The Chinese medicine Panax notoginseng (sanqi) is the dry root and rhizome of the Araliaceae plant Panax notoginseng (Burk.) F.H. Chen. The description of Panax notoginseng is mainly found in “The Compendium of Materia Medica”, “Collected Essentials of Species of Materia Medica”, “Roasting Encyclopedia” and so on. Panax notoginseng total saponins (PNS), as commonly used effective ingredients in clinical practice in China, are the main active ingredients of Panax notoginseng, including notoginsenoside R1 (NGR1), ginsenoside Rg1 (GRg1), ginsenoside Re (GRe), ginsenoside Rb1 (GRb1) and ginsenoside Rd (GRd). It has significant therapeutic effects on body pain, soft tissue injury, fracture, bone and joint injury and limb function recovery (Dragos et al., 2017, Ji et al., 2015). However, its clinical dosage forms are mainly ordinary tablets or injections via oral administration or intravenous injection, which are not ideal routes for the treatment of local tissue injury because of the low drug distribution in the local disease site. In general, transdermal administration can significantly increase the drug concentration in local tissue under the skin of the application site and consequently improve the effectiveness of the treatment of local diseases and reduce the side effects caused by the systemic distribution of the drug (Zhang et al., 2020). However, the large molecular weight, low lipophilicity, and high melting point (eg. GRg1 with a melting point of 194 ∼ 196.5 °C) restrict their transdermal permeation rate. Therefore, it is a challenge to improve the transdermal absorption rate in the design of transdermal drug delivery preparations for PNS.

As a carrier for transdermal drug delivery, transfersomes (TFSs) have been widely and intensively studied. They are composed of phospholipids, other lipid materials, and edge-activating agents (EA). The latter makes them deformable. At the same time, the natural hydration gradient of the skin can drive intact TFSs to enter and permeate the skin (Cevc et al., 2008, Zhang and Michniak-Kohn, 2020, El Maghraby et al., 2008). Furthermore, the cargo (therapeutic agents) in some TFSs can be delivered to deeper parts of the skin (Cevc, 2004, Peralta et al., 2018). In our previous study, we also found that sinomenine hydrochloride-loaded TFSs with mixed monoterpenes used as EA induced much higher steady-state drug concentrations in local tissues under the skin than ordinary liposomes (LPSs). (Zheng et al., 2020). Based on our previous work, we further attempted to make effective extracts of PNS from Chinese medicine sanqi into TFSs to realize transdermal and local tissue drug delivery. The formulation of PNS-TFSs was prepared and optimized in our group, and a pharmacodynamic study showed that compared with PNS-LPSs, PNS-TFSs had a stronger therapeutic effect on acute soft tissue injury in rats, while blank TFSs had no therapeutic effect (Chen et al., 2015).

However, TFSs are colloidal particle dispersion systems and are prone to particle aggregation and sedimentation, phospholipid oxidation, and hydrolysis. As a result, TFSs typically exhibit limited stability during storage, usually for a short duration. Hence, the stability of TFSs urgently needs to be improved. Vacuum freeze-drying technology can effectively improve the stability of liquid pharmaceutical preparations. The freeze-dried preparations have low water content and are prone to be preserved stably for a long time. In addition, the unique loose porous structure of freeze-dried preparations makes them redisperse quickly and restore the features of liquid preparations.

Therefore, to enhance the stability of PNS-TFSs, in this study, a freeze-drying process was developed, the stability of lyophilized PNS-TFSs (PNS-FD-TFSs) was investigated, and the ex vivo skin permeation/deposition and in vivo pharmacokinetics of the reconstituted dispersion of PNS-FD-TFSs were also evaluated. In our study, TFSs were prepared by the thin film hydration-dispersion method after screening the optimal levels of some formulation factors, and the optimal freeze-drying process was determined by investigating factors such as the type and quantity of lyoprotectants and the addition method of lyoprotectants. The stability of PNS-FD-TFSs within 6 months at different temperatures was investigated based on the appearance, reconstitution time, particle size, PDI, zeta potential, and EE of five index saponins as indices. The stability of phospholipids in the vesicles at different temperatures was studied by the thiobarbituric acid reactive substance assay (TBARS) method and ferric thiocyanate (FTC) method. In addition, the in vitro skin permeation/deposition of the drug loaded in PNS-TFSs, PNS-FD-TFSs, and PNS-LPSs was measured by a Franz diffusion cell, and the effect of freeze-drying on the transdermal permeation of TFSs was investigated. Finally, the pharmacokinetic characteristics in skin and blood after transdermal administration of PNS-FD-TFSs were investigated with PNS-LPSs as the control. This study provides an experimental basis for the design and development of PNS transdermal and local tissue-targeted delivery preparations.