Exosomes are nanoscale membrane bound vesicles secreted by almost all types of cells. Their unique attributes, such as minimal immunogenicity and compatibility with biological systems, make them novel carriers for drug delivery. These native exosomes harbor proteins, nucleic acids, small molecule compounds, and fluorogenic agents. Moreover, through a combination of chemical and bioengineering methodologies, exosomes are tailored to transport precise therapeutic payloads to designated cells or tissues. In this review, we summarize the strategies for exosome modification and drug loading modalities in engineered exosomes. In addition, we provide an overview of the advances in the use of engineered exosomes for targeted drug delivery. Lastly, we discuss the merits and limitations of chemically engineered versus bioengineered exosome-mediated target therapies. These insights offer additional options for refining engineered exosomes in pharmaceutical development and hold promise for expediting the successful translation of engineered exosomes from the bench to the bedside.

Protein compartments offer definitive structures with a large potential design space that are of particular interest for green chemistry and therapeutic applications. One family of protein compartments, encapsulins, are simple prokaryotic nanocompartments that self-assemble from a single monomer into selectively permeable cages of between 18 and 42 nm. Over the past decade, encapsulins have been developed for a diverse application portfolio utilizing their defined cargo loading mechanisms and repetitive surface display. Although it has been demonstrated that encapsulation of non-native cargo proteins provides protection from protease activity, the thermal effects arising from enclosing cargo within encapsulins remain poorly understood. This study aimed to establish a methodology for loading a reporter protein into thermostable encapsulins to determine the resulting stability change of the cargo. Building on previous in vitro reassembly studies, we first investigated the effectiveness of in vitro reassembly and cargo-loading of two size classes of encapsulins Thermotoga maritima T = 1 and Myxococcus xanthus T = 3, using superfolder Green Fluorescent Protein. We show that the empty T. maritima capsid reassembles with higher yield than the M. xanthus capsid and that in vitro loading promotes the formation of the M. xanthus T = 3 capsid form over the T = 1 form, while overloading with cargo results in malformed T. maritima T = 1 encapsulins. For the stability study, a Förster resonance energy transfer (FRET)-probed industrially relevant enzyme cargo, transketolase, was then loaded into the T. maritima encapsulin. Our results show that site-specific orthogonal FRET labels can reveal changes in thermal unfolding of encapsulated cargo, suggesting that in vitro loading of transketolase into the T. maritima T = 1 encapsulin shell increases the thermal stability of the enzyme. This work supports the move toward fully harnessing structural, spatial, and functional control of in vitro assembled encapsulins with applications in cargo stabilization.

Engineered calcium carbonate (CaCO3) particles are extensively used as drug delivery systems due to their availability, biological compatibility, biodegradability, and cost-effective production. The synthesis procedure of CaCO3 particles, however, suffers from poor reproducibility. Furthermore, reducing the size of CaCO3 particles to <100 nm requires the use of additives in the reaction, which increases the total reaction time. Here we propose on-chip synthesis and loading of nanoscaled CaCO3 particles using microfluidics. After the development and fabrication of a microfluidic device, we optimized the synthesis of CaCO3 NPs by varying different parameters such as flow rates in the microfluidic channels, concentration of reagents, and the reaction time. To prove the versatility of the used synthesis route, we performed single and double loading of CaCO3 NPs with various compounds (Doxorubicin, Cy5 or FITC conjugated with BSA, and DNA) using the same microfluidic device. Further, the on-chip loaded CaCO3 NPs were used as carriers to transfer compounds to model cells. We have developed a microfluidic synthesis method that opens up a new pathway for easy on-chip fabrication of functional nanoparticles for clinical use.

Extracellular vesicles (EVs) have mostly been investigated as carriers of biological therapeutics such as proteins and RNA. Nevertheless, small-molecule drugs of natural or synthetic origin have also been loaded into EVs, resulting in an improvement of their therapeutic properties. A few methods have been employed for EV cargo loading, but poor yield and drastic modifications of vesicles remain unsolved challenges. We tested a different strategy based on temporary pH alteration through incubation of EVs with alkaline sodium carbonate, which resulted in conspicuous exogenous molecule incorporation. In-depth characterization showed that vesicle size, morphology, composition, and uptake were not affected. Our method was more efficient than gold-standard electroporation, particularly for a potential therapeutic toxin: the plant Ribosome Inactivating Protein saporin. The encapsulated saporin resulted protected from degradation, and was efficiently conveyed to receiving cancer cells and triggered cell death. EV-delivered saporin was more cytotoxic compared to the free toxin. This approach allows both the structural preservation of vesicle properties and the transfer of protected cargo in the context of drug delivery.

The use of extracellular vesicles (EVs) for drug delivery is being widely explored by scientists from several research fields. To fully exploit their therapeutic potential, multiple methods for loading EVs have been developed. Although exogenous methods have been extensively utilized, in recent years the endogenous method has gained significant attention. This approach, based on parental cell genetic engineering, is suitable for loading large therapeutic biomolecules such as proteins and nucleic acids. We review the most commonly used EV loading methods and emphasize the inherent advantages of the endogenous method over the others. We also examine the most recent advances and applications of this innovative approach to inform on the diverse therapeutic opportunities that lie ahead in the field of EV-based therapies.

Protein nanocages have emerged as promising candidates for enzyme immobilization and cargo delivery in biotechnology and nanotechnology. Carboxysomes are natural proteinaceous organelles in cyanobacteria and proteobacteria and have exhibited great potential in creating versatile nanocages for a wide range of applications given their intrinsic characteristics of self-assembly, cargo encapsulation, permeability, and modularity. However, how to program intact carboxysome shells with specific docking sites for tunable and efficient cargo loading is a key question in the rational design and engineering of carboxysome-based nanostructures. Here, we generate a range of synthetically engineered nanocages with site-directed cargo loading based on an α-carboxysome shell in conjunction with SpyTag/SpyCatcher and Coiled-coil protein coupling systems. The systematic analysis demonstrates that the cargo-docking sites and capacities of the carboxysome shell-based protein nanocages could be precisely modulated by selecting specific anchoring systems and shell protein domains. Our study provides insights into the encapsulation principles of the α-carboxysome and establishes a solid foundation for the bioengineering and manipulation of nanostructures capable of capturing cargos and molecules with exceptional efficiency and programmability, thereby enabling applications in catalysis, delivery, and medicine.

Plant-derived extracellular vesicles (PDEVs) have shown remarkable potential as sustainable, green, and efficient drug delivery nanocarriers. As natural nanoparticles containing lipids, protein, nucleic acids and secondary metabolites, they have received widespread attention as a replacement for mammalian exosomes in recent years. In this review, the advances in isolation, identification, composition, therapeutic effect, and clinical application prospect were comprehensively reviewed, respectively. In addition, the specific modification strategies have been listed focusing on the inherent drawbacks of the raw PDEVs like low targeting efficiency and poor homogeneity. With emphasis on their biology mechanism in terms of immune regulation, regulating oxidative stress and promoting regeneration in the anti-inflammatory field and application value demonstrated by citing some typical examples, this review about PDEVs would provide a broad and fundamental vision for the in-depth exploration and development of plant-derived extracellular vesicles in the in-vivo anti-inflammation and even other biomedical applications.

Nanoparticles can encapsulate a range of therapeutics, from small molecule drugs to sensitive biologics, to significantly improve their biodistribution and biostability. Whilst the regulatory approval of several of these nanoformulations has proven their translatability, there remain several hurdles to the translation of future nanoformulations, leading to a high rate of candidate nanoformulations failing during the drug development process. One barrier is that the difficulty in tightly controlling nanoscale particle synthesis leads to particle-to-particle heterogeneity, which hinders manufacturing and quality control, and regulatory quality checks. To understand and mitigate this heterogeneity requires advancements in nanoformulation characterisation beyond traditional bulk methods to more precise, single particle techniques. In this review, we compare commercially available single particle techniques, with a particular focus on single particle Raman spectroscopy, to provide a guide to adoption of these methods into development workflows, to ultimately reduce barriers to the translation of future nanoformulations.

Extracellular vesicles (EVs) such as ectosomes and exosomes have gained attention as promising natural carriers for drug delivery. Exosomes, which range from 30 to 100 nm in diameter, possess a lipid bilayer and are secreted by various cells. Due to their high biocompatibility, stability, and low immunogenicity, exosomes are favored as cargo carriers. The lipid bilayer membrane of exosomes also offers protection against cargo degradation, making them a desirable candidate for drug delivery. However, loading cargo into exosomes remains to be a challenge. Despite various strategies such as incubation, electroporation, sonication, extrusion, freeze-thaw cycling, and transfection that have been developed to facilitate cargo loading, inadequate efficiency still persists. This review offers an overview of current cargo delivery strategies using exosomes and summarizes recent approaches for loading small-molecule, nucleic acid, and protein drugs into exosomes. With insights from these studies, we provide ideas for more efficient and effective delivery of drug molecules by using exosomes.

Exosomes are nanovesicles 40-120 nm in diameter secreted by almost all cell types and providing humoral intercellular interactions. Given the natural origin and high biocompatibility, the potential for loading various anticancer molecules and therapeutic nucleic acids inside, and the surface modification possibility for targeted delivery, exosomes are considered to be a promising means of delivery to cell cultures and experimental animal organisms. Milk is a unique natural source of exosomes available in semi-preparative and preparative quantities. Milk exosomes are highly resistant to the harsh conditions of the gastrointestinal tract. In vitro studies have demonstrated that milk exosomes have an affinity to epithelial cells, are digested by cells by endocytosis mechanism, and can be used for oral delivery. With milk exosome membranes containing hydrophilic and hydrophobic components, exosomes can be loaded with hydrophilic and lipophilic drugs. This review covers a number of scalable protocols for isolating and purifying exosomes from human, cow, and horse milk. Additionally, it considers passive and active methods for drug loading into exosomes, as well as methods for modifying and functionalizing the surface of milk exosomes with specific molecules for more efficient and specific delivery to target cells. In addition, the review considers various approaches to visualize exosomes and determine cellular localization and bio-distribution of loaded drug molecules in tissues. In conclusion, we outline new challenges for studying milk exosomes, a new generation of targeted delivery agents.