Dao, H. M. | Search bioRxiv

bioRxiv2022

Aerosolizable plasmid DNA dry powders engineered by thin-film freezing

This study was designed to test the feasibility of using thin-film freezing (TFF) to prepare aerosolizable dry powders of plasmid DNA (pDNA) for pulmonary delivery. Dry powders of pDNA formulated with mannitol/leucine (70/30, w/w) at various of drug loadings, solid contents, and solvents were prepared using TFF, their aerosol properties (i.e., mass median aerodynamic diameter (MMAD) and fine particle fraction (FPF)) determined, and selected powders were used for further characterization. Of the nine dry powders prepared, their MMAD values were about 1-2 mm, with FPF values (delivered) of 40-80%. The aerosol properties of the powders were inversely correlated with the pDNA loading and the solid content in the pDNA solution before thin-film freezing. Powders prepared with Tris-EDTA (TE) buffer or cosolvents (i.e., 1,4 dioxane or t-butanol in water), instead of water, showed slightly reduced aerosol properties. Ultimately, powders prepared with pDNA loading at 5% (w/w), 0.25% of solid content, with or without TE were selected for further characterization due to their overall good aerosol performance. The pDNA powders exhibited a porous matrix, crystalline structure, with a moisture content of <2% (w/w). Agarose gel electrophoresis confirmed the chemical integrity of the pDNA after it was subjected to TFF and after the TFF powder was actuated. A cell transfection study confirmed the activity of the pDNA after it was subjected to TFF. In conclusion, it is feasible to use TFF to produce aerosolizable pDNA dry powder for pulmonary delivery, while preserving the integrity and activity of the pDNA.

pharmacology and toxicology↗

bioRxiv2021

Degradation of lactoferrin caused by droplet atomization process via two fluid nozzle: The detrimental effect of air water interfaces

Biological macromolecules, especially therapeutic proteins, are delicate and highly sensitive to degradation from stresses encountered during the manufacture of dosage forms. Thin-film freeze-drying (TFFD) and spray freeze-drying (SFD) are two processes used to convert liquid forms of protein into dry powders. In the production of inhalable dry powders that contain proteins, these potential stressors fall into three categories based on their occurrence during the primary steps of the process: (1) droplet formation (e.g., the mechanism of droplet formation, including spray atomization), (2) freezing, and (3) frozen water removal (e.g., sublimation). This study compares the droplet formation mechanism used in TFFD and SFD by investigating the effects of spraying on the stability of proteins, using lactoferrin as a model. This study considers various perspectives on the degradation (e.g., conformation) of lactoferrin after subjecting the protein solution to the atomization process using a pneumatic two-fluid nozzle (employed in SFD) or a low-shear drop application through the nozzle. The surface activity of lactoferrin was examined to explore the interfacial adsorption tendency, diffusion, and denaturation process. Subsequently, this study also investigates the secondary and tertiary structure of lactoferrin, the quantification of monomers, oligomers, and ultimately, aggregates. The spraying process affected the tertiary structure more negatively than the tightly woven secondary structure, resulting in a 1.5 nm red shift in peak position corresponding to the Tryptophan (Trp) residues. This conformational change can either (a) be reversed at low concentrations via relaxation or (b) proceed to form irreversible aggregates at higher concentrations. Interestingly, when the sample was allowed to progress into micron-sized aggregates, such a dramatic change was not detected using methods such as size-exclusion chromatography, polyacrylamide gel electrophoresis, and dynamic light scattering at 173{degrees}. A more complete understanding of the heterogeneous protein sample was achieved only through a combination of 173{degrees} and 13{degrees} backward and forward scattering, a combination of derived count rate measurements, and micro-flow imaging (MFI). Finally, compared to the low-shear dripping used in the TFFD process, lactoferrin underwent a relatively fast conformational change upon exposure to the high air-water interface of the two-fluid atomization nozzle used in the SFD process as compared to the low shear dripping used in the TFFD process. The interfacial induced denaturation that occurred during spraying was governed primarily by the size of the atomized droplets, regardless of the duration of exposure to air.

pharmacology and toxicology↗