Nanoparticle-based delivery systems are attracting interest for vaccine and immunotherapy applications due to their potential to enhance antigen stability, protect bioactive cargo from enzymatic degradation, and modulate immune responses. However, many synthetic nanoparticle platforms have limitations such as immunogenicity, poor biocompatibility, and limited targeting efficiency [1], [2], [3]. In this context, biologically derived nanocarriers, particularly extracellular vesicles (EVs), have gained attention for antigen delivery [4]. Owing to their native membrane composition, EVs can preserve the structural and functional integrity of antigens, facilitate efficient antigen presentation, and interact with immune cells, thereby offering distinct advantages over conventional synthetic nanocarriers [5], [6], [7].

Extracellular vesicles are membrane-bound nanoscale vesicles secreted by almost all cell types and play a fundamental role in intercellular communication by transferring proteins, lipids, and nucleic acids between cells [8], [9], [10]. Based on their biogenesis and size, EVs are generally classified into ectosomes, which are formed through the outward budding mechanism involving the plasma membrane, while exosomes, which are generated through the endosomal pathway and typically range from 30 to 150 nm in diameter [11], [12]. Due to their natural origins, low immunogenicity, prolonged circulation time, and inherent targeting capabilities, exosomes have gained increasing attention as delivery platforms for therapeutic agents, vaccines, and antigens [6], [13], [14]. Despite these advantages, challenges related to scalable production, efficient isolation, and reproducible loading strategies continue to limit their clinical translation [15], [16].

A scalable and reproducible production and isolation process together with a high-yield cell source are required for the use of exosomes as a delivery system [6], [15], [17]. Almost all cell types produce exosomes, but the amount of production varies greatly. In small-scale laboratory production, exosomes are generally harvested from conventional culture flasks or small-volume systems, allowing tight control of culture parameters and relatively straightforward handling. However, these approaches inherently yield limited amounts of exosomes and often require repetitive culturing and labor-intensive isolation steps, which can reduce throughput and reproducibility [18], [19], [20]. In contrast, large-scale production using bioreactors aims to increase yield but presents additional challenges, including maintaining long-term cell viability, minimizing shear stress, and preventing nutrient and oxygen gradients that may alter exosome composition and bioactivity [21]. Moreover, isolation methods that are efficient in small volumes often become less scalable when adapted to industrial-scale, leading to reduced purity or increased costs. Batch-to-batch variability, process standardization, and the need for GMP-compliant, automated systems represent further bottlenecks in large-scale manufacturing of exosomes [20], [22]. Therefore, the transition from small-scale culture systems to bioreactor-based large-scale production requires the selection of an isolation strategy that is not only scalable and reproducible but also compatible with continuous processing and clinical-level production requirements. Moreover, high-volume optimized strategies, for instance, ultrafiltration methods, can prove to be helpful for overcoming limitations created by conventional methods as regards laboratory-scale production.

Various methods are available for exosome isolation, including ultracentrifugation, ultrafiltration, chromatography methods, etc., and the selected isolation method affects the purity, quantity, and physicochemical properties of the exosome to be obtained [23], [24], [25]. While ultracentrifugation remains one of the most commonly used techniques for small-scale studies in the laboratory, it is labor-intensive, time-consuming, and difficult to scale up production. In contrast, ultrafiltration-based approaches, such as cross-flow and tangential flow filtration, hold great advantages for large-scale exosome purification due to their ability to enable continuous processing, reducing the time of isolation and easily integrating into automated and GMP-compliant manufacturing [26], [27]. Importantly, these methods better preserve vesicle integrity and cargo bioactivity, making them particularly suitable for downstream therapeutic and vaccine-related applications. When selecting an isolation method, various factors such as automation, ease of use, speed, efficiency, purity, suitability for large-scale operations, flexibility, and reliability should be considered [13], [16], [25], [28].

Cargoes such as drugs, vaccines, and mRNA can be loaded onto exosomes, which are used as delivery systems, either in vivo or in vitro [24], [29], [30]. The loading that occurs during the biosynthesis of exosomes is called in vivo loading. The cargo is loaded in the donor cell before the exosomes are released into the extracellular space by methods such as transfection, electroporation, and co-incubation [14], [23], [31]. After the isolation of exosomes, loading of the cargo by physical methods such as electroporation, co-incubation, sonication or chemical methods such as transfection is called in vitro loading [6], [32], [33]. Despite their promise, both strategies face notable limitations. In vivo loading offers limited control over the amount and specificity of the cargo, and manipulation of donor cells may alter exosome composition. Meanwhile, in vitro methods can cause membrane disruption, aggregation, or reduced bioactivity, and often yield low or variable loading efficiency. Moreover, uniform cargo distribution, prevention of cargo leakage, and scalability for clinical-grade production remain challenges. The loading efficiency and stability of the cargo into exosomes differ depending on the selected loading method [14], [29], [34].

Hepatocellular carcinoma (HCC) accounts for approximately 90% of primary liver cancers. Chronic viral hepatitis (hepatitis B and hepatitis C viruses) is the primary cause of HCC, but alcohol and non-alcoholic steatohepatitis (NASH) enhance the risk of this malignancy [35], [36]. Current research on HCC focuses on the development of appropriate model systems using carcinoma cell lines derived from differentiated hepatocytes, such as HepG2, Hep3B and Huh 7, which can be used to determine disease mechanisms and develop therapeutic tools [37], [38]. Hepatitis B virus (HBV) is an important pathogenic factor for the occurrence and development of HCC. In order to reduce the incidence and mortality of HBV-associated HCC, prevention and treatment of viral infection are essential. In 1991, the World Health Organization (WHO) recommended the integration of HBV vaccination into national immunization programs [39], [40]. Currently, vaccination of newborns and infants is a key intervention for the prevention of HBV infections because infections that occur during perinatal infancy or early childhood have a higher chance of becoming chronic [41]. In the study by Jesus et al., exosomes were shown to induce a cellular immune response in mice when used as an adjuvant for hepatitis B surface antigens (HBsAg) and to trigger an immunomodulatory effect on the cellular immune response by increasing IFN-γ concentration and accelerate the emergence of IgG antibody production [42].

This study aimed to obtain a large number of exosomes to address the scalability problem and shorten the isolation time for industrial purposes. For this, THP-1 cells were selected as a model and produced in a controlled bioreactor for high-throughput production. Subsequently, the secreted exosomes were obtained and purified by the cross-flow ultrafiltration method. Then, the isolated exosomes were characterized by further analysis including morphology, size, concentration and specific marker determination via standardized techniques in nanoparticle studies. Furthermore, Hepatitis B surface antigen was loaded into these exosomes and investigated in both 2D and 3D HCC models to evaluate their potential as an antigen delivery platform for vaccine-related applications.