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1. Metabolic tagging of extracellular vesicles and development of enhanced extracellular vesicle based cancer vaccines

   https://doi.org/10.1038/s41467-023-43914-8  

   Bhatta, Rimsha; Han, Joonsu; Liu, Yusheng; Bo, Yang; Lee, David; Zhou, Jiadiao; Wang, Yueji; Nelson, Erik Russell; Chen, Qian; Zhang, Xiaojia Shelly; et al (December 2023, Nature Communications)

   Abstract As key mediators of cellular communication, extracellular vesicles (EVs) have been actively explored for diagnostic and therapeutic applications. However, effective methods to functionalize EVs and modulate the interaction between EVs and recipient cells are still lacking. Here we report a facile and universal metabolic tagging technology that can install unique chemical tags (e.g., azido groups) onto EVs. The surface chemical tags enable conjugation of molecules via efficient click chemistry, for the tracking and targeted modulation of EVs. In the context of tumor EV vaccines, we show that the conjugation of toll-like receptor 9 agonists onto EVs enables timely activation of dendritic cells and generation of superior antitumor CD8⁺T cell response. These lead to 80% tumor-free survival against E.G7 lymphoma and 33% tumor-free survival against B16F10 melanoma. Our study yields a universal technology to generate chemically tagged EVs from parent cells, modulate EV-cell interactions, and develop potent EV vaccines. 

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2. Click Conjugation of Boron Dipyrromethene (BODIPY) Fluorophores to EGFR-Targeting Linear and Cyclic Peptides

   https://doi.org/10.3390/molecules26030593  

   Williams, Tyrslai M.; Kaufman, Nichole E.; Zhou, Zehua; Singh, Sitanshu S.; Jois, Seetharama D.; Vicente, Maria da (February 2021, Molecules)

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   Through a simple 1,3-cycloaddition reaction, three BODIPY-peptide conjugates that target the extracellular domain of the epidermal growth factor receptor (EGFR) were prepared and their ability for binding to EGFR was investigated. The peptide ligands K(N3)LARLLT and its cyclic analog cyclo(K(N3)larllt, previously shown to have high affinity for binding to the extracellular domain of EGFR, were conjugated to alkynyl-functionalized BODIPY dyes 1 and 2 via a copper-catalyzed click reaction. This reaction produced conjugates 3, 4, and 5 in high yields (70–82%). In vitro studies using human carcinoma HEp2 cells that overexpress EGFR demonstrated high cellular uptake, particularly for the cyclic peptide conjugate 5, and low cytotoxicity in light (~1 J·cm−2) and darkness. Surface plasmon resonance (SPR) results show binding affinity of the three BODIPY-peptide conjugates for EGFR, particularly for 5 bearing the cyclic peptide. Competitive binding studies using three cell lines with different expressions of EGFR show that 5 binds specifically to EGFR-overexpressing colon cancer cells. Among the three conjugates, 5 bearing the cyclic peptide exhibited the highest affinity for binding to the EGFR protein. 

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3. The Synthesis of BODIPY-TKI Conjugates and Investigation of Their Ability to Target the Epidermal Growth Factor Receptor

   https://doi.org/10.3390/targets1010005  

   Dhingra, Simran; Shrestha, Prajesh; Chowdhury, Arpan; Zhou, Zehua; Jois, Seetharama D; Vicente, Maria_da_Graça H (September 2023, Targets)

   A near-IR BODIPY was covalently conjugated via its isothiocyanate groups to one or two Erlotinib molecules, a known tyrosine kinase inhibitor (TKI), via triethylene glycol spacers, to produce two novel BODIPY-monoTKI and BODIPY-diTKI conjugates. The ability of these conjugates to target the intracellular domain of the epidermal growth factor receptor (EGFR) was investigated using molecular modeling, surface plasma resonance (SPR), EGFR kinase binding assay, time-dependent cellular uptake, and fluorescence microscopy. While both the BODIPY-monoTKI and the BODIPY-diTKI conjugates were shown to bind to the EGFR kinase by SPR and accumulated more efficiently within human HEp2 cells that over-express EGFR than BODIPY alone, only the BODIPY-monoTKI exhibited kinase inhibition activity. This is due to the high hydrophobic character and aggregation behavior of the BODIPY-diTKI in aqueous solutions, as shown by fluorescence quenching. Furthermore, the competition of the two Erlotinibs in the diTKI conjugate for the active site of the kinase, as suggested by computational modeling, might lead to a decrease in binding relative to the monoTKI conjugate. Nevertheless, the efficient cellular uptake and intracellular localization of both conjugates with no observed cytotoxicity suggest that both could be used as near-IR fluorescent markers for cells that over-express EGFR. 

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4. The isolation of VCAM-1+ endothelial cell-derived extracellular vesicles using microfluidics

   https://doi.org/10.20517/evcna.2023.51  

   Akbar, Naveed; Luciani, Evelyn Grace; Ahmad, Raheel; Lee, Dasol; Veiga, Sara; Rabe, Daniel Christopher; Stott, Shannon Leigh (January 2023, Extracellular Vesicles and Circulating Nucleic Acids)

   Background: Vascular cell adhesion molecule-1 (VCAM-1+) endothelial cell-derived extracellular vesicles (EC-EVs) are augmented in cardiovascular disease, where they can signal the deployment of immune cells from the splenic reserve. Endothelial cells in culture activated with pro-inflammatory tumor necrosis factor-α (TNF-a) also release VCAM-1+ EC-EVs. However, isolating VCAM-1+ EC-EVs from conditioned cell culture media for subsequent in-depth analysis remains challenging. Aim: We utilized the extracellular vesicles (EV) microfluidics herringbone chip (EVHB-Chip), coated with anti-VCAM-1 antibodies, for selective capture of VCAM-1+ cells and EC-EVs. Methods and Results: Engineered EA.hy926 endothelial cells overexpressing VCAM-1 (P < 0.001 versus control) showed increased binding to the VCAM-1- EVHB-Chip versus an IgG device. TNF-α-stimulated human umbilical cord vein endothelial cells (HUVECs) exhibited elevated VCAM-1 protein levels (P < 0.001) and preferential binding to the VCAM-1- EVHB-Chip versus the IgG device. HUVECs stimulated with TNF-α showed differential gene expression of intercellular adhesion molecule-1 (ICAM-1) (P < 0.001) and VCAM-1 (P < 0.001) by digital droplet PCR versus control cells. HUVEC-derived EC-EVs were positive for CD9, CD63, HSP70, and ALIX and had a modal size of 83.5 nm. Control and TNF-α-stimulated HUVEC-derived EC-EV cultures were captured on the VCAM-1- EVHB-Chip, demonstrating selective capture. VCAM-1+ EC-EV were significantly enriched for ICAM-1 (P < 0.001) mRNA transcripts. Conclusion: This study presents a novel approach using the EVHB-Chip, coated with anti-VCAM-1 antibodies and digital droplet PCR for the study of VCAM-1+ EC-EVs. Isolation of VCAM-1+ EC-EV from heterogeneous sources such as conditioned cell culture media holds promise for subsequent detailed characterization, and may facilitate the study of VCAM-1+ EC-EVs in cardiovascular and metabolic diseases, for disease monitoring and therapeutic insights. 

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5. Preparation of Nanoparticle-Loaded Extracellular Vesicles Using Direct Flow Filtration

   https://doi.org/10.3390/pharmaceutics15051551  

   Mansur, Shomit; Habib, Shahriar; Hawkins, Mikayla; Brown, Spenser R.; Weinman, Steven T.; Bao, Yuping (May 2023, Pharmaceutics)

   Extracellular vesicles (EVs) have shown great potential as cell-free therapeutics and biomimetic nanocarriers for drug delivery. However, the potential of EVs is limited by scalable, reproducible production and in vivo tracking after delivery. Here, we report the preparation of quercetin-iron complex nanoparticle-loaded EVs derived from a breast cancer cell line, MDA-MB-231br, using direct flow filtration. The morphology and size of the nanoparticle-loaded EVs were characterized using transmission electron microscopy and dynamic light scattering. The SDS-PAGE gel electrophoresis of those EVs showed several protein bands in the range of 20–100 kDa. The analysis of EV protein markers by a semi-quantitative antibody array confirmed the presence of several typical EV markers, such as ALIX, TSG101, CD63, and CD81. Our EV yield quantification suggested a significant yield increase in direct flow filtration compared with ultracentrifugation. Subsequently, we compared the cellular uptake behaviors of nanoparticle-loaded EVs with free nanoparticles using MDA-MB-231br cell line. Iron staining studies indicated that free nanoparticles were taken up by cells via endocytosis and localized at a certain area within the cells while uniform iron staining across cells was observed for cells treated with nanoparticle-loaded EVs. Our studies demonstrate the feasibility of using direct flow filtration for the production of nanoparticle-loaded EVs from cancer cells. The cellular uptake studies suggested the possibility of deeper penetration of the nanocarriers because the cancer cells readily took up the quercetin-iron complex nanoparticles, and then released nanoparticle-loaded EVs, which can be further delivered to regional cells. 

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