Extracellular Vesicles from Highly Metastatic Osteosarcoma Cells Induce Pro-Tumorigenic Macrophage Phenotypes.

Metastasis is the principal factor in poor prognosis for individuals with osteosarcoma (OS). Understanding the events that lead to metastasis is critical to develop better interventions for this disease. Alveolar macrophages are potentially involved in priming the lung microenvironment for OS metastasis, yet the mechanisms involved in this process remain unclear. Since extracellular vesicles (EVs) are a known actor in primary tumor development, their potential role in OS metastagenesis through macrophage modulation is explored here. The interaction of EVs isolated from highly metastatic (K7M2) and less metastatic (K12) osteosarcoma cell lines is compared with a peritoneal macrophage cell line. An EV concentration that reproducibly induced macrophage migration is identified first, then used for later experiments. By confocal microscopy, both EV types associated with M0 or M1 macrophages; however, only K7M2-EVs are associated with M2 macrophages, an interaction that is abrogated by EV pre-treatment with anti-CD47 antibody. Interestingly, all interactions appeared to be surface binding, not internalized. In functional studies, K7M2-EVs polarized fewer macrophages to M1. Together, these data suggest that K7M2-EVs have unique interactions with macrophages that can contribute to the production of a higher proportion of pro-tumor type macrophages, thereby accelerating metastasis.

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches.

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.

A Multi-Fluorophore Staining Scheme for Identification and Quantification of Vomocytosis.

Vomocytosis is a process by which fungal pathogens, for instance, Cryptococcus neoformans (CN), escape from the digestive phagolysosome of phagocytic cells after ingestion. Interestingly, this expulsion leaves both the pathogen and phagocyte unharmed, and is believed to be an important mechanism by which CNs disseminate throughout infected hosts. This phenomenon was discovered in 2006, and research to date has relied almost entirely on quantification via manual counting of vomocytosis events in time-lapse microscopy videos. This archaic method has the significant disadvantages of requiring excessive labor in manual analysis, limited throughput capabilities, and low accuracy due to subjectivity. Here, we present an alternative method to measure vomocytosis rates using a multi-fluorophore reporter system comprised of two in situ staining steps during infection and a flow cytometry readout. This approach overcomes the limitations of conventional time lapse microscopy methods, with key advantages of high throughput capability, simple procedural steps, and accurate objective readouts. This study rigorously characterizes this vomocytosis reporter system in CN-infected MΦ and DC cultures via fluorescence microscopy, confocal microscopy, and flow cytometry. Here, this fluorescent tool is used to observe differences in expulsion rates after phagosome-modifying drug treatments and additionally utilized to distinguish differences in biochemical compositions among fluorescence-activated cell sorted fungal populations via Raman spectroscopy. Furthermore, this reporter scheme is demonstrated to be adaptable for use in measuring potential biomaterial particle expulsion events. Ultimately, the fluorescent reporter system presented here provides a universal tool for vomocytosis rate measurement of phagocytosed material. This facile approach opens the door to previously unfeasible types of vomocytosis-related studies such as high throughput treatment mechanistic screening and downstream characterization of expelled material.

Enhanced pericyte-endothelial interactions through NO-boosted extracellular vesicles drive revascularization in a mouse model of ischemic injury.

Despite improvements in medical and surgical therapies, a significant portion of patients with critical limb ischemia (CLI) are considered as "no option" for revascularization. In this work, a nitric oxide (NO)-boosted and activated nanovesicle regeneration kit (n-BANK) is constructed by decorating stem cell-derived nanoscale extracellular vesicles with NO nanocages. Our results demonstrate that n-BANKs could store NO in endothelial cells for subsequent release upon pericyte recruitment for CLI revascularization. Notably, n-BANKs enable endothelial cells to trigger eNOS activation and form tube-like structures. Subsequently, eNOS-derived NO robustly recruits pericytes to invest nascent endothelial cell tubes, giving rise to mature blood vessels. Consequently, n-BANKs confer complete revascularization in female mice following CLI, and thereby achieve limb preservation and restore the motor function. In light of n-BANK evoking pericyte-endothelial interactions to create functional vascular networks, it features promising therapeutic potential in revascularization to reduce CLI-related amputations, which potentially impact regeneration medicine.

Convection and extracellular matrix binding control interstitial transport of extracellular vesicles.

Extracellular vesicles (EVs) influence a host of normal and pathophysiological processes in vivo. Compared to soluble mediators, EVs can traffic a wide range of proteins on their surface including extracellular matrix (ECM) binding proteins, and their large size (∼30-150 nm) limits diffusion. We isolated EVs from the MCF10 series-a model human cell line of breast cancer progression-and demonstrated increasing presence of laminin-binding integrins α3ß1 and α6ß1 on the EVs as the malignant potential of the MCF10 cells increased. Transport of the EVs within a microfluidic device under controlled physiological interstitial flow (0.15-0.75 µm/s) demonstrated that convection was the dominant mechanism of transport. Binding of the EVs to the ECM enhanced the spatial concentration and gradient, which was mitigated by blocking integrins α3ß1 and α6ß1. Our studies demonstrate that convection and ECM binding are the dominant mechanisms controlling EV interstitial transport and should be leveraged in nanotherapeutic design.

Recent developments in biosensing methods for extracellular vesicle protein characterization.

Research into extracellular vesicles (EVs) has grown significantly over the last few decades with EVs being widely regarded as a source of biomarkers for human health and disease with massive clinical potential. Secreted by every cell type in the body, EVs report on the internal cellular conditions across all tissue types. Their presence in readily accessible biofluids makes the potential of EV biosensing highly attractive as a noninvasive diagnostic platform via liquid biopsies. However, their small size (50-250 nm), inherent heterogeneity, and the complexity of the native biofluids introduce challenges for effective characterization, thus, limiting their clinical utility. This has led to a surge in the development of various novel EV biosensing techniques, with capabilities beyond those of conventional methods that have been directly transferred from cell biology. In this review, key detection principles used for EV biosensing are summarized, with a focus on some of the most recent and fundamental developments in the field over the last 5 years. This article is categorized under: Diagnostic Tools > Biosensing Diagnostic Tools > In Vitro Nanoparticle-Based Sensing.

Development and Characterization of Bioinspired Lipid Raft Nanovesicles for Therapeutic Applications.

Lipid rafts are highly ordered regions of the plasma membrane enriched in signaling proteins and lipids. Their biological potential is realized in exosomes, a subclass of extracellular vesicles (EVs) that originate from the lipid raft domains. Previous studies have shown that EVs derived from human placental mesenchymal stromal cells (PMSCs) possess strong neuroprotective and angiogenic properties. However, clinical translation of EVs is challenged by very low, impure, and heterogeneous yields. Therefore, in this study, lipid rafts are validated as a functional biomaterial that can recapitulate the exosomal membrane and then be synthesized into biomimetic nanovesicles. Lipidomic and proteomic analyses show that lipid raft isolates retain functional lipids and proteins comparable to PMSC-EV membranes. PMSC-derived lipid raft nanovesicles (LRNVs) are then synthesized at high yields using a facile, extrusion-based methodology. Evaluation of biological properties reveals that LRNVs can promote neurogenesis and angiogenesis through modulation of lipid raft-dependent signaling pathways. A proof-of-concept methodology further shows that LRNVs could be loaded with proteins or other bioactive cargo for greater disease-specific functionalities, thus presenting a novel type of biomimetic nanovesicles that can be leveraged as targeted therapeutics for regenerative medicine.

Homogenous high enhancement surface-enhanced Raman scattering (SERS) substrates by simple hierarchical tuning of gold nanofoams.

Surface enhanced Raman scattering (SERS) is a powerful tool for vibrational spectroscopy, providing orders of magnitude increase in chemical sensitivity compared to spontaneous Raman scattering. Yet it remains a challenge to synthesize robust, uniform SERS substrates quickly and easily. Lithographic approaches to produce substrates can achieve high, uniform sensitivity but are expensive and complex, thus difficult to scale. Facile solution-phase chemical approaches often result in unreliable SERS substrates due to heterogeneous arrangement of "hot spots" throughout the material. Here we demonstrate the synthesis and characterization of a homogeneous gold nanofoam (AuNF) substrate produced by a rapid, one-pot, four-ingredient synthetic approach. AuNFs are rapidly nucleated with macroscale porosity and then chemically roughened to produce nanoscale features that confer homogeneous and high signal enhancement (~109) across large areas, a comparable performance to lithographically produced substrates.

Fused Raman spectroscopic analysis of blood and saliva delivers high accuracy for head and neck cancer diagnostics.

As a rapid, label-free, non-destructive analytical measurement requiring little to no sample preparation, Raman spectroscopy shows great promise for liquid biopsy cancer detection and diagnosis. We carried out Raman analysis and mass spectrometry of plasma and saliva from more than 50 subjects in a cohort of head and neck cancer patients and benign controls (e.g., patients with benign oral masses). Unsupervised data models were built to assess diagnostic performance. Raman spectra collected from either biofluid provided moderate performance to discriminate cancer samples. However, by fusing together the Raman spectra of plasma and saliva for each patient, subsequent analytical models delivered an impressive sensitivity, specificity, and accuracy of 96.3%, 85.7%, and 91.7%, respectively. We further confirmed that the metabolites driving the differences in Raman spectra for our models are among the same ones that drive mass spectrometry models, unifying the two techniques and validating the underlying ability of Raman to assess metabolite composition. This study bolsters the relevance of Raman to provide additive value by probing the unique chemical compositions across biofluid sources. Ultimately, we show that a simple data augmentation routine of fusing plasma and saliva spectra provided significantly higher clinical value than either biofluid alone, pushing forward the potential of clinical translation of Raman spectroscopy for liquid biopsy cancer diagnostics.

Extracellular Vesicles (EVs) Are Copurified with Feline Calicivirus, yet EV-Enriched Fractions Remain Infectious.

Feline calicivirus (FCV) is a major cause of upper respiratory disease in cats and is often used as a model for human norovirus, making it of great veterinary and human medical importance. However, questions remain regarding the route of entry of FCV in vivo. Increasing work has shown that extracellular vesicles (EVs) can be active in viral infectivity, yet there is no work examining the role of EVs in FCV infection. Here, we begin to address this knowledge gap by characterizing EVs produced by a feline mammary epithelial cell line (FMEC). We have confirmed that EVs are produced by infected and mock-infected FMECs and that both virions and EVs are coisolated with standard methods of virus purification. We also show that they can be enriched differentially by continuous iodixanol density gradient. EVs were enriched at a density of 1.10 g/mL confirmed by tetraspanin expression, size profile, and transmission electron microscopy (TEM). Maximum enrichment of FCV at a density of 1.18 g/mL was confirmed by titration, quantitative reverse transcriptase PCR (q-RT PCR), and TEM. However, infectious virus was recovered from nearly all samples. When used to infect in vitro epithelium, both EV-rich and virus-rich fractions had the same levels of infectiousness as determined by percentage of wells infected or titer achieved postinfection. These findings highlight the importance of coisolates during viral purification, showing that EVs may represent a parallel route of entry that has previously been overlooked. Additional experiments are necessary to explore the role of EVs in FCV infection. IMPORTANCE Feline calicivirus (FCV) is a common cause of upper respiratory infection in cats. Both healthy and infected cells produce small particles called extracellular vesicles (EVs), which are nanoparticles that act as messengers between cells and can be hijacked during viral infection. Historically, the role of EVs in viral infection has been overlooked, and subsequently no group has studied the role of EVs in FCV infection. We hypothesized that EVs may play a role in FCV infection. Here, we show that EVs are copurified with FCV when collecting virus. To study their individual effects, we successfully enrich for viral particles and EVs separately by taking advantage of their different densities. Our initial studies show that EV-enriched versus virus-enriched fractions are equally able to infect cells in culture. These findings highlight the need to both consider the purity of virus after purification and to further study EVs' role in natural FCV infection.

Novel Stilbene-Nitroxyl Hybrid Compounds Display Discrete Modulation of Amyloid Beta Toxicity and Structure.

Several neurodegenerative diseases are driven by misfolded proteins that assemble into soluble aggregates. These "toxic oligomers" have been associated with a plethora of cellular dysfunction and dysregulation, however the structural features underlying their toxicity are poorly understood. A major impediment to answering this question relates to the heterogeneous nature of the oligomers, both in terms of structural disorder and oligomer size. This not only complicates elucidating the molecular etiology of these disorders, but also the druggability of these targets as well. We have synthesized a class of bifunctional stilbenes to modulate both the conformational toxicity within amyloid beta oligomers (AßO) and the oxidative stress elicited by AßO. Using a neuronal culture model, we demonstrate this bifunctional approach has the potential to counter the molecular pathogenesis of Alzheimer's disease in a powerful, synergistic manner. Examination of AßO structure by various biophysical tools shows that each stilbene candidate uniquely alters AßO conformation and toxicity, providing insight towards the future development of structural correctors for AßO. Correlations of AßO structural modulation and bioactivity displayed by each provides insights for future testing in vivo. The multi-target activity of these hybrid molecules represents a highly advantageous feature for disease modification in Alzheimer's, which displays a complex, multifactorial etiology. Importantly, these novel small molecules intervene with intraneuronal AßO, a necessary feature to counter the cycle of dysregulation, oxidative stress and inflammation triggered during the earliest stages of disease progression.

Machine Learning-Assisted Sampling of Surfance-Enhanced Raman Scattering (SERS) Substrates Improve Data Collection Efficiency.

Surface-enhanced Raman scattering (SERS) is a powerful technique for sensitive label-free analysis of chemical and biological samples. While much recent work has established sophisticated automation routines using machine learning and related artificial intelligence methods, these efforts have largely focused on downstream processing (e.g., classification tasks) of previously collected data. While fully automated analysis pipelines are desirable, current progress is limited by cumbersome and manually intensive sample preparation and data collection steps. Specifically, a typical lab-scale SERS experiment requires the user to evaluate the quality and reliability of the measurement (i.e., the spectra) as the data are being collected. This need for expert user-intuition is a major bottleneck that limits applicability of SERS-based diagnostics for point-of-care clinical applications, where trained spectroscopists are likely unavailable. While application-agnostic numerical approaches (e.g., signal-to-noise thresholding) are useful, there is an urgent need to develop algorithms that leverage expert user intuition and domain knowledge to simplify and accelerate data collection steps. To address this challenge, in this work, we introduce a machine learning-assisted method at the acquisition stage. We tested six common algorithms to measure best performance in the context of spectral quality judgment. For adoption into future automation platforms, we developed an open-source python package tailored for rapid expert user annotation to train machine learning algorithms. We expect that this new approach to use machine learning to assist in data acquisition can serve as a useful building block for point-of-care SERS diagnostic platforms.

Surface enhanced Raman scattering of extracellular vesicles for cancer diagnostics despite isolation dependent lipoprotein contamination.

Given the emerging diagnostic utility of extracellular vesicles (EVs), it is important to account for non-EV contaminants. Lipoprotein present in EV-enriched isolates may inflate particle counts and decrease sensitivity to biomarkers of interest, skewing chemical analyses and perpetuating downstream issues in labeling or functional analysis. Using label free surface enhanced Raman scattering (SERS), we confirm that three common EV isolation methods (differential ultracentrifugation, density gradient ultracentrifugation, and size exclusion chromatography) yield variable lipoprotein content. We demonstrate that a dual-isolation method is necessary to isolate EVs from the major classes of lipoprotein. However, combining SERS analysis with machine learning assisted classification, we show that the disease state is the main driver of distinction between EV samples, and largely unaffected by choice of isolation. Ultimately, this study describes a convenient SERS assay to retain accurate diagnostic information from clinical samples by overcoming differences in lipoprotein contamination according to isolation method.

Identification of amyloid beta in small extracellular vesicles via Raman spectroscopy.

One of the hallmarks of Alzheimer's disease (AD) pathogenesis is believed to be the production and deposition of amyloid-beta (Aß) peptide into extracellular plaques. Existing research indicates that extracellular vesicles (EVs) can carry Aß associated with AD. However, characterization of the EVs-associated Aß and its conformational variants has yet to be realized. Raman spectroscopy is a label-free and non-destructive method that is able to assess the biochemical composition of EVs. This study reports for the first time the Raman spectroscopic fingerprint of the Aß present in the molecular cargo of small extracellular vesicles (sEVs). Raman spectra were measured from sEVs isolated from Alzheimer's disease cell culture model, where secretion of Aß is regulated by tetracycline promoter, and from midbrain organoids. The averaged spectra of each sEV group showed considerable variation as a reflection of the biochemical content of sEVs. Spectral analysis identified more intense Raman peaks at 1650 cm-1 and 2930 cm-1 attributable to the Aß peptide incorporated in sEVs produced by the Alzheimer's cell culture model. Subsequent analysis of the spectra by principal component analysis differentiated the sEVs of the Alzheimer's disease cell culture model from the control groups of sEVs. Moreover, the results indicate that Aß associated with secreted sEVs has a α-helical secondary structure and the size of a monomer or small oligomer. Furthermore, by analyzing the lipid content of sEVs we identified altered fatty acid chain lengths in sEVs that carry Aß that may affect the fluidity of the EV membrane. Overall, our findings provide evidence supporting the use of Raman spectroscopy for the identification and characterization of sEVs associated with potential biomarkers of neurological disorders such as toxic proteins.

Tetraspanins are unevenly distributed across single extracellular vesicles and bias sensitivity to multiplexed cancer biomarkers.

BACKGROUND: Tetraspanin expression of extracellular vesicles (EVs) is often used as a surrogate for their detection and classification, a practice that typically assumes their consistent expression across EV sources. RESULTS: Here we demonstrate that there are distinct patterns in colocalization of tetraspanin expression of EVs enriched from a variety of in vitro and in vivo sources. We report an optimized method for the use of single particle antibody-capture and fluorescence detection to identify subpopulations according to tetraspanin expression and compare our findings with nanoscale flow cytometry. We found that tetraspanin profile is consistent from a given EV source regardless of isolation method, but that tetraspanin profiles are distinct across various sources. Tetraspanin profiles measured by flow cytometry do not totally agree, suggesting that limitations in subpopulation detection significantly impact apparent protein expression. We further analyzed tetraspanin expression of single EVs captured non-specifically, revealing that tetraspanin capture can bias the apparent multiplexed tetraspanin profile. Finally, we demonstrate that this bias can have significant impact on diagnostic sensitivity for tumor-associated EV surface markers. CONCLUSION: Our findings may reveal key insights into protein expression heterogeneity of EVs that better inform EV capture and detection platforms for diagnostic or other downstream use.

Hybrid Nanoplasmonic Porous Biomaterial Scaffold for Liquid Biopsy Diagnostics Using Extracellular Vesicles.

For more effective early-stage cancer diagnostics, there is a need to develop sensitive and specific, non- or minimally invasive, and cost-effective methods for identifying circulating nanoscale extracellular vesicles (EVs). Here, we report the utilization of a simple plasmonic scaffold composed of a microscale biosilicate substrate embedded with silver nanoparticles for surface-enhanced Raman scattering (SERS) analysis of ovarian and endometrial cancer EVs. These substrates are rapidly and inexpensively produced without any complex equipment or lithography. We extensively characterize the substrates with electron microscopy and outline a reproducible methodology for their use in analyzing EVs from in vitro and in vivo biofluids. We report effective chemical treatments for (i) decoration of metal surfaces with cysteamine to nonspecifically pull down EVs to SERS hotspots and (ii) enzymatic cleavage of extraluminal moieties at the surface of EVs that prevent localization of complementary chemical features (lipids/proteins) to the vicinity of the metal-enhanced fields. We observe a major loss of sensitivity for ovarian and endometrial cancer following enzymatic cleavage of EVs' extraluminal domain, suggesting its critical significance for diagnostic platforms. We demonstrate that the SERS technique represents an ideal tool to assess and measure the high heterogeneity of EVs isolated from clinical samples in an inexpensive, rapid, and label-free assay.

Discovery and mechanistic characterization of a structurally-unique membrane active peptide.

Membrane active peptides (MAPs) have gained wide interest due to their far reaching applications in drug discovery and drug delivery. The search for new MAPs, however, has been largely skewed with bias selecting for physicochemical parameters believed to be important for membrane activity, such as alpha helicity, cationicity and hydrophobicity. Here we carry out a search-and-find strategy to screen a 100,000-membered one-bead-one-compound (OBOC) combinatorial peptide library for lead compounds, agnostic of those physicochemical constraints. Such a synthetic strategy also permits expansion of our peptide repertoire to include unnatural amino acids. Using this approach, we discovered a structurally unique lead peptide LBF14, a linear 14-mer peptide, that induces gross morphological disruption of membranes, irrespective of membrane composition. Further, we demonstrate that the unique insertion mechanism of the peptide, visualized by spinning disc confocal microscopy and further analyzed by electron paramagnetic resonance measurements, may be the cause of this large scale membrane deformation. We also demonstrate the robustness, reproducibility, and potential application of this technique to discover and characterize new membrane active peptides that display activity by local insertion and subsequent allosteric effects leading to global membrane disruption.

Correction to: High-affinity peptide ligand LXY30 for targeting α3ß1 integrin in non-small cell lung cancer.

The original article [1] contains an error in Fig. 2 whereby Fig. 2D has mistakenly been omitted. Fig. 2 can be viewed in its entirety - including Fig. 2D - in this Correction article.

High-affinity peptide ligand LXY30 for targeting α3ß1 integrin in non-small cell lung cancer.

BACKGROUND: α3ß1 integrin is a promising cancer biomarker and drug target. We previously identified a 9-amino-acid cyclic peptide LXY30 for detecting α3ß1 integrin on the surface of live tumor cells. This study was undertaken to characterize LXY30 in the detection, cellular function, imaging, and targeted delivery of in vitro and in vivo non-small cell lung cancer (NSCLC) models. METHODS: The whole-cell binding assay was performed by incubating NSCLC cells, extracellular vesicles (EVs), and peripheral blood mononuclear cells (PBMCs) with TentaGel resin beads coated with LXY30. In this study, we defined the nanosize EVs as exosomes, which were characterized by flow cytometry, transmission electron microscopy, dynamic light scattering, and Western blots. The function of LXY30 was determined by modulating the epidermal growth factor receptor (EGFR) signaling pathway by growth inhibition and Western blots. For in vivo biodistribution, mice bearing subcutaneous and intracranial NSCLC xenograft tumors were administrated intraveneously with LXY30-biotin/streptavidin-Cy5.5 complex and then analyzed for in vivo and ex vivo optical imaging and histopathology. RESULTS: We showed that LXY30 specifically and sensitively detected α3ß1 integrin-expressing NSCLC cells and tumor-derived exosomes. Tumor DNA isolated from LXY30-enriched plasma exosomes might be used to detect driver oncogenic mutations in patients with metastatic NSCLC. LXY30 only enriches tumor cells but not neutrophils, macrophages, or monocytes in the malignant pleural effusion of NSCLC patients for detecting genomic alterations by next-generation sequencing. LXY30 detected increased α3ß1 integrin expression on the EGFR-mutant NSCLC cells with acquired resistance to erlotinib compared to parental erlotinib-sensitive EGFR-mutant NSCLC cells. We further showed that LXY30 modulated the EGFR signaling pathway independently from another peptide ligand LXW64 targeting αvß3 integrin in erlotinib-resistant, EGFR-mutant H1975 cells. Analysis of The Cancer Genome Atlas (TCGA) revealed high α3 integrin expression was associated with poor prognosis in lung squamous cell carcinoma. LXY30-biotin/streptavidin-Cy5.5 complex had higher uptakes in the subcutaneous and intracranial xenografts of various α3ß1 integrin-expressing lung adenocarcinoma and patient-derived lung squamous cell carcinoma xenografts while sparing the surrounding normal tissues. CONCLUSION: LXY30 is a promising peptide for the cancer diagnosis and in vivo targeted delivery of imaging agents and cancer drugs in NSCLC, independent of histology and tumor genotype.

Nanoplasmonic Approaches for Sensitive Detection and Molecular Characterization of Extracellular Vesicles.

All cells release a multitude of nanoscale extracellular vesicles (nEVs) into circulation, offering immense potential for new diagnostic strategies. Yet, clinical translation for nEVs remains a challenge due to their vast heterogeneity, our insufficient ability to isolate subpopulations, and the low frequency of disease-associated nEVs in biofluids. The growing field of nanoplasmonics is poised to address many of these challenges. Innovative materials engineering approaches based on exploiting nanoplasmonic phenomena, i.e., the unique interaction of light with nanoscale metallic materials, can achieve unrivaled sensitivity, offering real-time analysis and new modes of medical and biological imaging. We begin with an introduction into the basic structure and function of nEVs before critically reviewing recent studies utilizing nanoplasmonic platforms to detect and characterize nEVs. For the major techniques considered, surface plasmon resonance (SPR), localized SPR, and surface enhanced Raman spectroscopy (SERS), we introduce and summarize the background theory before reviewing the studies applied to nEVs. Along the way, we consider notable aspects, limitations, and considerations needed to apply plasmonic technologies to nEV detection and analysis.