Liposomes have emerged as highly promising carriers for pulmonary drug delivery, a potential recently highlighted by the U.S. Food and Drug Administration (FDA) approval of Arikayce® in 2018, a liposomal amikacin suspension for inhalation [1], [2]. Their biocompatibility, capacity to encapsulate both hydrophilic and lipophilic drugs, membrane-like structure, tunable size, and ability to enhance retention and enable controlled release highlight their strong pharmacological potential [3], [4]. Additionally, liposomes can penetrate the mucus layer and overcome mucociliary clearance, addressing a major barrier to efficient pulmonary drug delivery and reinforcing their promise as advanced respiratory drug carriers [5], [6]. When combined with the intrinsic advantages of pulmonary administration, namely targeted lung delivery, rapid onset, limited drug degradation, and minimal systemic side effects, liposomal formulations offer significant opportunities for more effective inhaled therapies [7].

Despite these beneficial attributes, aqueous liposomal suspensions are prone to aggregation and fusion over time, which can reduce therapeutic efficacy [8]. To address this limitation, liposomes can be converted into dry formulations, with dry powder inhalers (DPIs) offering a particularly promising approach in the field of inhalation therapy [9]. Furthermore, unlike conventional inhalation devices such as nebulizers, metered-dose inhalers (pMDIs) and soft-mist inhalers (SMIs), DPIs bypass propellant gases and nebulization, thereby reducing the risk of liposomal disruption during delivery while extending shelf-life [10], [11]. Beyond improved stability, DPIs enable precise particle engineering, allowing control over size, surface charge, moisture content, and morphology, which critically influence dispersibility, stability, and deep lung deposition [[12], [13], [14], [15]].

Liposomal dispersions can be converted into dry powders using well-established techniques such as freeze-drying (FD) and spray-drying (SD) [13]. Among these, SD is particularly suited for DPIs production, allowing fine control over particle attributes. Indeed, when carefully optimized, this approach yields powders with consistent aerodynamic properties and enhanced pulmonary deposition [16], [17], whereas FD typically produces an irregular, lyophilized cake with inadequate flowability [18]. Furthermore, from an industrial perspective, SD is also considerably faster, more cost-effective, and highly reproducible [19]. Yet, while FD has been extensively documented for liposomal drying across a wide range of applications [[20], [21], [22]], the use of SD for liposomal powder formulation remains comparatively less investigated, largely due to the additional challenges of preserving liposomal integrity under this atomizing process. These challenges arise from the structural characteristics that make liposomes attractive carriers, most notably their flexible bilayer and aqueous core, which simultaneously make them highly vulnerable to shear, thermal, and dehydration stresses. Such stresses (high inlet temperatures and atomization pressures) can compromise membrane integrity, leading to drug leakage, vesicle fusion, or even collapse [23], [24]. In this context, a suitable carbohydrate matrix is crucial to protect liposomes during drying and ensure efficient aerosolization.

In the pulmonary context, early work by Goldbach et al. [25] showed that multilamellar soybean phosphatidylcholine (SPC) liposomes spray-dried with 10% lactose retained vesicle size and phospholipid stability upon reconstitution, establishing SD as a viable approach for producing stable pulmonary liposomal powders. However, subsequent studies revealed that SD can induce vesicle shrinkage or transitions from uni- to bi-lamellar structures, underscoring the need for protective excipients [26]. In this regard, cyclodextrins, specifically HPβCD, have been shown to stabilize PEGylated liposomal membranes during SD, preventing bilayer disruption and drug leakage [27]. Trehalose similarly enhances product yield while preserving liposome size and drug encapsulation, and, with or without L-leucine, enables the production of stable, re-dispersible lipid-based powders [23], [28]. Furthermore, Meenach et al. demonstrated that PEGylation facilitates the SD of particles, preserving the bilamellar phospholipid structure post-drying while ensuring favorable aerodynamic performance suitable for pulmonary delivery [29].

Taken together, these studies provide important proof that liposomes can be successfully processed by SD when appropriately formulated and protected. However, most investigations have primarily focused on end-point characterization, offering limited insight into the underlying formulation and process relationships required to derive transferable design principles. As a result, the successful development of liposomal DPIs demands not only confirmation of liposomal integrity, but also a systematic understanding of how formulation and process parameters collectively govern vesicle preservation while enabling inhalation-relevant powder properties. This gap has been explicitly highlighted in the literature, as Ingvarsson et al. [30] noted that liposome integrity following SD is frequently assumed rather than systematically investigated, despite being a critical quality attribute for inhalable liposomal products.

To address this, the present study systematically investigates how SD parameters and formulation strategies influence liposome integrity and DPI quality. A Quality by Design approach based on Design of Experiments (DOE) was applied, integrating formulation development with scale-up-oriented processing. Liposomes were prepared using supercritical fluid technology (PGSS), a solvent-free, one-step process that overcomes key limitations of conventional solvent-based methods, thereby supporting industry-relevant manufacturing [31], [32], and subsequently dried via SD. An initial DOE evaluated the influence of drying parameters at three distinct levels and compared two carbohydrate matrices, trehalose and HPβCD, for their stabilizing effects on liposomes. Despite the lack of current approval for pulmonary administration, the restricted excipient landscape for inhalation motivates the investigation of well-characterized carbohydrates to broaden the formulation design space and inform regulatory assessment. Subsequently, a second DOE examined the impact of liposomes variables, including active pharmaceutical ingredients (APIs) hydrophobicity, lipid composition (varying the percentage of PEGylated lipid), and carbohydrate-to-liposome ratio. By incorporating APIs with markedly different physicochemical properties, the robustness and transferability of the formulation-process relationships were specifically assessed. Following the identification of optimal drying conditions, structural stability of the liposomes was evaluated across the industrial processes (PGSS and SD), while in vitro aerodynamic performance was assessed using a Next Generation Impactor (NGI) to determine their suitability for pulmonary delivery.

Overall, this study aimed to develop optimized DPIs containing PEGylated liposomes capable of delivering multiple APIs. Formulations were designed to preserve liposome integrity while achieving physicochemical properties favorable for inhalation and increased deep lung deposition. This strategy enables the co-encapsulation of agents such as inhaled corticosteroids and β2-agonists, which are recommended for asthma and COPD therapy. To demonstrate the robustness of this approach, clinically used APIs, namely salbutamol (SAL), budesonide (BUD), formoterol (FOR), ciclesonide (CIC), and indacaterol (IND), were deliberately selected to span a broad range of physicochemical properties, particularly in terms of hydrophilicity/lipophilicity and molecular structure, thereby enabling assessment of the transferability of the formulation–process relationships across diverse APIs. The inclusion of underexplored combinations such as CIC with the ultra-long-acting β2-agonist IND further underscores the translational potential of these liposomal DPIs for future combination therapies.