Evaluating UiO-66 Metal-Organic Framework Nanoparticles as Acid-Sensitive Carriers for Pulmonary Drug Delivery Applications

Abstract

Developing novel drug carriers for pulmonary delivery is necessary to achieve higher efficacy and consistency for treating pulmonary diseases while limiting off-target side effects that occur from alternative routes of administration. Metal-organic frameworks (MOFs) have recently emerged as a class of materials with characteristics well-suited for pulmonary drug delivery, with chemical tunability, high surface area, and pore size, which will allow for efficient loading of therapeutic cargo and deep lung penetration. UiO-66, a zirconium and terephthalic acid-based MOF, has displayed notable chemical and physical stability and potential biocompatibility; however, its feasibility for use as a pulmonary drug delivery vehicle has yet to be examined. Here, we evaluate the use of UiO-66 nanoparticles (NPs) as novel pulmonary drug delivery vehicles and assess the role of missing linker defects in their utility for this application. We determined that missing linker defects result in differences in NP aerodynamics but have minimal effects on the loading of model and therapeutic cargo, cargo release, biocompatibility, or biodistribution. This is a critical result, as it indicates the robust consistency of UiO-66, a critical feature for pulmonary drug delivery, which is plagued by inconsistent dosage because of variable properties. Not only that, but UiO-66 NPs also demonstrate pH-dependent stability, with resistance to degradation in extracellular conditions and breakdown in intracellular environments. Furthermore, the carriers exhibit high biocompatibility and low cytotoxicity in vitro and are well-tolerated in in vivo murine evaluations of orotracheally administered NPs. Following pulmonary delivery, UiO-66 NPs remain localized to the lungs before clearance over the course of seven days. Our results demonstrate the feasibility of using UiO-66 NPs as a novel platform for pulmonary drug delivery through their tunable NP properties, which allow for controlled aerodynamics and internalization-dependent cargo release while displaying remarkable pulmonary biocompatibility.

Keywords: UiO-66; aerosols; defectiveness; metal−organic frameworks; nanoparticles; pulmonary drug delivery.

Figure 1:. SEM images of UiO-66 NPs.

Representative SEM images of the A) 1% defective, B) 8% defective, C) 12% defective, and D) 15% defective UiO-66 NPs. The scale bars in all images are 100 nm.

Figure 2:. Loading of RhB and dex into UiO-66 NPs.

Loading amounts of A) RhB and B) dex cargo for 1%, 8%, 12%, and 15% defective UiO-66 NP samples at varied incubation ratios. Bars represent the mean, and error bars represent standard deviation (n=3).

Figure 3:. RhB Cargo release from UiO-66 NPs.

Percentage of cargo release of RhB from A) 1% defective, B) 8% defective, C) 12% defective, and D) 15% defective UiO-66 NPs over the course of 6 days (144 h). The diamond symbols are for samples incubated in ALF and the circles are for those incubated in PBS. Points represent the mean, and error bars represent standard deviation (n=3).

Figure 4:. Degradation of UiO-66 NPs in ALF and PBS.

Degradation of A) 1%, B) 8%, C) 12%, and D) 15% defective UiO-66 NPs in ALF (diamonds) and in PBS (circles) over time. SEM images of 1% defective UiO-66 NPs in PBS after E) 0 h, F) 8 h, and G) 48 h and in ALF after H) 0 h, I) 8 h, and J) 48 h. Points represent the mean, and error bars represent standard deviation (n=3).

Figure 5:. Aerodynamic characterization of UiO-66 NPs.

A) MMAD of unloaded and loaded UiO-66 NPs with varying defectiveness (1%, 8%, 12%, and 15%). Error bars represent standard deviation (n =3, ** p < 0.05, * p < 0.10, ns not significant as determined via Tukey’s multiple comparisons as part of ANOVA). B) MMAD of unloaded UiO-66 with varying defectiveness after dry powder aerosolization. C) Mass deposition profile for 1% defective UiO-66 NPs from NGI impaction after dry powder aerosolization. D) Mass deposition profile for 15% defective UiO-66 NPs from NGI impaction after dry powder aerosolization. Bars represent the mean, and error bars indicate standard deviation (n = 3), where *p < 0.05 as determined by Tukey’s multiple comparisons as part of a two-way ANOVA.

Figure 6:. Cell Viability of cell lines following UiO-66 NP treatment.

24 h cell viability of A) A549 and B) MH-S cell lines. Solid bars in the 1%, 8%, 12%, and 15% conditions represent treatment with 1 μg/mL of UiO-66 NPs, while patterned bars represent treatment with 50 μg/mL of UiO-66 NPs. Bars represent the mean and error bars represent standard error (n=3). All groups are not statistically significant compared to the Untreated conditions (p>0.05) as determined by Dunnett’s multiple comparisons test as part of a two-way ANOVA.

Figure 7:. MH-S Cellular Uptake of UiO-66 NPs.

Fluorescent imaging of cells treated for 24 h with 50 μg/mL UiO-66 NPs of A) 1% defectiveness or B) 15% defectiveness. C) Histograms of flow cytometric NP uptake analysis of cells treated with 50 μg/mL of UiO-66 NPs of various levels of defectiveness determined using median fluorescence intensity of UiO-66. Treatment conditions are indicated on the plot to the left of the histograms D) Quantitative uptake with flow cytometry at two concentrations of UiO-66 NPs. Bars represent the mean and error bars represent standard error (n=3). *p-value<0.05, **p-value<0.01 as determined by Tukey’s multiple comparisons test as part of a two-way ANOVA.

Figure 8:. Inflammatory Cytokine Production in MH-S cells.

24 h secretion of A) TNF-α and B) IL-6. Solid bars in the 1%, 8%, 12%, and 15% conditions represent treatment with 1 μg/mL of UiO-66 NPs, while patterned bars represent treatment with 50 μg/mL of UiO-66 NPs. Bars represent the mean and error bars represent standard error (n=3). All groups are not statistically significant compared to the Untreated conditions (p>0.05) as determined by Dunnett’s multiple comparisons test as part of a two-way ANOVA.

Figure 9:. BALF alveolar macrophage uptake and activation markers.

A) Histograms of flow cytometric NP uptake analysis of alveolar macrophages determined using median fluorescence intensity of UiO-66. Treatment conditions are indicated on the plot to the left of the histograms B) Quantitative UiO-66 NP uptake by alveolar macrophages using flow cytometry. C) CD86 expression of alveolar macrophages. D) MHCII expression of alveolar macrophages. Lines represent the mean and error bars represent standard error (n=4). *p-value<0.05, **p-value<0.01, ****p-value<0.0001, and n.s. not significant as determined by Tukey’s multiple comparisons test as part of a one-way ANOVA.

Figure 10:. Inflammatory Analysis of BALF.

24 h secretion of A) TNF-α and B) IL-6 inflammatory cytokines in the BALF. C) Percentage of neutrophils of all CD45+ populations in the BALF. Lines represent the mean and error bars represent standard error (n=4). *p-value<0.05, **p-value<0.01, and n.s. not significant compared to the PBS conditions as determined by Dunnett’s multiple comparisons test as part of a one-way ANOVA.

Figure 11:. Histological analysis of lungs from mice treated with UiO-66 NPs.

H&E-stained lung sections of mice treated with A) PBS B) LPS C) 1% UiO-66 NPs D) 15% UiO-66 NPs. The top row represents images of lung sections taken at a 4x magnification, and the bottom row represents images of lung sections taken at a 20x magnification.

Figure 12:. Biodistribution of equivalent mass of UiO-66 in various organs and blood.

The two treatments shown are for 1% defective UiO-66-treated and PBS-treated mice at 1-day and 7-day time points. Error bars represent standard error (n=4). *** p-value < 0.001 via Sidak’s multiple comparisons test.