Lectin microarray based glycan profiling of exosomes for dynamic monitoring of colorectal cancer progression.

BACKGROUND: Exosomes, as emerging biomarkers in liquid biopsies in recent years, offer profound insights into cancer diagnostics due to their unique molecular signatures. The glycosylation profiles of exosomes have emerged as potential biomarkers, offering a novel and less invasive method for cancer diagnosis and monitoring. Colorectal cancer (CRC) represents a substantial global health challenge and burden. Thus there is a great need for the aberrant glycosylation patterns on the surface of CRC cell-derived exosomes, proposing them as potential biomarkers for tumor characterization. RESULTS: The interactions of 27 lectins with exosomes from three CRC cell lines (SW480, SW620, HCT116) and one normal colon epithelial cell line (NCM460) have been analyzed by the lectin microarray. The result indicates that Ulex Europaeus Agglutinin I (UEA-I) exhibits high affinity and specificity towards exosomes derived from SW480 cells. The expression of glycosylation related genes within cells has been analyzed by high-throughput quantitative polymerase chain reaction (HT-qPCR). The experimental result of HT-qPCR is consistent with that of lectin microarray. Moreover, the limit of detection (LOD) of UEA-I microarray is calculated to be as low as 2.7 × 105 extracellular vehicles (EVs) mL-1 (three times standard deviation (3σ) of blank sample). The UEA-I microarray has been successfully utilized to dynamically monitor the progression of tumors in mice-bearing SW480 CRC subtype, applicable in tumor sizes ranging from 2 mm to 20 mm in diameter. SIGNIFICANCE: The results reveal that glycan expression pattern of exosome is linked to specific CRC subtypes, and regulated by glycosyltransferase and glycosidase genes of mother cells. Our findings illuminate the potential of glycosylation molecules on the surface of exosomes as reliable biomarkers for diagnosis of tumor at early stage and monitoring of cancer progression.

Exosome for mRNA delivery: strategies and therapeutic applications.

Messenger RNA (mRNA) has emerged as a promising therapeutic molecule with numerous clinical applications in treating central nervous system disorders, tumors, COVID-19, and other diseases. mRNA therapies must be encapsulated into safe, stable, and effective delivery vehicles to preserve the cargo from degradation and prevent immunogenicity. Exosomes have gained growing attention in mRNA delivery because of their good biocompatibility, low immunogenicity, small size, unique capacity to traverse physiological barriers, and cell-specific tropism. Moreover, these exosomes can be engineered to utilize the natural carriers to target specific cells or tissues. This targeted approach will enhance the efficacy and reduce the side effects of mRNAs. However, difficulties such as a lack of consistent and reliable methods for exosome purification and the efficient encapsulation of large mRNAs into exosomes must be addressed. This article outlines current breakthroughs in cell-derived vesicle-mediated mRNA delivery and its biomedical applications.

Extraction and characterization of exosomes from the exhaled breath condensate and sputum of lung cancer patients and vulnerable tobacco consumers-potential noninvasive diagnostic biomarker source.

Noninvasive sample sources of exosomes, such as exhaled breath and sputum, which are in close proximity to the tumor microenvironment and may contain biomarkers indicative of lung cancer, are far more permissive than invasive sample sources for biomarker screening. Standardized exosome extraction and characterization approaches for low-volume noninvasive samples are critically needed. We isolated and characterized exhaled breath condensate (EBC) and sputum exosomes from healthy nonsmokers (n= 30), tobacco smokers (n= 30), and lung cancer patients (n= 40) and correlated the findings with invasive sample sources. EBC samples were collected by using commercially available R-Tubes. To collect sputum samples the participants were directed to take deep breaths, hold their breath, and cough in a collection container. Dynamic light scattering, nanoparticle tracking analysis, and transmission electron microscopy were used to evaluate the exosome morphology. Protein isolation, western blotting, exosome quantification via EXOCET, and Fourier transform infrared spectroscopy were performed for molecular characterization. Exosomes were successfully isolated from EBC and sputum samples, and their yields were adequate and sufficiently pure for subsequent downstream processing and characterization. The exosomes were confirmed based on their size, shape, and surface marker expression. Remarkably, cancer exosomes were the largest in size not only in the plasma subgroups, but also in the EBC (p < 0.05) and sputum (p= 0.0036) subgroups, according to our findings. A significant difference in exosome concentrations were observed between the control sub-groups (p < 0.05). Our research confirmed that exosomes can be extracted from noninvasive sources, such as EBC and sputum, to investigate lung cancer diagnostic biomarkers for research, clinical, and early detection in smokers.

Magnetic lanthanide sensor with self-ratiometric time-resolved luminescence for accurate detection of epithelial cancerous exosomes.

Fluorescence-based LB (liquid biopsy) offers a rapid means of detecting cancer non-invasively. However, the widespread issue of sample loss during purification steps will diminish the accuracy of detection results. Therefore, in this study, we introduce a magnetic lanthanide sensor (MLS) designed for sensitive detection of the characteristic protein, epithelial cell adhesion molecule (EpCAM), on epithelial tumor exosomes. By leveraging the inherent multi-peak emission and time-resolved properties of the sole-component lanthanide element, combined with the self-ratiometric strategy, MLS can overcome limitations imposed by manual operation and/or sample complexity, thereby providing more stable and reliable output results. Specifically, terbium-doped NaYF4 nanoparticles (NaYF4:Tb) and deformable aptamers terminated with BHQ1 were sequentially introduced onto superparamagnetic silica-decorated Fe3O4 nanoparticles. Prior to target binding, emission from NaYF4:Tb at 543 nm was partially quenched due to the fluorescence resonance energy transfer (FRET) from NaYF4:Tb to BHQ1. Upon target binding, changes in the secondary structure of aptamers led to the fluorescence intensity increasing since the deconfinement of distance-dependent FRET effect. The characteristic emission of NaYF4:Tb at 543 nm was then utilized as the detection signal (I1), while the less changed emission at 583 nm served as the reference signal (I2), further reporting the self-ratiometric values of I1 and I2 (I1/I2) to illustrate the epithelial cancerous features of exosomes while ignoring possible sample loss. Consequently, over a wide range of exosome concentrations (2.28 × 102-2.28 × 108 particles per mL), the I1/I2 ratio exhibited a linear increase with exosome concentration [Y(I1/I2) = 0.166 lg (Nexosomes) + 3.0269, R2 = 0.9915], achieving a theoretical detection limit as low as 24 particles per mL. Additionally, MLS effectively distinguished epithelial cancer samples from healthy samples, showcasing significant potential for clinical diagnosis.

Modulating Visible-Light Driven NIR Lanthanide Polymer Photocatalysis for Amplification Detection of Exosomal Proteins and Cancer Diagnosis.

Near-infrared (NIR) luminescent lanthanide materials hold great promise for bioanalysis, as they have anti-interference properties. The approach of efficient luminescence is sensitization through a reasonable chromophore to overcome the obstacle of the aqueous phase. The involvement of the surfactant motif is an innovative strategy to arrange the amphiphilic groups to be regularly distributed near the polymer to form a closed sensitized space. Herein, a lanthanide polymer (TCPP-PEI70K-FITC@Yb/SDBS) is designed in which the meso-tetra(4-carboxyphenyl)porphine (TCPP) ligand serves as both a sensitizer and photocatalytic switch. The surfactant sodium dodecyl benzenesulfonate (SDBS) wraps the photosensitive polymers to form a hydrophobic layer, which augments the light-harvesting ability and expedites its photocatalysis. TCPP-PEI70K-FITC@Yb/SDBS is subsequently applied as an amplified photocatalysis toolbox for universally regulating the generation of reactive oxygen species (ROS). Boosting 3,3',5,5'-tetramethylbenzidine (TMB) oxidation to produce blue products, a dual-mode biosensor is fabricated for improving the diagnosis of programmed death ligand-1-positive (PDL1) cancer exosomes. Exosomes were captured by Fe3O4 modified by the PDL1 aptamer, enabling replacement of alkaline phosphatase (ALP)-labeled multiple hybridized chains; then, the isolated ALP triggered a hydrolysis reaction to block the generation of oxTMB. Detection sensitivity improves by 1 order of magnitude through SDBS modulation, down to 104 particles/mL. The sensor performed well clinically in distinguishing cancer patients from healthy individuals, expanding physiological applications of near-infrared lanthanide luminescence.

Integration of photomagnetic bimodal imaging to monitor an autogenous exosome loaded platform: unveiling strong targeted retention effects for guiding the photothermal and magnetothermal therapy in a mouse prostate cancer model.

BACKGROUND: Prostate cancer (PCa) is the most prevalent cancer among males, emphasizing the critical need for precise diagnosis and treatment to enhance patient prognosis. Recent studies have extensively utilized urine exosomes from patients with cancer for targeted delivery. This study aimed to employ highly sensitive magnetic particle imaging (MPI) and fluorescence molecular imaging (FMI) to monitor the targeted delivery of an exosome-loaded platform at the tumour site, offering insights into a potential combined photothermal and magnetic thermal therapy regime for PCa. RESULTS: MPI and FMI were utilized to monitor the in vivo retention performance of exosomes in a prostate tumour mouse model. The exosome-loaded platform exhibited robust homologous targeting ability during imaging (SPIONs@EXO-Dye:66·48%±3·85%; Dye-SPIONs: 34·57%±7·55%, **P<0·01), as verified by in vitro imaging and in vitro tissue Prussian blue staining. CONCLUSIONS: The experimental data underscore the feasibility of using MPI for in vivo PCa imaging. Furthermore, the exosome-loaded platform may contribute to the precise diagnosis and treatment of PCa.

A lateral flow assay strip for simultaneous detection of miRNA and exosomes in liver cancer.

By employing an aptamer as the bridge and combining catalytic hairpin assembly with the Au aggregation amplification effect, a lateral flow assay (LFA) is designed for simultaneous detection of liver cancer-associated miRNA and exosomes. The LFA can differentiate between liver cancer patients and healthy individuals with simple operation and high accuracy.

The ECL enhancement of MBene QDs with nanoparticle-on-mirror structure for sensitive detection of exosomal miRNA.

Thyroid cancer is the most prevalent endocrine malignancy. The development of sensitive and reliable methods to detect the thyroid cancer is the currently urgent requirement. Herein, we developed an electrochemiluminescence (ECL) biosensor based on MBene derivative quantum dots (MoB QDs) and Ag NP-on-mirror (NPoM) nanocavity structure. On the one hand, MBene QDs as a novel luminescent material in the ECL process was reported for the first time, which can react with H2O2 as co-reactant. On the other hand, the NPoM nanostructure was successfully constructed with the Ag mirror and Ag NPs to provide highly localized hot spots. The NPoM structure had high degree of light field confinement and electromagnetic field enhancement, which can amplify the ECL signal as the signal modulator. Therefore, the synergistic effect of the nanocavity and localized surface plasmon resonance (LSPR) mode in the NPoM facilitated the enhancement of the ECL signal of MoB QDs over 21.7 times. Subsequently, the proposed ECL biosensing system was employed to analyze the expression level of miRNA-222-3p in the thyroid cancer exosome. The results indicated the relative association between miRNA-222-3p and BRAFV600E mutation. The MoB QDs/NPoM biosensor displayed the ideal potential in assessing thyroid cancer progression for advancing clinical diagnosis applications.

"Three-in-One" Plasmonic Au@PtOs Nanocluster Driven Lateral Flow Assay for Multimodal Cancer Exosome Biosensing.

Exploiting the multiple properties of nanozymes for the multimode lateral flow assay (LFA) is urgently required to improve the accuracy and versatility. Herein, we developed a novel plasmonic Au nanostar@PtOs nanocluster (Au@PtOs) as a multimode signal tag for LFA detection. Based on the PtOs bimetallic nanocluster doping strategy, Au@PtOs can indicate both excellent SERS enhancement and nanozyme catalytic activity. Meanwhile, Au@PtOs displays a better photothermal effect than that of Au nanostars. Therefore, catalytic colorimetric/SERS/temperature three-mode signals can be read out based on the Au@PtOs nanocomposite. The Au@PtOs was combined with LFA and applied for breast cancer exosome detection. The detection limit for the colorimetric/SERS/temperature mode was 2.6 × 103/4.1 × 101/4.6 × 102 exosomes/µL, respectively, which was much superior to the common Au nanoparticles LFA (∼105 exosomes/µL). Moreover, based on the fingerprint molecular recognition ability of the SERS mode, exosome phenotypes derived from different breast cancer cell lines can be discriminated easily. Based on the convenient visual colorimetric mode and sensitive SERS/temperature quantitative modes, Au@PtOs driven LFA can satisfy the requirements of accurate and flexible multimodal sensing in different application scenarios.

A proximity ligation hybridization triggered structure-switching based signal amplification strategy for sensitive and accurate exosome detection.

Exosomes have significant functions in intercellular communication, as well as in tumor migration and invasion. Nevertheless, the precise identification of exosomes poses a significant obstacle due to their low abundance in biofluids and potential disruption caused by free protein molecules, such as CD63 protein. In this study, we have developed a signal amplification method for precise detection of exosomes using a proximity ligation hybridization triggered structure-switching approach. The method involves the dual-recognition of exosomes by two probes: an aptamer probe that recognizes the exosomal surface protein CD63 (L1 probe), and a cholesterol probe that targets the biolipid layer of the exosomes (L2 probe). Based on the dual-recognition of exosomes, we have successfully developed an accurate and sensitive approach that integrates the proximity ligation hybridization technique with a structure-switching based signal cycle. This approach allows for the simultaneous analysis of two biomarkers, enabling both quantification and tracing of exosomes without the need for enzymes. Eventually, the proposed method exhibits a wide detection range of 5 orders of magnitude and a low limit of detection of 36 particles per µL, making it suitable for a wide range of applications in the fields of biological science, biomedical engineering, and personalized medicine.

Synergistic Effects of Shed-Derived Exosomes, Cu2+, and an Injectable Hyaluronic Acid Hydrogel on Antibacterial, Anti-inflammatory, and Osteogenic Activity for Periodontal Bone Regeneration.

The primary pathology of periodontitis involves the gradual deterioration of periodontal tissues resulting from the inflammatory reaction triggered by bacterial infection. In this study, a novel drug for periodontal pocket injection, known as the Shed-Cu-HA hydrogel, was developed by incorporating copper ions (Cu2+) and Shed-derived exosomes (Shed-exo) inside the hyaluronic acid (HA) hydrogel. Suitable concentrations of Cu2+ and Shed-exo released from Shed-Cu-HA enhanced cell viability and cell proliferation of human periodontal ligament stem cells. Additionally, the Shed-Cu-HA demonstrated remarkable antibacterial effects against the key periodontal pathogen (Aa) owing to the synergistic effect of Cu2+ and HA. Furthermore, the material effectively suppressed macrophage inflammatory response via the IL-6/JAK2/STAT3 pathway. Moreover, the Shed-Cu-HA, combining the inflammation-regulating properties of HA with the synergistic osteogenic activity of Shed-exo and Cu2+, effectively upregulated the expression of genes and proteins associated with osteogenic differentiation. The experimental findings from a mouse periodontitis model demonstrated that the administration of Shed-Cu-HA effectively reduced the extent of inflammatory cell infiltration and bacterial infections in gingival tissues and facilitated the regeneration of periodontal bone tissues and collagen after 2 and 4 weeks of injection. Consequently, it holds significant prospects for future applications in periodontitis treatment.

Mechanical properties of blood exosomes and lipoproteins after the rat whole blood irradiation with X-rays in vitro explored by atomic force microscopy.

Blood is a two-component system with two levels of hierarchy: the macrosystem of blood formed elements and the dispersed system of blood nanoparticles. Biological nanoparticles are the key participants in communication between the irradiated and non-irradiated cells and inducers of the non-targeted effects of ionizing radiation. The work aimed at studying by atomic force microscopy the structural, mechanical, and electrical properties of exosomes and lipoproteins (LDL/VLDL) isolated from rat blood after its exposure to X-rays in vitro. MATERIALS AND METHODS: The whole blood of Wistar rats fed with a high-fat diet was irradiated with X-rays (1 and 100 Gy) in vitro. The structural and mechanical properties (the elastic modulus and nonspecific adhesion force) of exosome and lipoprotein isolates from the blood by ultracentrifugation method were studied using Bruker Bioscope Resolve atomic force microscope in PF QNM mode, their electric properties (the zeta-potential) was measured by electrophoretic mobility. RESULTS: Lipoproteins isolated from non-irradiated blood were softer (Me(LQ; UQ): 7.8(4.9;12.1) MPa) compared to blood nanoparticles of its exosome fraction (34.8(22.6;44.9) MPa) containing both exosomes and non-membrane nanoparticles. X-ray blood irradiation with a dose of 1 Gy significantly weakened the elastic properties of lipoproteins. Exposure of the blood to 100 Gy X-rays made lipoproteins stiffer and their nonspecific adhesive properties stronger. The radiation effects on the mechanical parameters of exosomes and non-membrane nanoparticles in exosome fractions differed. The significant radiation-induced change in electric properties of the studied nanoparticles was detected only for lipoproteins in the blood irradiated with 1 Gy X-rays. The low-dose radiation-induced changes in zeta-potential and increase in lipoprotein size with the appearance of a soft thick surface layer indicate the formation of the modified lipoproteins covered with a corona from macromolecules of irradiated blood. CONCLUSION: Our data obtained using the nanomechanical mapping mode of AFM are the first evidence of the significant radiation-induced changes in the structural and mechanical properties of the dispersed system of blood nanoparticles after the X-ray irradiation of the blood.

Enhancing bone regeneration and immunomodulation via gelatin methacryloyl hydrogel-encapsulated exosomes from osteogenic pre-differentiated mesenchymal stem cells.

Mesenchymal stem cell-derived exosomes (MSC-Exos) have emerged as promising candidates for cell-free therapy in tissue regeneration. However, the native osteogenic and angiogenic capacities of MSC-Exos are often insufficient to repair critical-sized bone defects, and the underlying immune mechanisms remain elusive. Furthermore, achieving sustained delivery and stable activity of MSC-Exos at the defect site is essential for optimal therapeutic outcomes. Here, we extracted exosomes from osteogenically pre-differentiated human bone marrow mesenchymal stem cells (hBMSCs) by ultracentrifugation and encapsulated them in gelatin methacryloyl (GelMA) hydrogel to construct a composite scaffold. The resulting exosome-encapsulated hydrogel exhibited excellent mechanical properties and biocompatibility, facilitating sustained delivery of MSC-Exos. Osteogenic pre-differentiation significantly enhanced the osteogenic and angiogenic properties of MSC-Exos, promoting osteogenic differentiation of hBMSCs and angiogenesis of human umbilical vein endothelial cells (HUVECs). Furthermore, MSC-Exos induced polarization of Raw264.7 cells from a pro-inflammatory phenotype to an anti-inflammatory phenotype under simulated inflammatory conditions, thereby creating an immune microenvironment conducive to osteogenesis. RNA sequencing and bioinformatics analysis revealed that MSC-Exos activate the p53 pathway through targeted delivery of internal microRNAs and regulate macrophage polarization by reducing DNA oxidative damage. Our study highlights the potential of osteogenic exosome-encapsulated composite hydrogels for the development of cell-free scaffolds in bone tissue engineering.

The potential mechanisms and treatment effects of stem cell-derived exosomes in cardiac reengineering.

Exosomes are extracellular vesicles of diverse compositions that are secreted by numerous cell types. Exosomes contain significant bioactive components, including lipids, proteins, mRNA, and miRNA. Exosomes play an important role in regulating cellular signaling and trafficking under both normal physiological and pathological circumstances. A multitude of factors, including thermal stress, ribosomal stress, endoplasmic reticulum stress, and oxidative stress influence the concentrations of exosomal mRNA, miRNA, proteins, and lipids. It has been stated that exosomes derived from stem cells (SCs) modulate a range of stresses by preventing or fostering cell balance. Exosomes derived from SCs facilitate recovery by facilitating cross-cellular communication via the transmission of information in the form of proteins, lipids, and other components. For this reason, exosomes are used as biomarkers to diagnose a wide variety of diseases. The focus of this review is the bioengineering of artificial exosomal cargoes. This process encompasses the control and transportation of particular exosomal cargoes, including but not limited to small molecules, recombinant proteins, immune modulators, and therapeutic medications. Therapeutic approaches of this nature have the potential to deliver therapeutic medications precisely to the intended site for the cure of a variety of disorders. Notably, our attention has been directed towards the therapeutic implementations of exosomes derived from SCs in the cure of cardiovascular ailments, including but not limited to ischemic heart disease, myocardial infarction, sepsis, heart failure, cardiomyopathy, and cardiac fibrosis. In general, researchers employ two methodologies when it comes to exosomal bioengineering. This review aims to explain the function of exosomes derived from SCs in the regulation of stress and present a novel therapeutic approach for cardiovascular disorders.

Cascade CRISPR/Cas12a and DSN for the electrochemical biosensing of miR-1246 in BC-derived exosomes.

MiR-1246 in breast cancer-derived exosomes was a promising biomarker for early diagnosis of breast cancer(BC). However, the low abundance, high homology and complex background interference make the accurate quantitative detection of miR-1246 facing great challenges. In this study, we developed an electrochemical biosensor based on the subtly combined of CRISPR/Cas12a, double-stranded specific nuclease(DSN) and magnetic nanoparticles(MNPs) for the detection of miR-1246 in BC-derived exosomes. Ascribed to the good synergistic effect of DSN, Cas12a and MNPs, the developed electrochemical biosensor exhibited excellent performance with the linear range from 500 aM to 5 pM, and the detection limit as low down to about 50 aM. The target-specific triggered enzyme-digest activity of DSN and Cas12a system, as well as the powerful separation ability of MNPs ensure the high specificity of developed electrochemical biosensor which can distinguish single base mismatches. In addition, the developed electrochemical biosensor has been successfully applied to detect miR-1246 in blood-derived exosomes and realize distinguishing the BC patients from the healthy individuals. It is expected that the well-designed biosensing platform will open up new avenues for clinical liquid biopsy and early screening of breast cancer, as well as provide deeper insights into clinical oncology treatment.

Automatic Electrochemiluminescence Method for the Detection of Cancerous Exosomes Incorporating Specific Aptamer-Magnetic Beads and Signal Nanoprobes.

Exosomes, as an emerging biomarker, have exhibited remarkable promise in early cancer diagnosis. Here, a highly sensitive, selective, and automatic electrochemiluminescence (ECL) method for the detection of cancerous exosomes was developed. Specific aptamer-(EK)4 peptide-tagged magnetic beads (MBs-(EK)4-aptamer) were designed as a magnetic capture probe in which the (EK)4 peptide was used to reduce the steric binding hindrance of cancerous exosomes with a specific aptamer. One new universal ECL signal nanoprobe (CD9 Ab-PEG@SiO2ϵRu(bpy)32+) was designed and synthesized by using microporous SiO2 nanoparticles as the carrier for loading ECL reagent Ru(bpy)32+, polyethylene glycol (PEG) layer, and anticluster of differentiation 9 antibody (CD9 Ab). A "sandwich" biocomplex was formed on the surface of the magnetic capture probe after mixing the capture probe, target exosomes, and ECL signal nanoprobe, and then it was introduced into an automated ECL analyzer for rapid and automatic ECL measurement. It was found that the designed signal nanoprobe shows a 270-fold improvement in the signal-to-noise ratio than that of the ruthenium complex-labeled CD9 antibody signal probe. The relative ECL intensity was proportional to MCF-7 exosomes as a model in the range of 102 to 104 particle/µL, with a detection limit of 11 particle/µL. Furthermore, the ECL method was employed to discriminate cancerous exosomes based on fingerprint responses using the designed multiple magnetic capture probes and the universal ECL signal nanoprobe. This work demonstrates that the utilization of a designed automated ECL tactic using the MBs-(EK)4-aptamer capture probe and the CD9 Ab-PEG@SiO2ϵRu(bpy)32+ signal nanoprobe will provide a unique and robust method for the detection and discrimination of cancerous exosomes.

Biomimetic Exosome-Sheathed Magnetic Mesoporous Anchor with Modification of Glucose Oxidase for Synergistic Targeting and Starving Tumor Cells.

Efficient protection and precise delivery of biomolecules are of critical importance in the intervention and therapy of various diseases. Although diverse specific marker-functionalized drug carriers have been developed rapidly, current approaches still encounter substantial challenges, including strong immunogenicity, limited target availability, and potential side effects. Herein, we developed a biomimetic exosome-sheathed magnetic mesoporous anchor modified with glucose oxidase (MNPs@mSiO2-GOx@EM) to address these challenges and achieve synergistic targeting and starving of tumor cells. The MNPs@mSiO2-GOx@EM anchor integrated the unique characteristics of different components. An external decoration of exosome membrane (EM) with high biocompatibility contributed to increased phagocytosis prevention, prolonged circulation, and enhanced recognition and cellular uptake of loaded particles. An internal coated magnetic mesoporous core with rapid responsiveness by the magnetic field guidance and large surface area facilitated the enrichment of nanoparticles at the specific site and provided enough space for modification of glucose oxidase (GOx). The inclusion of GOx in the middle layer accelerated the energy-depletion process within cells, ultimately leading to the starvation and death of target cells with minimal side effects. With these merits, in vitro study manifested that our nanoplatform not only demonstrated an excellent targeting capability of 94.37% ± 1.3% toward homotypic cells but also revealed a remarkably high catalytical ability and cytotoxicity on tumor cells. Assisted by the magnetic guidance, the utilization of our anchor obviously inhibits the tumor growth in vivo. Together, our study is promising to serve as a versatile method for the highly efficient delivery of various target biomolecules to intended locations due to the fungibility of exosome membranes and provide a potential route for the recognition and starvation of tumor cells.

Comparison of morphology, protein concentration, and size distribution of bone marrow and Wharton's jelly-derived mesenchymal stem cells exosomes isolated by ultracentrifugation and polymer-based precipitation techniques.

Exosomes which are tiny extracellular vesicles (30-150 nm), transport vital proteins and gene materials such as miRNA, mRNA, or DNA, whose role in cell communication and epithelia regulation is critical. Many techniques have been developed as a result of studying exosomes' biochemical and physicochemical properties, although there is still no standard method to isolate exosomes simply with high yield. Commercial kits have gained popularity for exosome extraction despite concerns about their effectiveness in scientific research. On the other hand, ultracentrifugation remains the gold standard isolation method. This study compares these two common exosome isolation methods to determine their impact on the quality and quantity of exosomes isolated from bone marrow (BM) and Wharton's jelly (WJ)-derived mesenchymal stem cells. Isolated exosomes from the two sources of the cell's conditioned medium by two methods (polymer kit and ultracentrifuge) were characterized using western blotting, scanning electron microscopy (SEM), dynamic light scattering (DLS), and the Bradford assay. Western blot analysis confirmed separation efficiency based on CD81 and CD63 markers, with the absence of calnexin serving as a negative control. The Morphology of exosomes studied by SEM image analysis revealed a similar round shape appearance and their sizes (30-150 nm) were the same in both isolation techniques. The DLS analysis of the sample results was consistent with the SEM ones, showing a similar size range and very low disparity. The exosome protein content concentration analysis revealed that exosomes isolated by the polymer-based kits contained higher protein concentration density and purity (p <0.001). In general, though the protein yield was higher when the polymer-based kits were used, there were no significant differences in morphology, or size between WJ-derived and BM-derived exosomes, regardless of the isolation method employed.

Ultrasensitive PD-L1-Expressing Exosome Immunosensors Based on a Chemiluminescent Nickel-Cobalt Hydroxide Nanoflower for Diagnosis and Classification of Lung Adenocarcinoma.

Programmed death ligand-1 (PD-L1)-expressing exosomes are considered a potential marker for diagnosis and classification of lung adenocarcinoma (LUAD). There is an urgent need to develop highly sensitive and accurate chemiluminescence (CL) immunosensors for the detection of PD-L1-expressing exosomes. Herein, N-(4-aminobutyl)-N-ethylisopropanol-functionalized nickel-cobalt hydroxide (NiCo-DH-AA) with a hollow nanoflower structure as a highly efficient CL nanoprobe was synthesized using gold nanoparticles as a "bridge". The resulting NiCo-DH-AA exhibited a strong and stable CL emission, which was ascribed to the exceptional catalytic capability and large specific surface area of NiCo-DH, along with the capacity of AuNPs to facilitate free radical generation. On this basis, an ultrasensitive sandwich CL immunosensor for the detection of PD-L1-expressing exosomes was constructed by using PD-L1 antibody-modified NiCo-DH-AA as an effective signal probe and rabbit anti-CD63 protein polyclonal antibody-modified carboxylated magnetic bead as a capture platform. The immunosensor demonstrated outstanding analytical performance with a wide detection range of 4.75 × 103-4.75 × 108 particles/mL and a low detection limit of 7.76 × 102 particles/mL, which was over 2 orders of magnitude lower than the reported CL method for detecting PD-L1-expressing exosomes. Importantly, it was able to differentiate well not only between healthy persons and LUAD patients (100% specificity and 87.5% sensitivity) but also between patients with minimally invasive adenocarcinoma and invasive adenocarcinoma (92.3% specificity and 52.6% sensitivity). Therefore, this study not only presents an ultrasensitive and accurate diagnostic method for LUAD but also offers a novel, simple, and noninvasive approach for the classification of LUAD.

A comprehensive review of engineered exosomes from the preparation strategy to therapeutic applications.

Exosomes exhibit high bioavailability, biological stability, targeted specificity, low toxicity, and low immunogenicity in shuttling various bioactive molecules such as proteins, lipids, RNA, and DNA. Natural exosomes, however, have limited production, targeting abilities, and therapeutic efficacy in clinical trials. On the other hand, engineered exosomes have demonstrated long-term circulation, high stability, targeted delivery, and efficient intracellular drug release, garnering significant attention. The engineered exosomes bring new insights into developing next-generation drug delivery systems and show enormous potential in therapeutic applications, such as tumor therapies, diabetes management, cardiovascular disease, and tissue regeneration and repair. In this review, we provide an overview of recent advancements associated with engineered exosomes by focusing on the state-of-the-art strategies for cell engineering and exosome engineering. Exosome isolation methods, including traditional and emerging approaches, are systematically compared along with advancements in characterization methods. Current challenges and future opportunities are further discussed in terms of the preparation and application of engineered exosomes.