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Extracellular vesicles (EVs) are a population of small vesicles secreted by essentially all cell types, containing a wide variety of biological macromolecules. Due to their intrinsic capabilities for efficient intercellular communication, they are involved in various aspects of cellular functioning. In the past decade, EVs derived from stem cells attracted interest in the field of regenerative medicine. Owing to their regenerative properties, they have great potential for use in tissue repair, in particular for tissues with limited regenerative capabilities such as cartilage. The maintenance of articular cartilage is dependent on a precarious balance of many different components that can be disrupted by the onset of prevalent rheumatic diseases. However, while cartilage is a tissue with strong mechanical properties that can withstand movement and heavy loads for years, it is virtually incapable of repairing itself after damage has occurred. Stem cell-derived EVs (SC-EVs) transport regenerative components such as proteins and nucleic acids from their parental cells to recipient cells, thereby promoting cartilage healing. Many possible pathways through which SC-EVs execute their regenerative function have been reported, but likely there are still numerous other pathways that are still unknown. This review discusses various preclinical studies investigating intra-articular injections of free SC-EVs, which, while often promoting chondrogenesis and cartilage repair in vivo, showed a recurring limitation of the need for multiple administrations to achieve sufficient tissue regeneration. Potentially, this drawback can be overcome by making use of an EV delivery platform that is capable of sustainably releasing EVs over time. With their remarkable versatility and favourable chemical, biological and mechanical properties, hydrogels can facilitate this release profile by encapsulating EVs in their porous structure. Ideally, the optimal delivery platform can be formed in-situ, by means of an injectable hydrogel that can be administered directly into the affected joint. Relevant research fulfilling these criteria is discussed in detail, including the steps that still need to be taken before injectable hydrogels for sustained delivery of EVs can be applied in the context of cartilage regeneration in the clinic.

The CRISPR-Cas9 technology represents a powerful tool for genome engineering in eukaryotic cells, advancing both fundamental research and therapeutic strategies. Despite the enormous potential of the technology, efficient and direct intracellular delivery of Cas9 ribonucleoprotein (RNP) complexes in target cells poses a significant hurdle, especially in refractive primary cells. In the present work, vapor nanobubble (VNB) photoporation was explored for Cas9 RNP transfection in a variety of cell types. Proof of concept was first demonstrated in H1299-EGFP cells, before proceeding to hard-to-transfect stem cells and T cells. Gene knock-out levels over 80% and up to 60% were obtained for H1299 cells and mesenchymal stem cells, respectively. In these cell types, the unique possibility of VNB photoporation to knock out genes according to user-defined spatial patterns was demonstrated as well. Next, effective targeting of the programmed cell death 1 receptor and Wiskott-Aldrich syndrome gene in primary human T cells was demonstrated, reaching gene knock-out levels of 25% and 34%, respectively. With a throughput of >200,000 T cells per second, VNB photoporation is a scalable and versatile intracellular delivery method that holds great promise for CRISPR-Cas9-mediated ex vivo engineering of cell therapy products.

Extracellular vesicles (EVs) are submicron cell-secreted structures containing proteins, nucleic acids and lipids. EVs can functionally transfer these cargoes from one cell to another to modulate physiological and pathological processes. Due to their presumed biocompatibility and capacity to circumvent canonical delivery barriers encountered by synthetic drug delivery systems, EVs have attracted considerable interest as drug delivery vehicles. However, it is unclear which mechanisms and molecules orchestrate EV-mediated cargo delivery to recipient cells. Here, we review how EV properties have been exploited to improve the efficacy of small molecule drugs. Furthermore, we explore which EV surface molecules could be directly or indirectly involved in EV-mediated cargo transfer to recipient cells and discuss the cellular reporter systems with which such transfer can be studied. Finally, we elaborate on currently identified cellular processes involved in EV cargo delivery. Through these topics, we provide insights in critical effectors in the EV-cell interface which may be exploited in nature-inspired drug delivery strategies.

The CRISPR-Cas9 technology represents a powerful tool for genome engineering in eukaryotic cells, advancing both fundamental research and therapeutic strategies. Despite the enormous potential of the technology, efficient and direct intracellular delivery of Cas9 ribonucleoprotein (RNP) complexes in target cells poses a significant hurdle, especially in refractive primary cells. In the present work, vapor nanobubble (VNB) photoporation was explored for Cas9 RNP transfection in a variety of cell types. Proof of concept was first demonstrated in H1299-EGFP cells, before proceeding to hard-to-transfect stem cells and T cells. Gene knock-out levels over 80% and up to 60% were obtained for H1299 cells and mesenchymal stem cells, respectively. In these cell types, the unique possibility of VNB photoporation to knock out genes according to user-defined spatial patterns was demonstrated as well. Next, effective targeting of the programmed cell death 1 receptor and Wiskott-Aldrich syndrome gene in primary human T cells was demonstrated, reaching gene knock-out levels of 25% and 34%, respectively. With a throughput of >200,000 T cells per second, VNB photoporation is a scalable and versatile intracellular delivery method that holds great promise for CRISPR-Cas9-mediated ex vivo engineering of cell therapy products.

Extracellular vesicles (EVs) form an endogenous transport system for intercellular transfer of biological cargo, including RNA, that plays a pivotal role in physiological and pathological processes. Unfortunately, whereas biological effects of EV-mediated RNA transfer are abundantly studied, regulatory pathways and mechanisms remain poorly defined due to a lack of suitable readout systems. Here, we describe a highly-sensitive CRISPR-Cas9-based reporter system that allows direct functional study of EV-mediated transfer of small non-coding RNA molecules at single-cell resolution. Using this CRISPR operated stoplight system for functional intercellular RNA exchange (CROSS-FIRE) we uncover various genes involved in EV subtype biogenesis that play a regulatory role in RNA transfer. Moreover we identify multiple genes involved in endocytosis and intracellular membrane trafficking that strongly regulate EV-mediated functional RNA delivery. Altogether, this approach allows the elucidation of regulatory mechanisms in EV-mediated RNA transfer at the level of EV biogenesis, endocytosis, intracellular trafficking, and RNA delivery., Extracellular vesicles (EV) facilitate intercellular transfer of biological material including RNA, but the regulatory mechanisms for their formation and transfer are incompletely known. Here the authors develop a CRISPR-based reporting system to detect the transfer of guide RNAs and identify genes not previously linked to EV-mediated RNA delivery.

Over the past decades, a multitude of synthetic drug delivery systems has been developed and introduced to the market. However, applications of such systems are limited due to inefficiency, cytotoxicity and/or immunogenicity. At the same time, the field of natural drug carrier systems has grown rapidly. One of the most prominent examples of such natural carriers are extracellular vesicles (EVs). EVs are cell-derived membranous particles which play important roles in intercellular communication. EVs possess a number of characteristics that qualify them as promising vehicles for drug delivery. In order to take advantage of these attributes, an in-depth understanding of why EVs are such unique carrier systems and how we can exploit their qualities is pivotal. Here, we review unique EV features that are relevant for drug delivery and highlight emerging strategies to make use of those features for drug loading and targeted delivery.

Intracellular delivery of membrane-impermeable cargo offers unique opportunities for biological research and the development of cell-based therapies. Despite the breadth of available intracellular delivery tools, existing protocols are often suboptimal and alternative approaches that merge delivery efficiency with both biocompatibility, as well as applicability, remain highly sought after. Here, a comprehensive platform is presented that exploits the unique property of cationic hydrogel nanoparticles to transiently disrupt the plasma membrane of cells, allowing direct cytosolic delivery of uncomplexed membrane-impermeable cargo. Using this platform, which is termed Hydrogel-enabled nanoPoration or HyPore, the delivery of fluorescein isothiocyanate (FITC)-dextran macromolecules in various cancer cell lines and primary bovine corneal epithelial cells is convincingly demonstrated. Of note, HyPore demonstrates efficient FITC-dextran delivery in primary human T cells, outperforming state-of-the-art electroporation-mediated delivery. Moreover, the HyPore platform enables cytosolic delivery of functional proteins, including a histone-binding nanobody as well as the enzymes granzyme A and Cre-recombinase. Finally, HyPore-mediated delivery of the MRI contrast agent gadobutrol in primary human T cells significantly improves their T1 -weighted MRI signal intensities compared to electroporation. Taken together, HyPore is proposed as a straightforward, highly versatile, and cost-effective technique for high-throughput, ex vivo manipulation of primary cells and cell lines.

Extracellular vesicles (EVs) are nanoparticles which are released by cells from all three domains of life: Archaea, Bacteria and Eukarya. They can mediate cell-cell communication by transferring cargoes such as proteins and nucleic acids between cells. EVs receive great interest in both academia and industry as they have the potential to be natural drug carriers or vaccine candidates. However, limitations to their clinical translation exist as efficient isolation, loading, labelling and surface-engineering methods are lacking. In this article, we investigate a ‘post-insertion’ approach, which is commonly used in the functionalization of liposomes in the pharmaceutical field, on two different EV types: mammalian cell-derived EVs and bacteria-derived EVs. We aimed to find an easy and flexible approach to functionalize EVs, thereby improving the labelling, isolation, and surface-engineering.

Intracellular delivery of membrane-impermeable cargo offers unique opportunities for biological research and the development of cell-based therapies. Despite the breadth of available intracellular delivery tools, existing protocols are often suboptimal and alternative approaches that merge delivery efficiency with both biocompatibility, as well as applicability, remain highly sought after. Here, a comprehensive platform is presented that exploits the unique property of cationic hydrogel nanoparticles to transiently disrupt the plasma membrane of cells, allowing direct cytosolic delivery of uncomplexed membrane-impermeable cargo. Using this platform, which is termed Hydrogel-enabled nanoPoration or HyPore, the delivery of fluorescein isothiocyanate (FITC)-dextran macromolecules in various cancer cell lines and primary bovine corneal epithelial cells is convincingly demonstrated. Of note, HyPore demonstrates efficient FITC-dextran delivery in primary human T cells, outperforming state-of-the-art electroporation-mediated delivery. Moreover, the HyPore platform enables cytosolic delivery of functional proteins, including a histone-binding nanobody as well as the enzymes granzyme A and Cre-recombinase. Finally, HyPore-mediated delivery of the MRI contrast agent gadobutrol in primary human T cells significantly improves their T1 -weighted MRI signal intensities compared to electroporation. Taken together, HyPore is proposed as a straightforward, highly versatile, and cost-effective technique for high-throughput, ex vivo manipulation of primary cells and cell lines.

RNA therapeutics have high potential that is yet to be fully realized, largely due to challenges involved in the appropriate delivery to target cells. Extracellular vesicles (EVs) are lipid bound nanoparticles released by cells of all types and possess numerous features that may help overcome this hurdle and have emerged as a promising RNA delivery vehicle candidate. Despite extensive research into the engineering of EVs for RNA delivery, it remains unclear how the intrinsic RNA delivery efficiency of EVs compares to currently used synthetic RNA delivery vehicles. Using a novel CRISPR/Cas9-based RNA transfer reporter system, we compared the delivery efficiency of EVs to clinically approved state-of-the-art DLin-MC3-DMA lipid nanoparticles and several in vitro transfection reagents. We found that EVs delivered RNA several orders of magnitude more efficiently than these synthetic systems. This finding supports the continued research into EVs as potential RNA delivery vehicles.

The expression of the transcription factor SOX4 is increased in many human cancers, however, the pro-oncogenic capacity of SOX4 can vary greatly depending on the type of tumor. Both the contextual nature and the mechanisms underlying the pro-oncogenic SOX4 response remain unexplored. Here, we demonstrate that in mammary tumorigenesis, the SOX4 transcriptional network is dictated by the epigenome and is enriched for pro-angiogenic processes. We show that SOX4 directly regulates endothelin-1 (ET-1) expression and can thereby promote tumor-induced angiogenesis both in vitro and in vivo. Furthermore, in breast tumors, SOX4 expression correlates with blood vessel density and size, and predicts poor-prognosis in patients with breast cancer. Our data provide novel mechanistic insights into context-dependent SOX4 target gene selection, and uncover a novel pro-oncogenic role for this transcription factor in promoting tumor-induced angiogenesis. These findings establish a key role for SOX4 in promoting metastasis through exploiting diverse pro-tumorigenic pathways.