Spray drying siRNA-lipid nanoparticles for dry powder pulmonary delivery

Abstract

While all the siRNA drugs on the market target the liver, the lungs offer a variety of currently undruggable targets which could potentially be treated with RNA therapeutics. Hence, local, pulmonary delivery of RNA nanoparticles could finally enable delivery beyond the liver. The administration of RNA drugs via dry powder inhalers offers many advantages related to physical, chemical and microbial stability of RNA and nanosuspensions. The present study was therefore designed to test the feasibility of engineering spray dried lipid nanoparticle (LNP) powders. Spray drying was performed using 5% lactose solution (m/V), and the targets were set to obtain nanoparticle sizes after redispersion of spray-dried powders around 150 nm, a residual moisture level below 5%, and RNA loss below 15% at maintained RNA bioactivity. The LNPs consisted of an ionizable cationic lipid which is a sulfur-containing analog of DLin-MC3-DMA, a helper lipid, cholesterol, and PEG-DMG encapsulating siRNA. Prior to the spray drying, the latter process was simulated with a novel dual emission fluorescence spectroscopy method to preselect the highest possible drying temperature and excipient solution maintaining LNP integrity and stability. Through characterization of physicochemical and aerodynamic properties of the spray dried powders, administration criteria for delivery to the lower respiratory tract were fulfilled. Spray dried LNPs penetrated the lung mucus layer and maintained bioactivity for >90% protein downregulation with a confirmed safety profile in a lung adenocarcinoma cell line. Additionally, the spray dried LNPs successfully achieved up to 50% gene silencing of the house keeping gene GAPDH in ex vivo human precision-cut lung slices at without increasing cytokine levels. This study verifies the successful spray drying procedure of LNP-siRNA systems maintaining their integrity and mediating strong gene silencing efficiency on mRNA and protein levels both in vitro and ex vivo. The successful spray drying procedure of LNP-siRNA formulations in 5% lactose solution creates a novel siRNA-based therapy option to target respiratory diseases such as lung cancer, asthma, COPD, cystic fibrosis and viral infections.

Keywords: Formulation screening; Human precision-cut lung slices; LNP; Lipid nanoparticles; Onpattro®; Pulmonary delivery; RNA therapeutics; Respiratory diseases; Spray drying; siRNA delivery.

Graphical Abstract

Figure 1. Box plots of the Δ R values observed by each LNP in different buffers (as indicated by colored legends) and hold temperatures).

Figure 2. Stability plot shows stability of LNPs in each excipient buffer condition.

The ΔR is plotted against initial ratio in each buffer condition, with the color indicating LNP and the size of marker stating the temperature the LNPs were stressed at. The top left quadrants represents the most stable environments.

Figure 3. Quantification of the losses of A) siRNA and B) cholesterol after spray drying of LNPs in 5% lactose solution (m/V) at an outlet temperatures of 62°C. A nLNP loss at outlet temperature of 72°C is shown in B). Each bar shown as mean ± standard deviation, n=3. SD stands for spray-dried.

Figure 4

A) DLS measurements of freshly prepared (full colored bars) and redispersed (shaded colored bars) LNPs. PDI is indicated by black squares. LNP formulations with neutral, positive or negative charge and spray dried (SD) 5% lactose were redispersed in HPW after spray drying at 62°C outlet temperature and compared to freshly prepared LNPs in 5% lactose solution. B) Zeta potential measurements of fresh and spray dried LNPs in 5% lactose solution via LDA. Mean ± standard deviation, n=3.

Figure 5. DSC measurements of lactose formulations spray dried at an outlet temperature of 62°C: 1.3) SD (-)LNP (brown), 2.3) SD 5% lactose (grey), 3.3) SD (+)LNP (green), 4.3) SD nLNP (blue).

Figure 6. SEM pictures of spray dried A) 5% lactose solution, B) nLNP formulation, C) (+)LNP formulation and D) (-)LNP formulation. All samples were spray dried in 5% lactose solution at 62°C outlet temperature.

Figure 7. Mucus penetration assay of fresh LNPs vs spray dried (SD) LNPs. The time points were chosen at 0h, 0.5h, 1h, 2h, 4h and 24h.

Figure 8. In vitro gene silencing effect of enhanced green fluorescent protein (eGFP) within a H1299-eGFP expressing cell line.

Different siRNA concentrations were tested: A) 1 μg/mL, B) 10 μg/mL. Samples are plotted as follow: freshly prepared LNPs in full colored bars, spray dried LNPs in shaded colored bars. Bars show the mean fluorescent intensities (MFI) of the eGFP as a percentage relative to the untreated sample (grey, unpatterned bar).

Figure 9. In vitro cytotoxicity evaluation via a MTT assay in H1299-eGFP cells.

The siRNA concentrations were set at 1 μg/mL and 10 ug/mL for all samples. Samples are plotted as follow: freshly prepared LNPs in full colored bars, spray dried LNPs in shaded colored bars.

Figure 10. Ex vivo knockdown of house-keeping gene GAPDH.

Human precision cut lung slices (hPCLS) were transfected at 10 μg siRNA / mL with spray dried LNPs encapsulating either siGAPDH or siGFP. All values were expressed as a percentage in comparison to the baseline values of samples treated with LNP-siGFP. Mean ± standard deviation, n=3.