônico/análise , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , Células Epiteliais/metabolismo , Humanos , Ácido Hialurônico/metabolismo , Microscopia de Força Atômica , Espectrofotometria Atômica , Tetraspanina 29/análiseRESUMO
Recently, surface-enhanced Raman scattering nanoprobes have shown tremendous potential in oncological imaging owing to the high sensitivity and specificity of their fingerprint-like spectra. As current Raman scanners rely on a slow, point-by-point spectrum acquisition, there is an unmet need for faster imaging to cover a clinically relevant area in real-time. Herein, we report the rational design and optimization of fluorescence-Raman bimodal nanoparticles (FRNPs) that synergistically combine the specificity of Raman spectroscopy with the versatility and speed of fluorescence imaging. DNA-enabled molecular engineering allows the rational design of FRNPs with a detection limit as low as 5 × 10-15 M. FRNPs selectively accumulate in tumor tissue mouse cancer models and enable real-time fluorescence imaging for tumor detection, resection, and subsequent Raman-based verification of clean margins. Furthermore, FRNPs enable highly efficient image-guided photothermal ablation of tumors, widening the scope of the NPs into the therapeutic realm.
Assuntos
Neoplasias Encefálicas/terapia , DNA/química , Nanopartículas Metálicas/química , Imagem Óptica/métodos , Neoplasias Ovarianas/terapia , Análise Espectral Raman/métodos , Animais , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/cirurgia , Linhagem Celular Tumoral , DNA/metabolismo , Portadores de Fármacos/síntese química , Portadores de Fármacos/farmacocinética , Feminino , Corantes Fluorescentes/química , Engenharia Genética , Humanos , Terapia a Laser/instrumentação , Terapia a Laser/métodos , Limite de Detecção , Terapia com Luz de Baixa Intensidade/instrumentação , Terapia com Luz de Baixa Intensidade/métodos , Nanopartículas Metálicas/administração & dosagem , Camundongos , Imagem Óptica/instrumentação , Neoplasias Ovarianas/diagnóstico por imagem , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/cirurgia , Imagens de Fantasmas , Prata/química , Análise Espectral Raman/instrumentação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The protein p300 is a multifunctional transcriptional coactivator that plays pivotal role in several cellular functions. Although structures of several domains have been solved in isolation, the structures of full-length protein (p300 FL) or its complexes with transcription activators are completely unknown. Herein, we applied atomic force microscopy to visualize p300 FL. We found that it is almost prolate ellipsoidal in shape, having several bulges. We further identified the functionally significant N-terminal and C-terminal regions, by applying domain-specific antibodies and found that they are located near one end and centre of the molecule, respectively. Importantly, we have visualized the complex between p300 FL and tumor suppressor protein p53. The relevance of these data in understanding dynamics of p300 during acetylation and transcription will be mentioned.