Development of a Spray-Dried Formulation of Peptide-DNA Nanoparticles into a Dry Powder for Pulmonary Delivery Using Factorial Design

Abstract

Background: Gene therapy via pulmonary delivery holds the potential to treat various lung pathologies. To date, spray drying has been the most promising method to produce inhalable powders. The present study determined the parameters required to spray dry nanoparticles (NPs) that contain the delivery peptide, termed RALA (N-WEARLARALARALARHLARALARALRACEA-C), complexed with plasmid DNA into a dry powder form designed for inhalation.

Methods: The spray drying process was optimised using full factorial design with 19 randomly ordered experiments based on the combination of four parameters and three centre points per block. Specifically, mannitol concentration, inlet temperature, spray rate, and spray frequency were varied to observe their effects on process yield, moisture content, a median of particle size distribution, Z-average, zeta potential, encapsulation efficiency of DNA NPs, and DNA recovery. The impact of mannitol concentration was also examined on the spray-dried NPs and evaluated via biological functionality in vitro.

Results: The results demonstrated that mannitol concentration was the strongest variable impacting all responses apart from encapsulation efficiency. All measured responses demonstrated a strong dependency on the experimental variables. Furthermore, spray drying with the optimal variables in combination with a low mannitol concentration (1% and 3%, w/v) produced functional RALA/pDNA NPs.

Conclusion: The optimal parameters have been determined to spray dry RALA/pDNA NPs into an dry powder with excellent biological functionality, which have the potential to be used for gene therapy applications via pulmonary delivery.

Keywords: cell-penetrating peptide; dry powder formulation; gene delivery; pulmonary delivery; spray drying.

Fig. 1

One Factor Plot Representing the Influence of (A) Mannitol Concentration on Process Yield, (B) Mannitol Concentration on DNA Recovery

Fig. 2

Contour Plots Presenting a Variation of (A) Median of Particle size Distribution Measured by Laser Light Diffraction Analyzer in Response to Spray Inlet Temperature, Mannitol Concentration and Spray Frequency; (B) Z-average in Response to Inlet Temperature, Mannitol Concentration, and Spray Rate; (C) Zeta Potential in Response to Inlet Temperature, Mannitol Concentration, and Spray Rate

Fig. 3

3D Plot Representing the Effect of the Mannitol Concentration and Inlet Temperature on Relative Moisture Content

Fig. 4

Micrographs of Spray-Dried RALA/pEGFP-N1 NPs as a Function of Mannitol Concentration. Spray-Dried RALA/pEGFP-N1 NPs were Sprinkled onto Sticky Carbon Tape Mounted on SEM Stubs and were Observed using SEM at Magnifications of 1000 × and 4000x

Fig. 5

DSC Thermogram of Spray-Dried RALA/pEGFP-N1 NPs. Spray-Dried RALA/pEGFP-N1 NPs were Sealed into a Hermetic Aluminium Pan, then Analysed using DSC Q50 with a Heat Rate of 5ºC/min from 25 to 200ºC. The Thermal Analysis Data were Analysed using Universal Analysis

Fig. 6

Overlay and fluorescence Image of in vitro Transfection Efficiency of Spray-Dried RALA/pEGFP-N1 NPs at Different Mannitol Concentrations in Opti-MEM Media Assessed in A549 cells. Spray-dried RALA/pEGFP-N1 complexes were reconstituted with 50 µL of DNase/RNase-free water and incubated for 3 h at room temperature prior to A549 cells transfection (density of 10⁴ cells per well) for 4–6 h in Opti-MEM media. Subsequently, cells were observed under an optical microscope after transfection. Experiments were performed as three independent replicates, and a representative image is shown for each cryoprotectant

Fig. 7

A In-vitro Transfection of Spray-Dried RALA/pEGFP-N1 complexes at different mannitol concentrations. Cells were trypsinised 48 h and transfection efficiency was measured by flow cytometry. B A549 cells viability after 48 h transfection with spray-dried RALA/pEGFP-N1 NPs in Opti-MEM. Spray-dried RALA/pEGFP-N1 complexes were reconstituted with 50 µL of DNase/RNase-free water and incubated for 3 h at room temperature prior to A549 cells transfection (density of 1 × 10⁴ cells per well) for 4–6 h in Opti-MEM media. 48 h post-transfection, cells were incubated with 10% Alamar blue reagent for 2–3 h and absorbance was measured in a plate reader. Results displayed as mean ± SEM, n = 3. (** p < 0.01, **** p < 0.0001; ANOVA)