Extracellular vesicles (EVs), which are nanocarriers with phospholipid bilayer structures released by most cells, play a key role in regulating physiological and pathological processes. EVs have been investigated due to their loading capacity, low toxicity, immunogenicity, and biofunctions. Although EVs have shown good potential as therapeutic vehicles, natural EVs have a poor targeting ability, which substantially reduces the therapeutic effect. Through the addition of a targeting unit into the membrane surface of EVs or inside EVs by engineering technology, the therapeutic agent can accumulate in specific cells and tissues. Here, we focus on mammalian EVs (MEVs) and bacterial EVs (BEVs), which are the two most common types of EVs in the biomedical field. In this review, we describe engineered MEVs and BEVs as promising nanocarriers for targeted therapy and summarize the biogenesis, isolation, and characterization of MEVs and BEVs. We then describe engineering techniques for enhancement of the targeting ability of EVs. Moreover, we focus on the applications of engineered MEVs and BEVs in targeted therapy, including the treatment of cancer and brain and bone disease. We believe that this review will help improve the understanding of engineered MEVs and BEVs, thereby promoting their application and clinical translation.

Extracellular vesicles (EVs) have been regarded as influential intracellular delivering parcels that possess tremendous potential because of their strict and complex secretion regulation processes. However, traditional detection methods cannot monitor the secretion of EVs due to their small particle diameters. Inspired by their peculiar diverse appearances and lipid membranes ingredients, we developed an innovative strategy to detect EVs in any kind of fluids by using rationally designed peptide probes that particularly recognize the highly curved surface of EVs. These peptide probes also serve as novel tools to selectively target cancerous cells with specific lipid compositions and distributions. With this strategy, we discovered a series of EV-secreting regulation mechanisms and identified their roles within physiological processes. Recently, we found that the transportation of oligodeoxynucleotides and cell division control protein 42 homolog from TLR9-activated macrophages to naïve cells via EVs exerts synergetic effects in the propagation of the intracellular immune response, which suggests a general mechanism for EV-mediated uptake of pathogen-associated molecular patterns.

Cell-secreted extracellular vesicles (EVs) are membranous particles highly heterogeneous in size and molecular cargo. Comprehensively, released EV sub-populations can show a wide range and selection of different protein, RNA, and lipid species, complementing cell communication signals. Recently, EVs represent a new source for developing targeted delivery systems. EVs are stable in biofluids, intrinsically biocompatible with low immunogenicity, and capable of transferring cargo molecules into “recipient” cells. The immune-mediated recognition represents a popular approach to functionalize and direct EVs towards receptor-positive cell populations. The human epidermal growth factor receptor 2 (HER2, also known as neu or ERBB2) is a tyrosine kinase of clinical relevance, targeted by several available antibodies, and a model receptor used to test the biodistribution and anticancer activity of bioformulations, including EVs. Here, we focus on recent strategies adopted for EV functionalization with fusion ligands able to recognize HER2, covering the enhanced expression of membrane-fusion proteins in “EV-donor” cells as well as post-isolation EV-surface modifications.

Mesenchymal stem cells-derived extracellular vesicles (MSC-EVs) have noticeably attracted clinicians’ attention in treating ocular diseases. As the paracrine factor of MSCs and an alternative for cell-free therapies, MSC-EVs can be conveniently dropped over the ocular surface or diffused through the retina upon intravitreal injection, without increasing the risks of cellular rejection and tumor formation. For clinical translation, a standardized and scalable production, as well as reprogramming the MSC-EVs, are highly encouraged. This review aims to assess the potential approaches for EV production and functional modification, in addition to summarizing the worldwide clinical trials initiated for various physiological systems and the specific biochemical effects of MSC-EVs on the therapy of eye diseases. Recent advances in the therapy of ocular diseases based on MSC-EVs are reviewed, and the associated challenges and prospects are discussed as well.

Aim: A peripheral inflammatory response can drive neuroinflammation in a number of infections including human immunodeficiency virus (HIV). Monocyte/macrophage (M/Mφ) activation is a hallmark of acute HIV infection and a source of chronic inflammation in a subset of HIV-infected individuals. We sought to decrease peripheral inflammation and M/Mφ transmigration after HIV infection by engineering extracellular vesicles (EV) to antagonize a microRNA (miR) associated with inflammation. We hypothesized that induced pluripotent stem cell (iPSC)-derived monocyte EVs (mEVs), engineered to contain an antagomir to miR-155 (αmiR mEV) would target monocyte inflammation and influence neuroinflammation in an HIV-infected humanized mice.

Methods: mEVs were characterized by tetraspanins, nanoparticle tracking analysis, electron microscopy, and their preferential entry into circulating monocytes as well as testing for endogenous selected miRNAs. HIV-infected humanized mice were treated with control or antagomir155 mEVs. Plasma viral load was measured plus activation markers on lymphocytes and monocytes and the number of macrophages in the brain was quantified.

Results: mEVs preferentially entered peripheral monocytes. HIV infection increased C-C chemokine receptor type 5 (CCR5) and major histocompatibility complex, class II, DR (HLA-DR) expression on T cells and monocytes. Treatments with mEVs did not decrease plasma HIV viral load; however, mEVs alone resulted in a decrease in %CCR5+ and %HLA-DR+ on T cells and an increase in %CCR5+ monocytes. αmiR mEVs decreased %CCR5 on M/Mφ. The mEV-treated HIV-infected mice did not show an increase in macrophage transmigration to the brain.

Conclusion: mEVs alone caused an unexpected decrease in lymphocyte activation and increase in monocyte %CCR5; however, this did not translate to an increase in macrophage transmigration to the brain.

Aim: Activation of microglial NLRP3 inflammasome is an essential contributor to neuroinflammation underlying HIV-associated neurological disorders (HAND). Under pathological conditions, microglia-derived-EVs (MDEVs) can affect neuronal functions by delivering neurotoxic mediators to recipient cells. However, the role of microglial NLRP3 in mediating neuronal synaptodendritic injury has remained unexplored to date. In the present study, we sought to assess the regulatory role of HIV-1 Tat induced microglial NLRP3 in neuronal synaptodendritic injury. We hypothesized that HIV-1 Tat mediated microglia EVs carrying significant levels of NLRP3 contribute to the synaptodendritic injury, thereby affecting the maturation of neurons.

Methods: To understand the cross-talk between microglia and neuron, we isolated EVs from BV2 and human primary microglia (HPM) cells with or without NLRP3 depletion using siNLRP3 RNA. EVs were isolated by differential centrifugation, characterized by ZetaView nanoparticle tracking analysis, electron microscopy, and western blot analysis for exosome markers. Purified EVs were exposed to primary rat neurons isolated from E18 rats. Along with green fluorescent protein (GFP) plasmid transfection, immunocytochemistry was performed to visualize neuronal synaptodendritic injury. Western blotting was employed to measure siRNA transfection efficiency and the extent of neuronal synaptodegeneration. Images were captured in confocal microscopy, and subsequently, Sholl analysis was performed for analyzing dendritic spines using neuronal reconstruction software Neurolucida 360. Electrophysiology was performed on hippocampal neurons for functional assessment.

Results: Our findings demonstrated that HIV-1 Tat induced expression of microglial NLRP3 and IL1β, and further that these were packaged in microglial exosomes (MDEV) and were also taken up by the neurons. Exposure of rat primary neurons to microglial Tat-MDEVs resulted in downregulation of synaptic proteins- PSD95, synaptophysin, excitatory vGLUT1, as well as upregulation of inhibitory proteins- Gephyrin, GAD65, thereby implicating impaired neuronal transmissibility. Our findings also showed that Tat-MDEVs not only caused loss of dendritic spines but also affected numbers of spine sub-types- mushroom and stubby. Synaptodendritic injury further affected functional impairment as evidenced by the decrease in miniature excitatory postsynaptic currents (mEPSCs). To assess the regulatory role of NLRP3 in this process, neurons were also exposed to Tat-MDEVs from NLRP3 silenced microglia. Tat-MDEVs from NLRP3 silenced microglia exerted a protective role on neuronal synaptic proteins, spine density as well as mEPSCs.

Conclusion: In summary, our study underscores the role of microglial NLRP3 as an important contributor to Tat-MDEV mediated synaptodendritic injury. While the role of NLRP3 in inflammation is well-described, its role in EV-mediated neuronal damage is an interesting finding, implicating it as a target for therapeutics in HAND.

Plant exosome-like nanovesicles (PELNVs) are membrane-encapsulated nanostructures released from cells into their surroundings. PELNVs have an important role in intercellular and interspecies communication in all three domains of life. They act as protective compartments for the long-distance transit of signal molecules like proteins, nucleic acids, lipids, and other metabolites. A range of plants and vegetables can emit PELNVs. The importance of PELNVs in interspecies communication stems from their concentration in biomolecules (lipids, proteins, and miRNAs), lack of toxicity, ease of internalization by cells, and anti-inflammatory, immune-modulatory, and regenerative characteristics. PELNVs derived from numerous fruits and vegetables are biocompatible, biodegradable, and abundant in various plant species. Moreover, their convincing physicochemical characteristics underpin their modulative role in physiological and pathological processes, all of which have fueled speculation that these nanovesicles could be particularly adept at developing future-generation bio-therapeutic platforms. The goal of this review was not only to present an overview of the identified roles of PELNVs in physiology and pathology, but also to provide new insight toward their engineering for effective therapeutics and drug delivery nanoplatforms, a clue for future direction to the ongoing research gaps.