Encapsulation and assessment of therapeutic cargo in engineered exosomes: a systematic review

Evaluation of drug loading into exosomes focuses on several aspects, including drug release efficiency, drug stability, drug delivery efficiency, cellular uptake efficiency, therapeutic efficacy, and biosafety evaluation. For Drug release efficiency, it refers to the percentage of drugs released from exosome and indicates the loading and controlled release capabilities. Methods to quantify drug release efficiency include UV–vis spectrophotometry, high performance liquid chromatography (HPLC), fluorescence staining, Tinopal staining, magnetic resonance imaging (MRI), and filtration. Chen et al. developed a graphene oxide-based nanocomposite coated with chitooligosaccharides and polyglutamic acid (GO-CO-PGA) as an exosome-based drug delivery system. Using UV–vis spectrophotometry, they confirmed sustained drug loading efficacy by 73% [71]. Similarly, Sun et al. loaded curcumin into exosomes and examined its efficacy using HPLC [72]. Değirmenci et al. electroporated exosomes derived from normal epithelial breast cells with lapatinib and verified the drug loading efficacy using HPLC, they found that compared to free drug, the lapatinib-loaded exosomes showed higher anti-proliferative effects and enhanced apoptosis induction ability in breast cancer cells [73].

Drug stability describes the ability of a loaded drug to maintain its molecular structure, activity, physicochemical properties, and biological function under certain conditions without being influenced by external factors over time. Methods to assess drug stability include high performance liquid chromatography-mass spectrometry (HPLC–MS), flow cytometry, electron microscopy, and molecular dynamic simulations. Sharma et al. used an immunoinformatic approach to design a multi-epitope tuberculosis vaccine from immunogenic exosomal proteins, and they demonstrated that the vaccine elicited cellular and humoral immunity and provided broad population coverage by compensating for genetic variations. In particular, molecular dynamics simulations were used to model, refine, and dock the vaccine structure to the TLR4 immune receptor [74].

The efficiency of drug delivery is gauged by the quantity of drug that exosomes successfully transport to target cells within a specified timeframe, which is influenced by several factors, including the properties of the exosome suspension, the concentration of the drug, its stability, and the target organ. Various methods are employed to assess drug delivery efficiency, such as fluorescent labeling, biofluorescence imaging, fractionation, transmission electron microscopy (TEM), and scanning electron microscopy (SEM). In a study aimed at promoting cartilage regeneration, Lee et al. utilized the freeze–thaw cycle method to load exosomes with miR-140 [75]. They found that these miR-140-loaded exosomes induced membrane fusion and released miRNA into the cytoplasm. Furthermore, TEM was used to confirm the morphology of the exosomes and the delivery efficiency of miR-140. On a related note, Zhu et al. engineered tumor-exocytosis exosome/AIE luminogenic hybrid nanovesicles for use in photodynamic therapy [76]. They used electroporation to load the photosensitizer DCPy into exosomes. The quantity of drugs packaged into the exosomes was then confirmed using fluorescent labeling.

Cellular uptake efficiency defines the efficacy of exosome drug absorption and utilization by target cells after uptake. It depends on various factors, such as drug properties, exosome loading capacity, exosome/cell membrane permeability, receptor affinity, and metabolic capacity. To assess cellular uptake efficiency, different methods can be used, such as fluorescent/radioisotope labeling, confocal microscopy, and cytological staining. For example, Zhu et al. developed an intraocular lens (IOL) surface modified with exosomes derived from lens epithelial cells (LECs) and loaded with doxorubicin (Dox), an anti-proliferative drug [77]. They used confocal microscopy to demonstrate that the exosome-functionalized IOL enhanced the cellular uptake of Dox by LECs due to the homologous targeting feature of exosome, which resulted in superior anti-proliferation effect and effective posterior capsular opacification (PCO) prevention. A recent development in the evaluation of exosome cellular uptake efficiency is the creation of fluorine-engineered exosomes (exo@FPG3), which are generated through surface engineering of exosomes with fluorinated peptide dendrimers (FPG3), and they are proved to enhance the intracellular delivery and biological activity of exosomes [78]. The study found that the intracellular uptake of exo@FPG3 was energy-dependent and clathrin-mediated endocytosis was a key pathway for exo@FPG3 to enter HUVECs. Moreover, Yang et al. investigated the cellular uptake efficiency of curcumae rhizoma exosome-like nanoparticles (CELNs) loaded with astragalus components (AC) in Caco-2 cell models, and they found that AC-CELNs exhibited superior uptake and transmembrane transport capacity compared to free AC [79].

Therapeutic efficacy assessment involves laboratory assessment of disease status in experimental subjects treated with drug-loaded exosomes to determine if the expected therapeutic outcomes are achieved, it is crucial for establishing the scientific merit, rationale, and safety of a treatment regimen. Better therapeutic efficacy indicates more potent disease treatment by the drug-loaded exosomes. Methods for evaluating therapeutic efficacy include cell viability assays, molecular profiling, animal studies, and clinical trials. Since MSCs have low retention and survival in infarcted hearts, Huang et al. investigated whether exosomes derived from MSCs could enhance treatment of acute myocardial infarction. They found that intramyocardial delivery of exosomes followed by intravenous MSC infusion significantly improved cardiac function, reduced infarct size, and increased neovascularization compared to exosome alone or MSC monotherapy [80]. Xie et al. presented a novel technique for label-free detection of cellular HER2 using machine learning-driven SERS, and they applied their method to dynamically monitor the therapeutic efficacy of drug-loaded exosomes targeting HER2+ breast cancer cells [81]. They showed that their method could capture the variations in HER2 expression and cell viability during the treatment, which could facilitate the therapeutic decision-making and management of breast cancer. Sana et al. investigated the use of exosomes as a natural delivery platform for bleomycin, where they prepared exosomes loaded with bleomycin (Exo-BLM) from cancer cells and tested their effects on tumor cells in vitro and in vivo [82]. Their findings showed that Exo-BLM had high cancer targeting ability and enhanced antitumor activity, and reduced toxicity compared to free bleomycin in a mouse model. Similarly, using a non-invasive liquid-biopsy-based assay, Ting et al. developed a novel approach for assessing the therapeutic efficacy of neoadjuvant chemotherapy (NACT) in patients with advanced gastric cancer (AGC), which was demonstrated to potentially facilitate precision treatment of NACT for patients with AGC [83].

Since exosomes are recognized as promising drug delivery vehicles regarding their ability to traverse biological barriers, a comprehensive biosafety evaluation is imperative prior to clinical translation. This rigorous assessment should encompass several areas: (1) In vitro cytotoxicity and functional assays can determine potential toxicity and adverse effects of exosomes on cells; (2) animal models enable evaluation of in vivo systemic toxicity, immunogenicity, and other safety risks; (3) biodistribution studies are critical for tracking exosome accumulation and clearance kinetics in organs and tissues following administration; (4) pharmacokinetic and pharmacodynamic analyses elucidate exosome stability, metabolism, elimination, and delivery of drug cargo to target sites; and (5) drug interaction studies reveal impacts of exosomes on the safety and efficacy of concomitant medications. For example, Kim et al. evaluated exosomes loaded with the chemotherapy drug paclitaxel and demonstrated that the drug-loaded exosomes are free of cytotoxicity or immunogenicity, but can accumulate in and deliver drug to tumor sites [84]. Jiang et al. loaded exosomes engineered to express TRAIL with the drug triptolide as targeted melanoma therapy. In vitro, the TRAIL-expressing exosomes enhanced tumor cell uptake, inhibited cancer cell proliferation, invasion, and migration, and induced apoptosis. In vivo, the TRAIL-expressing exosomes suppressed tumor progression and reduced triptolide toxicity with ideal biosafety [85].

TEM enables direct visualization of exosomes such as their morphology, size distribution, cargo abundance, and other physical characteristics. Zhu et al. imaged triple-negative breast cancer cell-derived exosomes by TEM and found proteins involved in extracellular matrix interactions and metastasis [86]. Also, Yu et al. visualized milk-derived and drug-loaded exosomes by TEM, they showed superior osteogenic potential of exosomes encapsulating icariin both in vitro and in vivo [87]. Liu et al. engineered MRI-trackable exosomes by expressing a ferritin-lactadherin fusion protein in parent cells, they demonstrated that the genetic modification had no effect on exosome morphology using TEM, and further MR imaging enabled in vitro tracking and in vivo monitoring of exosomes [88].

To detect drugs encapsulated in exosomes, researchers commonly employ UV–vis spectroscopy. Tanziela et al. (2022) reported a novel drug delivery platform based on exosomes isolated from glioblastoma cells (U87), where they detected the UV–vis absorption and fluorescence spectra from prepared solution of AgNCs [89]. Additionally, Zheng et al. confirmed the assembly of gold nanorods and aptamers on exosomes for targeted cancer photothermal therapy using UV–vis spectroscopy, among which the UV–vis characterization enabled investigation of the photothermal properties of modified exosome [90].

Protein quantification method can rapidly detect the total protein content loaded in exosomes. Although these methods do not provide information on specific protein cargoes, they allow quick measurement of the overall protein loading. By quantifying total protein, these methods enable fast detection of the presence of protein-based drugs in exosomes. Haney et al. studied exosome delivery of the antioxidant enzyme catalase as a potential therapy for Parkinson’s disease. They first isolated exosomes from macrophages using differential centrifugation. To load the exosomes with catalase, they tested passive incubation, freeze–thaw, sonication, and extrusion methods. Western blot analysis showed that sonication and extrusion loading resulted in higher levels of catalase protein in the exosomes compared to passive incubation [91].

Prior to loading into exosomes, the drug can be fluorescently labeled, then fluorescence microscopy was employed to visualize the intensity within exosomes and further determine the successful loading and quantify/visualize cargo distribution. This method is applicable to fluorescently taggable drugs but costly, and it can also visualize interactions between labeled exosomes and cells to elucidate delivery mechanisms. For example, He et al. used fluorescence microscopy to visualize the uptake of PKH26-labeled exosomes by chondrocytes in vitro [69], they found that the injected labeled exosomes accumulated in the joint and attenuated cartilage damage and pain versus controls, and these exosomes could increase collagen II synthesis and improve pain thresholds over 6 weeks.

Stability testing evaluates exosome stability and drug release kinetics by measuring the drug release over time, thereby this method can confirm the drug loading, stability, and guides storage recommendations for drug-loaded exosomes. In area of breast cancer treatment, Moumita et al. performed stability testing of docetaxel-loaded exosomes by measuring the release kinetics, particle size, zeta-potential, and encapsulation efficiency. As a result, they demonstrated promising anticancer efficacy of docetaxel-loaded exosomes against 4T1 breast cancer cells [92]. Other studies found exosomes protected cargoes from enzymatic degradation in blood and from environmental stressors like high temperature and low pH [93,94,95,96]. Furthermore, exosomes were found to have the ability in improving drug pharmacokinetics through increased circulation half-life, and these exosomes could be maintained for 24 h at 37 °C (Table 3 and Fig. 3)[97].

Laboratory detection and evaluation of engineered exosomes. After harvesting drug-loaded exosomes, the total protein is first quantified to determine exosome yield. Next, UV absorbance of the exosome suspension is measured to quantify drug loading efficacy. The engineered exosomes are then fluorescently labeled, enabling evaluation of their biodistribution and drug release capabilities in vitro and in vivo. Finally, storage conditions are optimized by studying the engineered exosomes after freezing for various periods

Cell experiments are fundamental for validating exosome drug loading capacity and assessing exosome pharmacological activity (Fig. 4). Methodology, drug-loaded exosomes are added to cultured cells, then the changes in drug levels, cell viability, function, and other endpoints are quantified to evaluate the drug delivery efficacy, which enables the investigation of drug affinity, pharmacology, and mechanisms. For instance, drug-loaded exosomes had shown promise for cancer therapy, and available studies had demonstrated that they could modulate cell viability, apoptosis, migration, proliferation, invasion, angiogenesis, metastasis, and drug resistance across cancer types [98,99,100,101,102]. However, as factors like cell type, density, timing, and detection methods can influence the evaluation results, standardizing assays improves reliability are urgently needed. For instance, immunomodulatory exosomes, such as those loaded with STING agonists or IL-12, can activate anti-tumor immunity and are currently in clinical trial [103], while exosomes delivering KRAS-G12D siRNA can also silence this oncogenic mutation and reduce mutant cancer cell growth []. Hopefully, exosomes loaded with paclitaxel or Dox were demonstrated to overcome drug resistance and improve cytotoxicity compared to free drugs.

Cellular experiments and animal models for testing engineered exosomes. In vitro cell studies assess exosome-mediated drug delivery by quantifying drug abundance within cells, measuring changes in cell viability or functionality after exosome treatment. In vivo animal studies establish disease models in rodents or other organisms to test exosome biodistribution and therapeutic efficacy

Animal experiments are crucial for evaluating drug-loaded exosomes before human testing, as preclinical studies enable assessment of pharmacokinetics, dynamics, and toxicity as required by regulations. Animal models, such as mice and rats, possess similar physiologies to humans and are generally cost-effective and easily managed. Usually, researchers administer drug-loaded exosomes into these testing animals, then they monitor the biodistribution of the drug-loaded exosomes and observe the effects on the animals, including physiological and behavioral changes as well as histological effects [104,105,106,107]. The use of small animal models thus allows for controlled evaluation of drug-loaded exosomes before advancing to human testing. For instance, exosomes loaded with chemotherapy drugs inhibited tumor growth in colon and glioblastoma cancer models, and those exosomes were found in tumors with enhanced efficacy over free drug [108, 109]. Moreover, using cardiovascular disease models, Cheng et al. revealed accumulation of angiogenic drug-loaded exosomes in heart tissue, which was demonstrated to promote vessel growth [110].