Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible interstitial lung disease with high mortality and poor prognosis, affecting about 3 million people worldwide. Its incidence is higher in men than women, with the highest incidence observed in people aged over 70 years old (Martinez, 2017, Olson, 2018). The median survival time of IPF patients after diagnosis has been reported to be only 2–3 years, and the 5-year survival rate is less than 30 %, which is lower than that of most cancers (Barratt, 2018, Maher, 2021). Currently, only pirfenidone (PFD) and nintedanib (NDB) are recommended for the treatment of pulmonary fibrosis (PF), but their efficacies remain limited. They can only delay the progression of PF but cannot reverse the course of the disease. In addition, large doses of the medications are often needed, leading to serious adverse reactions and hindering the possibility of long-term treatment (Galli, 2017). With the worsening of environmental pollution and the aging society, the rising incidence of PF poses a serious threat to human life and health (Podolanczuk, 2023, Taskar and Coultas, 2006). Moreover, the emergence of complications of PF in severe COVID-19 patients highlights the urgent need for effective prevention and treatment of PF (George et al., 2020). Hence, the search for newer, safer and more efficient therapeutic drugs is of paramount significance.

Cryptotanshinone (CTS) is a traditional Chinese medicine (TCM) that has been clinically used for thousands of years. With its wide range of pharmacological effects, CTS has been shown to have clear efficacy and good safety, making it a promising candidate for the prevention and treatment of various diseases (Wu, 2020, Li, 2021). Previous studies in our laboratory demonstrated promising anti-fibrotic effects of oral CTS in alleviating PF (Zhang, 2019). In addition, CTS possesses distinctive advantages as a natural plant extract, such as low side effects, high safety and reliability compared to conventional chemotherapy drugs, making it suitable for long-term treatment and improving patients’ quality of life (Zhang, 2021). However, its low oral bioavailability and wide biodistribution greatly hinder further applications (Kobryń et al., 2021). Hence, developing efficient drug delivery strategies is critical.

Pulmonary administration offers a direct route for delivering drugs to the lungs, resulting in high local lung bioavailability and reduced risk of systemic adverse reactions, making it particularly suitable for treating local lung diseases. Dry powder inhalant (DPI) is a current research focus of pulmonary drug delivery systems, characterized by ease of use and storage, free from the influence of drug solubility, fewer interference factors, good drug stability and accurate administration dosage (Liang, 2015). Lung clearance of foreign substances is primarily achieved by airway mucociliary clearance and alveolar macrophage phagocytosis, which pose significant challenges to the design of pulmonary drug delivery systems by hindering effective drug delivery by inhalation. Notably, frequent administration may cause many adverse reactions for local lung diseases requiring long-term treatment (Ruge et al., 2013). Therefore, it is of crucial to develop effective delivery strategies that can overcome the clearance ability of the lung defense system, prolong drug action duration, reduce administration frequency, and improve patient compliance and adaptability.

In our previous study, we developed a traditional Chinese medicine combined with sustained pulmonary drug delivery regimen (CTS nanocrystals-loaded CS swellable microparticles, SM(NC)) for the treatment of PF, and obtained encouraging results compared to oral administration of CTS and positive control drug PFD (Wang, 2022). Although conventional pulmonary drug delivery systems may directly deliver drugs to the lungs, efficient drug delivery to lung lesions and target cells cannot be achieved. Another major challenge in pulmonary drug delivery is targeted delivery (Ruge et al., 2013). In general, the degree of targeting can be divided into three levels: the first level involves delivering the drug to the center of the lung or surrounding areas, the right or left lung; the second level refers to accurate drug delivery to lung lesions; and the third level involves specific drug delivery to target cells. The second and third levels of targeting enable the specific delivery of drugs to lung lesions and target cells via rational active targeting strategies, thus achieving more accurate, efficient and safe therapeutic effects. This is particularly important for local lung disease treatment and is likely to be a key area of focus in future research.

Liposomes are similar in composition to the endogenous substances in the lungs with good biocompatibility, and are the only available carrier currently used for sustained pulmonary drug delivery. Liposomes can improve the solubility of drugs, enhance drug absorption, extend drug release, and effectively avoid phagocytosis of macrophages. They can also be easily modified to enable precise therapy, exhibiting great clinical application potential (Sarvepalli, 2022). However, their poor stability, such as oxidation, hydrolysis, rupture, aggregation sedimentation and drug leakage, has greatly limited liposome development as drug carriers (Peng, 2022). Pulmonary myofibroblasts are an important factor in PF pathogenesis and are known to overexpress fibronectin (FN) during the formation of PF, resulting in abnormal deposition of extracellular cell matrix (ECM) (Sava, 2017, Moore and Herzog, 2013). The inhibition of myofibroblast proliferation and activation is an effective treatment strategy for PF. Studies have shown that CREKA peptide can specifically bind to FN to target myofibroblasts (Zhou, 2015). Rui Li et al. designed CREKA-coupled liposomes containing tripterine and confirmed that intravenous injection of prepared liposomes could effectively relieve mouse renal fibrosis induced by unilateral ureteral obstruction by specifically targeting activated renal interstitial muscle fibroblasts and significantly reducing systemic toxicity compared to free drugs (Li, 2020). However, CREKA peptide has not been reported in the treatment of PF. TAT peptide is a positively charged cell-penetrating peptide (CPPs) that can significantly enhance infiltration through the matrix barrier in the focal area of PF and improve the ability of drugs to enter cells (Zong, 2014, Torchilin, 2008). Therefore, based on our previous research, this study constructed a CTS-loaded modified liposome-CS microspheres system on the basis of the advantages of dual carriers to achieve specific targeting to pulmonary myofibroblasts, enhance stability, sustain drug release ability and promote absorption, thereby significantly improving the utilization of drugs and resulting in more accurate, efficient and safe anti-PF treatment.

Exosomes represent a kind of small endogenous vesicles secreted by living cells with lipid bilayer membrane structures that are efficient in accumulating and penetrating highly dense matrix barriers formed by PF to reach target cells (Jeppesen, 2019, El Andaloussi, 2013). In addition, exosomes exhibit specific homing effects on their parent cells, thereby reducing off-target effects in vivo (Vader, 2016, Batrakova and Kim, 2015). They have low immunogenicity and can effectively avoid being phagocytosis by macrophages. However, low drug loading efficiency limits their application as drug delivery carriers (Lu and Huang, 2020). Therefore, based on the above studies of the liposome-CS microspheres system, exosomes were first extracted from normal MRC-5 cells and TGF-β1 activated MRC-5 cells by ultra-high speed centrifugal method. Based on the similarity of membrane composition, liposome-exosome hybrid bionic nanovesicles were prepared by membrane fusion method to combine the advantages of liposomes and exosomes and achieved enhanced drug loading ability and pulmonary myofibroblasts’ specific targeting efficiency (Piffoux, 2018, Zhou, 2020). Then, CS microspheres were used as an effective carrier for pulmonary delivery of the developed hybrid bionic vesicles.

Altogether, we designed two kinds of nano-in-micro particles to integrate the advantages of carriers aiming to enhance the targeting efficiency and sustained-release capability of conventional pulmonary drug delivery systems to achieve more accurate, efficient and safe anti-PF therapy and offer novel strategies for local pulmonary diseases that require long-term treatment.