Engineering Extracellular Vesicles to Target Pancreatic Tissue <i>In Vivo</i>

Komuro, Hiroaki; Kawai‐Harada, Yuki; Aminova, Shakhlo; Pascual, Nathaniel; Malik, Anshu; Contag, Christopher H.; Harada, Masahiko

doi:10.7150/ntno.54879

Cited by 21 publications

(29 citation statements)

References 80 publications

(121 reference statements)

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“…Direct EV imaging allows for an evaluation of cell-specific binding and EV uptake. As described previously, we used the endogenous loading method of EV labeling by co-transfection of fluorescence (mCherry) or bioluminescence (gaussia luciferase/gLuc) molecule–C1C2 fusion and the targeting constructs to generate dual-functional EVs [ 23 ]. The equivalent activity of imaging molecules per set of EV samples was tested prior to each experiment to avoid bias from the EV source.…”

Section: Resultsmentioning

confidence: 99%

“…However, due to the risk of immortalization gene packaging, such cells are excluded from a potential therapeutic EV source. In addition, while we and others have previously successfully displayed the designed peptide display on the HEK293T cell-derived EV surfaces by encoding plasmid transfection, the plasmid DNA packaging was inevitable [ 23 , 39 , 40 , 54 ]. Unwanted gene transcription from the strong promoter from such constructs limits their clinical applications.…”

Section: Discussionmentioning

confidence: 99%

“…The EV monobody display constructs were created using Seamless Ligation Cloning Extract (SLiCE) assembly, as previously described [ 23 ]. PCR fragment of EV display backbone from pcS-p88-C1C2 using the primer sets listed in Table S1 was fused with the synthetic double stranded DNA fragments coding for monobody (E626 or RDG) and G4S-PAS linker [ 41 ] including 15 bp overhangs.…”

Section: Methodsmentioning

confidence: 99%

“…Our previous work described the EV surface protein engineering method [ 23 ] using protein fusion with a C1C2 domain of human lactadherin (milk fat globule EGF factor VIII/MFG-E8) fused with a pancreas-specific peptide [ 23 , 24 , 25 ]. We engineered HEK293T-derived EVs to target pancreatic cells in vitro and in vivo and demonstrated pancreas targeting and delivery of cargo DNA.…”

Section: Introductionmentioning

confidence: 99%

“…We engineered HEK293T-derived EVs to target pancreatic cells in vitro and in vivo and demonstrated pancreas targeting and delivery of cargo DNA. EV surface protein modification enables EVs to display a protein with a high affinity towards a specific cell type [ 23 ]. In this report, we used the same EV surface approach with the use of an engineered monobody (E626), a clinically approved binding molecule with high affinity towards EGFR tumors [ 26 ].…”

Section: Introductionmentioning

confidence: 99%

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Design and Evaluation of Engineered Extracellular Vesicle (EV)-Based Targeting for EGFR-Overexpressing Tumor Cells Using Monobody Display

et al. 2022

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Background: Extracellular vesicles (EVs) are attracting interest as a new class of drug delivery vehicles due to their intrinsic nature of biomolecular transport in the body. We previously demonstrated that EV surface modification with tissue-specific molecules accomplished targeted EV-mediated DNA delivery. Methods: Here, we describe reliable methods for (i) generating EGFR tumor-targeting EVs via the display of high-affinity monobodies and (ii) in vitro measurement of EV binding using fluorescence and bioluminescence labeling. Monobodies are a well-suited class of small (10 kDa) non-antibody scaffolds derived from the human fibronectin type III (FN3) domain. Results: The recombinant protein consists of the EGFR-targeting monobody fused to the EV-binding domain of lactadherin (C1C2), enabling the monobody displayed on the surface of the EVs. In addition, the use of bioluminescence or fluorescence molecules on the EV surface allows for the assessment of EV binding to the target cells. Conclusions: In this paper, we describe methods of EV engineering to generate targeted delivery vehicles using monobodies that will have diverse applications to furnish future EV therapeutic development, including qualitative and quantitative in vitro evaluation for their binding capacity.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Design and Evaluation of Engineered Extracellular Vesicle (EV)-Based Targeting for EGFR-Overexpressing Tumor Cells Using Monobody Display

et al. 2022

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Considerations for extracellular vesicle and lipoprotein interactions in cell culture assays

Busatto

Yang

Iannotta

et al. 2022

J of Extracellular Vesicle

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With an exponential increase in extracellular vesicle (EV) studies in the past decade, focus has been placed on standardization of experimental design to ensure inter‐study comparisons and validity of conclusions. In the case of in vitro assays, the composition of cell culture media is important to consider for EV studies. In particular, levels of lipoproteins, which are critical components of the interstitial fluid, should be taken into consideration. Results from this study reveal that lipoprotein levels in cell culture medium impact the effects that EVs have on recipient cells. Additionally, evidence of EV binding and fusion to lipoprotein‐like structures in plasma is provided. However, it is unclear whether the impact of lipoproteins in cell culture is due to direct interactions with EVs, indirect effects, or a combination of both mechanisms. Taken together, cell culture studies performed in the absence of physiological levels of lipoproteins are unlikely to reflect interactions that occur between EVs and recipient cells in an in vivo environment.

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Exploring the role of polymers to overcome ongoing challenges in the field of extracellular vesicles

Simon,

Lapinte,

Morille

2023

J of Extracellular Vesicle

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Extracellular vesicles (EVs) are naturally occurring nanoparticles released from all eucaryotic and procaryotic cells. While their role was formerly largely underestimated, EVs are now clearly established as key mediators of intercellular communication. Therefore, these vesicles constitute an attractive topic of study for both basic and applied research with great potential, for example, as a new class of biomarkers, as cell‐free therapeutics or as drug delivery systems. However, the complexity and biological origin of EVs sometimes complicate their identification and therapeutic use. Thus, this rapidly expanding research field requires new methods and tools for the production, enrichment, detection, and therapeutic application of EVs. In this review, we have sought to explain how polymer materials actively contributed to overcome some of the limitations associated to EVs. Indeed, thanks to their infinite diversity of composition and properties, polymers can act through a variety of strategies and at different stages of EVs development. Overall, we would like to emphasize the importance of multidisciplinary research involving polymers to address persistent limitations in the field of EVs.

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Engineering Extracellular Vesicles to Target Pancreatic Tissue In Vivo

Cited by 21 publications

References 80 publications

Design and Evaluation of Engineered Extracellular Vesicle (EV)-Based Targeting for EGFR-Overexpressing Tumor Cells Using Monobody Display

Design and Evaluation of Engineered Extracellular Vesicle (EV)-Based Targeting for EGFR-Overexpressing Tumor Cells Using Monobody Display

Considerations for extracellular vesicle and lipoprotein interactions in cell culture assays

Exploring the role of polymers to overcome ongoing challenges in the field of extracellular vesicles

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