Investigation of Factors Influencing the Effectiveness of Deformable Nanovesicles for Insulin Nebulization Inhalation.

Nebulized inhalation offers a noninvasive method for delivering drugs to treat both local respiratory and systemic diseases. In this study, insulin was used as a model drug to design a series of deformable nanovesicles (DNVs) with key quality attributes, including particle size, deformability, and drug load capacity. We investigated the effects of these properties on aerosol generation, macrophage phagocytosis, and bloodstream penetration. The results showed that deformability improved nebulization performance and reduced macrophage phagocytosis, benefiting local and systemic delivery. However, the advantage of DNVs for transmembrane penetration was not evident in the alveolar epithelium. Within the size range of 80-490 nm, the smaller the particle size of IPC-DNVs, the easier it is to evade clearance by macrophages and the more effective the in vivo hypoglycemic efficacy will be. In the drug load range of 3-5 mg/mL, a lower drug load resulted in better hypoglycemic efficacy. The area above the blood glucose decline curve with time (AAC) of nebulized DNVs was 2.32 times higher than that of the insulin solution, demonstrating the feasibility and advantages of DNVs in the pulmonary delivery of biomacromolecule drugs. This study provides insights into the construction and formulation optimization of pulmonary delivery carriers.

Deformable Nanovesicle-Loaded Gel for Buccal Insulin Delivery.

Deformable nanovesicles (DNVs) have been widely used in oral mucosal delivery studies of biomolecular drugs. However, their development for oral mucosal preparations has been limited by their physical and chemical instability, the need for small oral volumes, and the complexity of the oral microenvironment. This study aimed to develop a more suitable buccal delivery system for DNVs with improved storage stability. Preliminary stability studies investigated different gel types, the effects of different hydrophilic gel matrices, and matrix temperature sensitivity using DNVs loaded with insulin-phospholipid complex (IPC-DNVs). A temperature-sensitive gel encapsulating IPC-DNVs (IPC-DNV-TSG) prepared with 2% w/v gelatin was stable at 4 °C for three months and maintained an excellent hypoglycemic effect. The delivery efficiency of IPC-DNVs and IPC-DNV-TSG was compared using a TR146 cell model, revealing that cell viability remained high. Cellular uptake was slightly lower for IPC-DNV-TSG than for IPC-DNVs, but total transport did not differ significantly between the two groups, which may have been related to the viscosity of IPC-DNV-TSG and the hydrophilicity, cell adhesion properties, and biocompatibility of gelatin. Moreover, neither IPC-DNVs nor IPC-DNV-TSG induced significant mucosal irritation in rabbit tongue tissue sections. The study findings demonstrate a promising method for possible use as oral mucosal delivery of peptide drugs.

Factors affecting the buccal delivery of deformable nanovesicles based on insulin-phospholipid complex: an in vivo investigation.

Deformable nanovesicles (DNVs) have been used in the buccal delivery of biomacromolecules due to their ability to enhance drug penetration. However, no breakthroughs have been made until now due to limited understanding of the factors affecting in vivo buccal delivery. In this study, we designed a series of DNVs, based on an insulin-phospholipid complex (IPC-DNVs), to investigate the influence of drug dose, buccal administration methods, and key quality characteristics of IPC-DNVs for buccal delivery. IPC-DNVs showed a non-linear dose-response relationship between 8 and 12 IU. There was no significant effect of drug delivery site (sublingual mucosa/buccal mucosa) or ligation time (15 or 30 min) on buccal absorption of IPC-DNVs. However, the area above the curve of reduction in blood glucose level overtime (AAC0-6h) for oral mucosa administration was significantly higher than that for buccal mucosa administration. Increasing the drug concentration in IPC-DNVs led to a decrease in AAC0-6h. This might be due to local leakage of DNVs, while squeezing through biological barriers with high concentration of insulin, thus hindering the subsequent delivery of DNVs. IPC-DNVs, measuring 80-220 nm in size, did not significantly affect AAC0-6h. However, when the size was increased to approximately 400 nm, AAC0-6h decreased, thus suggesting that IPC-DNVs with reasonable size were more effective. Additionally, increased deformability of IPC-DNVs might cause drugs to leak easily, thus reducing the promoting effect of buccal absorption. Our results clarified the effect of characteristics of IPC-DNVs on buccal delivery in vivo and provided meaningful support for the design of dosage form of DNVs.

Cell-penetrating peptide enhanced insulin buccal absorption.

Non-injectable delivery of peptides and proteins is not feasible due to the limitations of large molecular mass, high hydrophilic properties, and gastrointestinal degradation. Therefore, proposing a new method to solve this problem is a burning issue. The objective of this study was to propose a novel protein delivery strategy to overcome the poor efficacy and irritation of buccal insulin delivery. In this study, we applied a conjugate of cell-penetrating peptides (LMWP) and insulin (INS-PEG-LMWP) for buccal delivery. INS-PEG-LMWP was prepared using insulin solution and mixture as references. The transport behaviour, in vivo bioactivity, hypoglycaemic effect, and safety of INS-PEG-LMWP were systematically characterised. An in vitro study demonstrated that the uptake and transportation of INS-PEG-LMWP across buccal mucosal multilayers significantly increased. By comparing the effects of different endocytic inhibitors on INS-PEG-LMWP uptake, the conjugate might be delivered via an energy independent, electrostatically adsorbed pathway. INS-PEG-LMWP's relative pharmacological bioavailability was high and its relative bioavailability was up to 26.86%, demonstrating no visible mucosal irritation. Cell-penetrating peptides are likely to become a reliable and safe tool for overcoming insulin's low permeability through the epithelial multilayers, the major barrier to buccal delivery.

Stabilization of Deformable Nanovesicles Based on Insulin-Phospholipid Complex by Freeze-Drying.

Deformable nanovesicles have been extensively investigated due to their excellent ability to penetrate biological barriers. However, suffering from serious physical and chemical instabilities, the wide use of deformable nanovesicles in medical applications is still limited. Moreover, far less work has been done to pursue the lyophilization of deformable nanovesicles. Here, we aimed to obtain stable deformable nanovesicles via freeze-drying technology and to uncover the underlying protection mechanisms. Firstly, the density of nanovesicles before freeze-drying, the effect of different kinds of cryoprotectants, and the types of different reconstituted solvents after lyophilization were investigated in detail to obtain stable deformable nanovesicles based on insulin-phospholipid complex (IPC-DNVs). To further investigate the underlying protection mechanisms, we performed a variety of analyses. We found that deformable nanovesicles at a low density containing 8% lactose and trehalose in a ratio of 1:4 (8%-L-T) have a spherical shape, smooth surface morphology in the lyophilized state, a whorl-like structure, high entrapment efficiency, and deformability after reconstitution. Importantly, the integrity of IPC, as well as the secondary structure of insulin, were well protected. Accelerated stability studies demonstrated that 8%-L-T remained highly stable during storage for 6 months at 25 °C. Based on in vivo results, lyophilized IPC-DNVs retained their bioactivity and had good efficacy. Given the convenience of preparation and long term stability, the use of combined cryoprotectants in a proper ratio to protect stable nanovesicles indicates strong potential for industrial production.