Continuous droplet freezing enables stable powder formulation of extracellular vesicles for therapeutic applications

Abstract

Extracellular vesicles (EVs) hold great promise as a novel therapeutic modality. While their use as certain formulations has been actively explored, the development of lyophilized EV formulations is essential for ensuring long-term stability and facilitating the clinical translation. However, conventional lyophilization methods often lead to nanoparticle aggregation upon reconstitution, which compromises formulation quality. Herein, we explored a continuous droplet freezing (CDF) approach using a precision droplet generation system, followed by lyophilization, to prepare spherical powder particles containing bovine milk-derived EVs (mEVs). A formulation composed of 0.01 wt% mEVs and 10% trehalose was processed using this CDF approach, followed by drum-type drying. The CDF-based process produced particles with smoother surfaces, higher sphericity, larger Brunauer-Emmett-Teller surface areas, and a lower angle of repose compared to classical tray-type freeze-drying. Notably, the CDF particles retained an amorphous state for over six months at 4°C, whereas the tray-dried particles exhibited crystallinity. The CDF-dried powders showed excellent flowability and redispersibility, with a more monodisperse size distribution upon reconstitution. Furthermore, cellular uptake of mEVs from the CDF powder was comparable to that of untreated mEVs. These findings demonstrate that CDF followed by drum-type lyophilization is a promising technique for producing stable and functional EV powder formulations.

Introduction

Extracellular vesicles (EVs), naturally occurring nanoscale vesicles secreted by cells, have recently garnered significant attention as promising drug delivery carriers owing to their high biocompatibility and intrinsic cell targeting capabilities [1]. Unlike synthetic nanocarriers such as liposomes, EVs contain membrane proteins and lipids derived from their parent cells, which allow EVs to selectively interact with specific tissues or cell types [2], [3]. These properties render EVs suitable candidates in developing precision therapeutics [4]. In line with this potential, various formulation strategies have been explored for using EVs in injectable, topical, and oral suspension forms [5], [6], [7]. However, the clinical translation of EV-based therapeutics requires robust solutions for long-term storage and transportation, which currently rely on cold-chain logistics (typically at 4 or −80°C) [8]. Most EV formulations are prepared as aqueous nanosuspensions with particle sizes ranging from tens to several hundred nanometers [9]. These suspensions are prone to aggregating and sedimenting, especially during storage or handling, which compromise their physicochemical stability and therapeutic efficacy [10], [11]. To address these challenges, dry powder formulations of EVs have emerged as a promising alternative. Powdered EVs offer improved stability, ease of handling, and potential for room-temperature storage. Moreover, recent advances have highlighted the feasibility of non-invasive delivery routes, such as pulmonary and nasal administration [12], [13], which further expands the applicability of EVs as next-generation drug delivery systems (DDS). These administration routes not only avoid first-pass metabolism but also enable local or systemic delivery via mucosal surfaces, which is particularly suitable for treating respiratory diseases and central nervous system disorders [14], [15]. Although not powder formulations, the utility of nebulizing formulations of EVs for treating infection and neurodegenerative diseases has been demonstrated [16], [17]. Given these considerations, EV powder formulations are expected to play important roles in overcoming the current limitations of EVs to achieve their therapeutic potential.

Freeze-drying (lyophilization) has long been employed as an effective technique for drying heat-sensitive and moisture-rich materials, particularly in the pharmaceutical field [18], [19], [20]. By freezing the material and subsequently applying low pressure to induce sublimation without thermal stress, freeze-drying preserves the structural integrity of delicate biomolecules such as proteins [21]. Tray-type freeze-dryers are widely used in pharmaceutical manufacturing, especially for injectable formulations, owing to their ability to dry active ingredients at low temperatures [22]. However, this conventional approach suffers from several limitations, such as non-uniform temperature distribution, batch-to-batch variability, and scalability issues, which restrict its use to certain formulations [22], [23], [24]. In response to these challenges, spray-freeze-drying (SFD) has attracted increasing attention as a promising particle engineering technique [25]. In this process, a liquid formulation is atomized into fine droplets that are rapidly frozen and subsequently lyophilized. This process minimizes thermal degradation and yields highly spherical particles with narrow size distributions, resulting in increased flowability, redispersibility, as well as porous structures that often enhance dissolution, compared with those produced via tray-type freeze-dryers [24], [26], [27]. SFD is thus particularly appealing for preparing nanoparticle-based formulations, such as biologics and advanced DDS [28], [29]. Nevertheless, SFD experiences some practical limitations, including complex equipment requirements, low throughput, and difficulties in controlling droplet size during atomization [30]. For sensitive materials such as EVs—naturally derived nanoparticles with therapeutic potential—these limitations are further compounded by the need to preserve membrane integrity and biological functionality throughout processing.

Herein, we apply a novel continuous droplet freezing (CDF) approach that uses a precision droplet generation system to produce uniform droplets that are rapidly frozen upon contact with a cryogenic medium. CDF provides gentler processing conditions, precisely controls droplet size, and is scalable compared with atomization-based methods. The frozen droplets are subsequently dried using a rotating drum-type freeze-dryer, which enables uniform sublimation and efficient powder recovery. This integrated process is particularly suitable for EV-based formulations, in which particle morphology and biological activity must be maintained.

In this study, we combined CDF and drum-type lyophilization to prepare spherical powder particles containing bovine milk-derived EVs (mEVs) and evaluated the physicochemical properties and biological performance of the mEVs to establish a robust platform for developing EV powder formulations.

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Tatsuya Fukuta, Masato Miyazaki, Kotone Yoshimura, Rikuto Ihara, Haruhiko Nakamura, Haruka Mogami, Wuxuan Liu, Mayumi Ikeda-Imafuku, Satoshi Kodama, Ko Matsui, Taiki Fujimoto, Kenjirou Higashi, Kazunori Kadota, Continuous droplet freezing enables stable powder formulation of extracellular vesicles for therapeutic applications, Advanced Powder Technology, Volume 37, Issue 2, 2026, 105174, ISSN 0921-8831, https://doi.org/10.1016/j.apt.2026.105174.

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