(M1030-10-59) Polymer Nanovectors for Treating Lung Inflammation

Purpose: Uncontrolled inflammation is a key factor in the pathogenesis of various lung diseases such as emphysema, COPD, and ARDS. Current therapies, including corticosteroids and bronchodilators, have significant side effects, limiting their long-term use. Small interfering RNA (siRNA) offers a promising alternative for targeted immunomodulation. However, efficient siRNA delivery faces challenges in cellular uptake and tissue localization.

Methods: This study investigates a series of guanidinium-functionalized polymers (Cn-Guan) designed to explore the effects of amphiphilicity on siRNA complexation and efficiency in vitro and in vivo. Nine polymers with varying side chain lengths (C3, C5, C7) and molecular weights (17 kDa, 30 kDa, 65 kDa) were synthesized, forming polyplexes with siRNA.

Results: Characterization revealed that C7-Guan polymers exhibited the smallest polyplex sizes and the tightest complexation with siRNA. In vitro studies showed that 65 kDa polymers had the highest gene knockdown efficiency, with C3 and C5-Guan achieving ~70% knockdown, while C7-Guan achieved ~30%. In vivo, all polymers demonstrated significant lung accumulation and ~70% TNF-α knockdown with a low dose of 0.14 mg/kg in a murine lung inflammation model. C7-Guan polymers, despite lower in vitro efficiency, performed excellently in vivo, likely due to enhanced stability.

Conclusion: Overall, the Cn-Guan/siRNA polyplexes effectively and safely attenuated LPS-induced pulmonary inflammation, highlighting their potential as therapeutic agents. The exploration of polymer side-chain lengths and their impact on siRNA transfection efficacy provides valuable insights for the design of future siRNA delivery systems. These findings underscore the importance of optimizing polymer characteristics to achieve efficient and targeted gene silencing in the treatment of inflammatory lung diseases.  

Acknowledgements: VR acknowledges support from the National Institutes of Health under EB EB022641. The content is solely the authors’ responsibility and does not necessarily represent the official views of the National Institutes of Health. The authors thank the Light Microscopy Core Facility and the Flow Cytometry Core Facility at the University of Massachusetts, Amherst, for establishing confocal microscopy and flow cytometric assays. The authors thank the Farkas group at the University of Massachusetts Amherst for sharing their RT-PCR instrument.

Schematic of study design. Cn-Guan polymers of varying chain lengths and molecular weights self-assemble with siRNA. Cn-Guan/siRNA knockdown TNF-α gene in inflamed macrophages in vitro and in vivo in inflamed lungs through systemic administration.

Evaluation of uptake and fluorescent reporter gene silencing in. a) Quantification of siRNA uptake in M1-polarized RAW 264.7 cells following treatment with Cn-Guan/AF488-siRNA (25 nM of siRNA) polyplexes in all molecular weights as a function of G/P ratio, as quantified by cytometry. b) Representative flow cytometry histogram for uptake for 65 kDa Cn-Guan/AF488-siRNA. c) Quantification of eGFP knockdown M1-polarized RAW 264.7:eGFP cells following treatment with Cn-Guan/si_GFP (75 nM of siRNA) polyplexes in all molecular weights as a function of G/P ratio, as quantified by cytometry. d) Representative CLSM images for eGFP knockdown in M1-polarized RAW 264.7:eGFP after treatment with Cn-Guan/si_GFP (75 nM of siRNA) with the 65 kDa polymer weight polymers. Cell nuclei stained with DAPI (blue). Scale bar: 50 μm.

Therapeutic efficacy of Cn-Guan/siRNA polyplex-mediated gene knockdown. a) Serum TNF-α measured by ELISA 24 h after polyplexes dosing. b) qRT-PCR for TNF-α mRNA levels. BALB/c mice were systematically injected with the polyplexes at a siRNA dose of 0.14 mg/kg, and the lungs were harvested after 24 h. c) Relative bronchial wall thickness analysis 24 h after systemic administration of polyplexes at a siRNA dose of 0.14 mg/kg. d) Representative hematoxylin and eosin-stained lung histology sections of mice. Yellow arrows indicate bronchial wall thickness (scale bar, 200 μm). e) in vivo determination of hepatic function using ALT analysis of the blood plasma. Error bars represent the mean ± the standard error of the mean (SEM) (n=4, one-way ANOVA and Tukey multiple comparisons, ****p < 0.001, 99.9% CI).