Therapeutic biologics such as peptides and proteins hold clinical benefits over conventional medicines in terms of strong specificity, high potency, good tolerability and low toxicity. A breakdown of marketed peptide products indicates that injection dosage forms (61 %) are predominant owing to their poorly absorbable feature [1]. Parenteral injection of therapies, however, accompany with numerous inconveniences, including pain, risk of infection and inferior patient acceptance, particularly when chronic care is needed. Hence, developing a non-invasive administration route is crucial for better delivery of biologics especially for chronic diseases therapy. Despite extensive efforts and nearly a century of studies on their oral delivery, the oral bioavailability of peptide and protein drugs remains low (typically less than 1 %) [2].

Pulmonary delivery of biological agents might be an attractive alternative to parenteral administration for systemic absorption. The unique physiological characteristics of lung, such as a large surface area (about 100 m2), thin alveolar epithelial layer (about 0.1 μm), extensive vasculature, together with low intrinsic enzymatic activity and avoidance of first-pass hepatic metabolism, enable rapid drug absorption thus fast onset of action [3]. However, the lung also presents specific barriers (mucociliary clearance and alveolar macrophage phagocytosis) for therapeutics. In particular, the limited epithelium permeability is the major challenge for systemic delivery of peptide and protein drugs via pulmonary route due to their large molecular size and high polarity [4].

Recent studies focusing on improvement of membrane permeability of inhalable biologics have persistently been performed with great efforts. Of which, absorption enhancers are considered as one of the most straightforward and economic methods for enhancing drug transmucosal permeation with a dose-dependent efficacy and potential toxicity [5]. Even so, few functional excipients have been translated into commercial products, taking into account the balance between drug potency and excipients safety. As a representative, salcaprozate sodium (SNAC) has been approved for enhanced semaglutide oral delivery, while the clinical application of absorption enhancers for pulmonary delivery has not yet been approved by authorities.

Phospholipids are the main constituent of pulmonary surfactants with good biological safety, accounting for approximately 90 % (w/w) of total composition [6]. Previous studies indicated that DPPC-based liposomes or microparticles could effectively enhance systemic absorption of inhalable insulin and parathyroid hormone [7], [8]. However, it is still unknown whether other endogenous phospholipids could also act as absorption enhancers to improve macromolecular absorption in the lung. As amphiphilic surfactant, phospholipids consist of hydrophilic head group and hydrophobic acyl tail chain, and their structural variation in head or tail groups could lead to functional diversity [9], and which structural characteristics of phospholipids could exhibit a more pronounced effect in promoting absorption is a question to be answered. Also, the mechanism underlying the absorption-enhancing action of phospholipids remains unclear.

Therefore, in the present study, Octreotide Acetate (OA), the first somatostatin analogue used for acromegaly and metastatic carcinoid tumors treatment in clinic, was selected as a model peptide drug. Given the higher membrane permeability of saturated phospholipids than unsaturated counterparts [10], herein, endogenous saturated phospholipids with different tail structures (lauroyl, myristoyl, palmitoyl and stearoyl), and head structures (phosphatidylserine, phosphatidylglycerol, phosphatidylcholine and phosphatidylethanolamine) were chosen to explore the influence of structural characteristics of phospholipids on pulmonary mucosal permeability of peptides. In order to make phospholipids suitable for mucosal delivery, an “inhalable reservoir” system consisting of octreotide acetate and phospholipids in the form of blank liposomes was designed, and the absorption-enhancing effect of phospholipids with distinct structures was investigated systematically utilizing 3D Calu-3 Transwell models and SD rats, as well as the possible mechanisms behind the absorption enhancement were further explored.