Extracellular vesicles (EVs) from stem cells have shown significant therapeutic potential to repair injured cardiac tissues and regulate pathological fibrosis. However, scalable generation of stem cells and derived EVs for clinical utility remains a huge technical challenge. Here, we report a rapid size-based extrusion strategy to generate EV-like membranous nanovesicles (NVs) from easily sourced human iPSCs in large quantities (yield 900× natural EVs). NVs isolated using density-gradient separation (buoyant density 1.13 g/mL) are spherical in shape and morphologically intact and readily internalised by human cardiomyocytes, primary cardiac fibroblasts, and endothelial cells. NVs captured the dynamic proteome of parental cells and include pluripotency markers (LIN28A, OCT4) and regulators of cardiac repair processes, including tissue repair (GJA1, HSP20/27/70, HMGB1), wound healing (FLNA, MYH9, ACTC1, ILK), stress response/translation initiation (eIF2S1/S2/S3/B4), hypoxia response (HMOX2, HSP90, GNB1), and extracellular matrix organization (ITGA6, MFGE8, ITGB1). Functionally, NVs significantly promoted tubule formation of endothelial cells (angiogenesis) (p < 0.05) and survival of cardiomyocytes exposed to low oxygen conditions (hypoxia) (p < 0.0001), as well as attenuated TGF-β mediated activation of cardiac fibroblasts (p < 0.0001). Quantitative proteome profiling of target cell proteome following NV treatments revealed upregulation of angiogenic proteins (MFGE8, MYH10, VDAC2) in endothelial cells and pro-survival proteins (CNN2, THBS1, IGF2R) in cardiomyocytes. In contrast, NVs attenuated TGF-β-driven extracellular matrix remodelling capacity in cardiac fibroblasts (ACTN1, COL1A1/2/4A2/12A1, ITGA1/11, THBS1). This study presents a scalable approach to generating functional NVs for cardiac repair.

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Scalable Generation of Nanovesicles from Human-Induced Pluripotent Stem Cells for Cardiac Repair

Background: Mesenchymal stem cell (MSC)-derived exosomes are well recognized immunomodulating agents for cardiac repair, while the detailed mechanisms remain elusive. The Pericardial drainage pathway provides the heart with immunosurveillance and establishes a simplified model for studying the mechanisms underlying the immunomodulating effects of therapeutic exosomes. Methods: Myocardial infarction (MI) models with and without pericardiectomy (corresponding to Tomy MI and NonTomy MI) were established to study the functions of pericardial drainage pathway in immune activation of cardiac-draining mediastinal lymph node (MLN). Using the NonTomy MI model, MSC exosomes or vehicle PBS was intrapericardially injected for MI treatment. Via cell sorting and RNA-seq (RNA-sequencing) analysis, the differentially expressed genes were acquired for integrated pathway analysis to identify responsible mechanisms. Further, through functional knockdown/inhibition studies, application of cytokines and neutralizing antibodies, western blot, flow cytometry, and cytokine array, the molecular mechanisms were studied. In addition, the therapeutic efficacy of intrapericardially injected exosomes for MI treatment was evaluated through functional and histological analyses. Results: We show that the pericardial draining pathway promoted immune activation in the MLN following MI. Intrapericardially injected exosomes accumulated in the MLN and induced regulatory T cell differentiation to promote cardiac repair. Mechanistically, uptake of exosomes by major histocompatibility complex (MHC)-II + antigen-presenting cells (APCs) induced Foxo3 activation via the protein phosphatase (PP)-2A/p-Akt/forkhead box O3 (Foxo3) pathway. Foxo3 dominated APC cytokines (IL-10, IL-33, and IL-34) expression and built up a regulatory T cell (Treg)-inducing niche in the MLN. The differentiation of Tregs as well as their cardiac deployment were elevated, which contributed to cardiac inflammation resolution and cardiac repair. Conclusions: This study reveals a novel mechanism underlying the immunomodulation effects of MSC exosomes and provides a promising candidate (PP2A/p-Akt/Foxo3 signaling pathway) with a favorable delivery route (intrapericardial injection) for cardiac repair. 

https://www.ahajournals.org/doi/pdf/10.1161/CIRCRESAHA.122.321384

Intrapericardial Exosome Therapy Dampens Cardiac Injury via Activating Foxo3

Extracellular vesicles (EVs), of which exosomes are a representative subgroup, are naturally secreted nanoparticles with a variety of payloads. With the intrinsic merits of stability, biocompatibility, low immunogenicity, and large capacity, EVs are widely regarded as effective carriers of drug delivery. However, disadvantages, such as low yield, complicated isolation procedures, and low loading efficiency, hinder its clinical translation. In this review, we systematically summarize the advances in EV (especially exosomes) engineering for clinical application, focusing on strategies toward high yield, facile isolation, efficient cargo loading, improved delivery, and optimized manufacturing, which might unleash the infinite power of EVs in clinical translation.

Multiplexed strategies toward clinical translation of extracellular vesicles

Cancer patients often face severe organ toxicity caused by chemotherapy. Among these, chemotherapy‐induced hepatotoxicity and cardiotoxicity are the main causes of death of cancer patients. Chemotherapy‐induced cardiotoxicity even creates a new discipline termed “cardio‐oncology”. Therefore, relieving toxicities induced by chemotherapy has become a key issue for improving the survival and quality of life in cancer patients. In this work, mesenchymal stem cell exosomes with the “G‐C” abundant tetrahedral DNA nanostructure (TDN) are modified to form a decoy exosome (Exo‐TDN). Exo‐TDN reduces DOX‐induced hepatotoxicity as the “G‐C” base pairs scavenge DOX. Furthermore, Exo‐TDN with cardiomyopathic peptide (Exo‐TDN‐PCM) is engineered for specific targeting to cardiomyocytes. Injection of Exo‐TDN‐PCM significantly reduces DOX‐induced cardiotoxicity. Interestingly, Exo‐TDN‐PCM can also promote macrophage polarization into the M2 type for tissue repair. In addition, those decoy exosomes do not affect the anticancer effects of DOX. This decoy exosome strategy serves as a promising therapy to reduce chemo‐induced toxicity.

Decoy Exosomes Offer Protection Against Chemotherapy‐Induced Toxicity (Adv. Sci. 32/2022)

Abstract Cancer patients often face severe organ toxicity caused by chemotherapy. Among these, chemotherapy‐induced hepatotoxicity and cardiotoxicity are the main causes of death of cancer patients. Chemotherapy‐induced cardiotoxicity even creates a new discipline termed “cardio‐oncology”. Therefore, relieving toxicities induced by chemotherapy has become a key issue for improving the survival and quality of life in cancer patients. In this work, mesenchymal stem cell exosomes with the “G‐C” abundant tetrahedral DNA nanostructure (TDN) are modified to form a decoy exosome (Exo‐TDN). Exo‐TDN reduces DOX‐induced hepatotoxicity as the “G‐C” base pairs scavenge DOX. Furthermore, Exo‐TDN with cardiomyopathic peptide (Exo‐TDN‐PCM) is engineered for specific targeting to cardiomyocytes. Injection of Exo‐TDN‐PCM significantly reduces DOX‐induced cardiotoxicity. Interestingly, Exo‐TDN‐PCM can also promote macrophage polarization into the M2 type for tissue repair. In addition, those decoy exosomes do not affect the anticancer effects of DOX. This decoy exosome strategy serves as a promising therapy to reduce chemo‐induced toxicity.

Decoy Exosomes Offer Protection Against Chemotherapy‐Induced Toxicity

Extracellular vesicles (EV) as drug delivery nanocarriers are under intense investigation. Although clinical-grade EVs have been produced on a large-scale, low yield and high production costs of natural EVs (nEV) limit the relevant industrial translation. Recent studies show that mechanical extrusion of cells can generate nEV-like cell-derived nanovesicles (CNV) which can also be used as drug nanocarriers. Moreover, in comparison with nEVs, CNVs have similar physicochemical properties. Nevertheless, a comprehensive comparison of cargo between nEVs and CNVs has not been investigated yet. Therefore, the aim of this study is to profile and compare CNVs to nEVs. Our results show that no significant difference was found in size, morphology, and classical markers between nEVs and CNVs derived from MDA-MB-231 cells. Protein sequencing data reveals the similarity of membrane proteins between the two groups was ∼71%, while it was ∼21% when pertaining to total protein cargo. Notably, a high similarity of membrane proteins was also found between nEVs and CNVs derived from eight additional cancer cell lines. Moreover, analysis of the top 1000 small RNAs with RNA sequencing showed a ∼65% similarity between the two groups. Altogether, we infer from the high similarity of membrane proteins and small RNA cargo that CNVs can be a good substitute for nEVs. In brief, our findings support previous studies with a notion that CNVs yield comparable performance with nEVs and could pave the way for clinical implementation of CNV-based therapeutics in the future. 

https://doi.org/10.1016/j.vesic.2022.100004

Cell-derived nanovesicles prepared by membrane extrusion are good substitutes for natural extracellular vesicles

Self-assembly peptide nanotechnology has attracted much attention due to its regular and orderly structure and diverse functions. Most of the existing self-assembly peptides can form aggregates with specific structures only under specific conditions and their assembly time is relatively long. They have good biocompatibility but no immunogenicity. To optimize it, a self-assembly peptide named DRF3 was designed. It contains a hydrophilic and hydrophobic surface, using two N-terminal arginines, leucine, and two c-terminal aspartate and glutamic acid. Meanwhile, the c-terminal of the peptide was amidated, so that peptide segments were interconnected to increase diversity. Its characterization, biocompatibility, controlled release effect on antigen, immune cell recruitment ability, and antitumor properties were examined here. Congo red/aniline blue staining revealed that peptide hydrogel DRF3 could be immediately gelled in PBS. The stable β-sheet secondary structure of DRF3 was confirmed by circular dichroism spectrum and IR spectra. The observation results of cryo-scanning electron microscopy, transmission electron microscopy, and atomic force microscopy demonstrated that DRF3 formed nanotubule-like and vesicular structures in PBS, and these structures interlaced with each other to form ordered three-dimensional nanofiber structures. Meanwhile, DRF3 showed excellent biocompatibility, could sustainably and slowly release antigens, recruit dendritic cells and promote the maturation of dendritic cells (DCs) in vitro. In addition, DRF3 has a strong inhibitory effect on clear renal cell carcinoma (786-0). These results provide a reliable basis for the application of peptide hydrogels in biomedical and preclinical trials.

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A Rapid Self-Assembly Peptide Hydrogel for Recruitment and Activation of Immune Cells

Patient-derived organoid (PDO) models have been widely used in precision medicine. The inability to standardize organoid creation in pre-clinical models has become apparent. The common mouse-derived extracellular matrix can no longer meet the requirements for the establishment of PDO models. Therefore, in order to develop effective methods for 3D cultures of organoids, we designed a self-assembling peptide, namely DRF3, which can be self-assembled into ordered fibrous scaffold structures. Here, we used the co-assembly of self-assembling peptide (SAP) and collagen type I, fibronectin, and laminin (SAP-Matrix) to co-simulate the extracellular matrix, which significantly reduced the culture time of PDO, improved the culture efficiency, and increased the self-assembly ability of cells. Compared with the results from the 2D cell line, the PDO showed a more significant expression of cancer-related genes. During organoid self-assembly, the expression of cancer-related genes is increased. These findings provide a theoretical basis for the establishment of precision molecular modeling platforms in the future.

/pdf/a-self-assembling-peptide-as-a-model-for-detection-of-2ixrlqoi.pdf

A Self-Assembling Peptide as a Model for Detection of Colorectal Cancer

Objective To investigate the effect of chiral self-assembled peptides SciobioII and Sciobio IV on the repair of anterior cruciate ligament injury. Methods The structure of the self-assembled peptides SciobioII and Sciobio IV was analyzed by circular dichroism, transmission electron microscopy, and aniline blue staining; the activity and morphology of human ligament fibroblasts (HLF) in 3D cell culture matrix were detected by acridine orange/ethidium bromide (AO/EB) staining and FITC-phalloidin staining. The rabbit model of anterior cruciate ligament injuries was established and the effects of self-assembled peptides on the ligament repair were analyzed by HE staining and immunohistochemistry. Results The self-assembled peptides SciobioII and Sciobio IV formed a stable β-sheet after self-assembling in PBS for 24 hours, and futher constructed a nanofiber network structure, which was suitable for 3D cell culture. Human ligament fibroblasts maintained a round shape and grew vigorously in the 3D cell culture media constructed by self-assembled peptides. Animal experiments showed that the self-assembled peptide SciobioII promoted the repair rate of anterior cruciate ligament injury in rabbit. Conclusion Chiral self-assembled peptides Sciobio II and Sciobio IV can be used for 3D cell culture and repair of anterior cruciate ligament injury in rabbit.

Yuan Wan

Papers

Cell-derived nanovesicles prepared by membrane extrusion are good substitutes for natural extracellular vesicles

A Rapid Self-Assembly Peptide Hydrogel for Recruitment and Activation of Immune Cells

A Self-Assembling Peptide as a Model for Detection of Colorectal Cancer

[Chiral self-assembled peptides Sciobio II and Sciobio IV for 3D cell culture and repair of anterior cruciate ligament injury in rabbit].