# Smart Inhalation Therapy: Boosting siRNA Efficacy with Inulin-Based Multifunctional Polymers

## TL;DR

This paper introduces a new inulin-based polymer that improves siRNA delivery to the lungs, helping treat respiratory diseases more effectively.

## Contribution

The study presents a novel inulin-based copolymer for efficient siRNA delivery via inhalation, overcoming lung barriers.

## Key findings

- The polymer forms stable siRNA complexes with a size below 30 nm and shows pH-dependent membrane destabilization.
- Polyplexes remain stable in mucus and surfactant, with high muco-diffusivity and over 80% cell viability in biocompatibility tests.
- siRNA-mediated gene silencing was demonstrated in MDA-MB-231-eGFP cells, showing therapeutic potential for respiratory diseases.

## Abstract

Therapeutic delivery of siRNA via inhalation holds significant
promise for managing severe pulmonary diseases. However, an effective
delivery platform capable of overcoming the lung’s physical
and biological barriers is essential to achieve efficient gene silencing
in the airway epithelium. Here, we describe the synthesis of an inulin
(INU)-based copolymer, INU-VS-g-(PMeOx; bAPAE), designed
for siRNA inhalation. A stepwise synthesis was employed: first, INU
was functionalized with divinyl sulfone to form INU-VS, allowing controlled
conjugation of 1,2-bis­(3-aminopropylamino)­ethane (bAPAE) and poly­(2-methyl-2-oxazoline)
(PMeOx) at, respectively, 25 and 5 mol % on the total INU repeat units.
The resulting copolymer exhibited protonatable amine groups essential
for nucleic acid complexation. Stable formation of siRNA polyplexes
was found at low polymer/siRNA weight ratios (R =
5), with a mean size below 30 nm. Potentiometric titrations confirmed
efficient buffering capacity while fluorescence microscopy demonstrated
pH-dependent membrane destabilization, indicating an enhanced endosomal
escape potential at low pH values. Stability studies in mucus and
pulmonary surfactant revealed that polyplexes remained intact even
at high mucin concentrations (5 mg mL–1) and exhibited
high muco-diffusivity. Biocompatibility assessments on 16-HBE showed
excellent cytocompatibility with over 80% cell viability even at high
polymer concentrations. Uptake studies confirmed polyplex internalization
and siRNA release. Experiments on MDA-MB-231-eGFP cells demonstrated
siRNA-mediated silencing. Overall, together with the excellent aerosol
performance of the polyplex aqueous dispersions, these findings highlight
the potential of INU-VS-g-(PMeOx; bAPAE) as a versatile
and effective siRNA carrier for pulmonary administration, paving the
way for future therapeutic applications in respiratory diseases.