Enhancing the Cellular Production of Extracellular Vesicles for Developing Therapeutic Applications.

Extracellular vesicles (EVs) have various advantageous properties, including a small size, high biocompatibility, efficient cargo loading, and precise cell targeting ability, making them promising tools for therapeutic development. EVs have been increasingly explored for applications like drug delivery. However, due to limited cellular secretion rates of EVs, wide-scale clinical applications are not achievable. Therefore, substantial strategies and research efforts have been devoted to increasing cellular secretion rates of EVs. This review describes various studies exploring different methods to increase the cellular production of EVs, including the application of electrical stimulus, pharmacologic agents, electromagnetic waves, sound waves, shear stress, cell starvation, alcohol, pH, heat, and genetic manipulation. These methods have shown success in increasing EV production, but careful consideration must be given as many of these strategies may alter EV properties and functionalities, and the exact mechanisms causing the increase in cellular production of EVs is generally unknown. Additionally, the methods' effectiveness in increasing EV secretion may diverge with different cell lines and conditions. Further advancements to enhance EV biogenesis secretion for therapeutic development is still a significant need in the field.

CRISPR-Cas9 Engineered Extracellular Vesicles for the Treatment of Dominant Progressive Hearing Loss.

Clinical translation of gene therapy has been challenging, due to limitations in current delivery vehicles such as traditional viral vectors. Herein, we report the use of gRNA:Cas9 ribonucleoprotein (RNP) complexes engineered extracellular vesicles (EVs) for in vivo gene therapy. By leveraging a novel high-throughput microfluidic droplet-based electroporation system (µDES), we achieved 10-fold enhancement of loading efficiency and more than 1000-fold increase in processing throughput on loading RNP complexes into EVs (RNP-EVs), compared with conventional bulk electroporation. The flow-through droplets serve as enormous bioreactors for offering millisecond pulsed, low-voltage electroporation in a continuous-flow and scalable manner, which minimizes the Joule heating influence and surface alteration to retain natural EV stability and integrity. In the Shaker-1 mouse model of dominant progressive hearing loss, we demonstrated the effective delivery of RNP-EVs into inner ear hair cells, with a clear reduction of Myo7ash1 mRNA expression compared to RNP-loaded lipid-like nanoparticles (RNP-LNPs), leading to significant hearing recovery measured by auditory brainstem responses (ABR).

Light-induced high-efficient cellular production of immune functional extracellular vesicles.

Extracellular vesicle (EV)-based therapies and vaccines are emerging. However, employment at the scale for population-based dose development is always a huge bottleneck. In order to overcome such a roadblock, we introduce a simple and straightforward approach for promoting cellular production of dendritic cell derived EVs (DEVs) by leveraging phototherapy based light induction. Under the optimization of light wavelengths, intensities, and exposure times, we achieved more than 13-fold enhancement in DEV production rate, while maintaining good integral quality and immune function from produced EVs. The LED light at 365 nm is optimal to reliably trigger enhanced cellular production of EVs no matter cell line types. Our observation and other reported studies support longer near UV wavelength does not impair cell growth. We conducted a series of investigations in terms of size, zeta potential, morphology, immune surface markers and cytokines, biocompatibility, cellular uptake behaviour, and immune-modulation ability on eliciting cellular responses in vitro. We also validated the biodistribution, immunogenicity, and administration safety using light-promoted DEVs in mice models from both male and female genders. Overall data supports that light promoted DEVs are highly immune functional with great biocompatibility for serving as good therapeutic platforms. The in vivo animal study also demonstrated light-promoted DEVs are as well tolerated as native DEVs, with no safety concerns. Taken together, the data supports that light promoted DEVs are in excellent quality, high biocompatibility, in vivo tolerant, and viable for serving as an ideal therapeutic platform in scalable production.

Extracellular Vesicles as an Advanced Delivery Biomaterial for Precision Cancer Immunotherapy.

In recent years, cancer immunotherapy has been observed in numerous preclinical and clinical studies for showing benefits. However, due to the unpredictable outcomes and low response rates, novel targeting delivery approaches and modulators are needed for being effective to more broader patient populations and cancer types. Compared to synthetic biomaterials, extracellular vesicles (EVs) specifically open a new avenue for improving the efficacy of cancer immunotherapy by offering targeted and site-specific immunity modulation. In this review, the molecular understanding of EV cargos and surface receptors, which underpin cell targeting specificity and precisely modulating immunogenicity, are discussed. Unique properties of EVs are reviewed in terms of their surface markers, intravesicular contents, intrinsic immunity modulatory functions, and pharmacodynamic behavior in vivo with tumor tissue models, highlighting key indications of improved precision cancer immunotherapy. Novel molecular engineered strategies for reprogramming and directing cancer immunotherapeutics, and their unique challenges are also discussed to illuminate EV's future potential as a cancer immunotherapeutic biomaterial.