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Endoplasmic reticulum stress induced autophagy alters cellular processing of cationic lipid delivered siRNAs

R Chauncey Splichal¹, Christina Chan^1,2,3,4, S Patrick Walton⁵

¹Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, MI, USA.

Drug Delivery and Translational Research

|March 17, 2026

Abstract:

siRNA therapeutics have achieved increased clinical success in the past decade. To achieve a therapeutic effect, siRNAs must enter the cytoplasm of the target cells. Thus, their function typically relies on proper endocytosis and trafficking. Endoplasmic reticulum (ER) stress is associated with diseases that are being studied for treatment with siRNAs (cancer, amyloids, mutations, etc.) and common comorbidities (obesity, smoking, hypertension, etc.). ER stress results in activation of the Unfolded Protein Response (UPR), which initiates changes in cellular function that include disruption to endosomal vesicle trafficking and processing. We used the N-glycosylation inhibitor, tunicamycin, to induce ER stress in HeLa cells during transfection with siRNAs targeting EGFP. Our results showed that ER stress resulted in an increased accumulation of siRNA but a reduction in silencing of the siRNA target (EGFP). Further, we used 3-methyladenine (autophagy inhibitor) and bafilomycin A1 (inhibits endosome maturation) to show that autophagy plays a role in increasing siRNA accumulation. Finally, we showed that the additional accumulated siRNA in ER-stressed cells remained functional, was retained in cells for longer, and prolonged silencing. Our results will inform the design of siRNA delivery vehicles and dosing schedules, by ensuring that disease complications, specifically ER stress/UPR activation, are considered.

Insights

Endoplasmic reticulum (ER) stress increases small interfering RNA (siRNA) accumulation in cells but reduces gene silencing. Autophagy inhibition enhances this siRNA accumulation, suggesting a role for ER stress in siRNA delivery optimization.

Area of Science:

Molecular Biology
Cell Biology
RNA Therapeutics

Background:

Small interfering RNA (siRNA) therapeutics require cellular uptake and trafficking for efficacy.
Endoplasmic reticulum (ER) stress, linked to diseases and comorbidities, activates the Unfolded Protein Response (UPR).
UPR activation disrupts cellular processes, including endosomal vesicle trafficking crucial for siRNA function.

Purpose of the Study:

To investigate the impact of ER stress on siRNA cellular accumulation and gene silencing.
To elucidate the role of autophagy and endosomal maturation in siRNA processing under ER stress.
To determine the functionality and retention of siRNA in ER-stressed cells.

Main Methods:

Tunicamycin was used to induce ER stress in HeLa cells during siRNA transfection targeting EGFP.
Autophagy inhibitor (3-methyladenine) and endosome maturation inhibitor (bafilomycin A1) were employed.
siRNA accumulation, EGFP silencing, siRNA functionality, and retention were analyzed.

Main Results:

ER stress led to increased siRNA accumulation but decreased EGFP target silencing.
Autophagy inhibition further enhanced siRNA accumulation in ER-stressed cells.
Accumulated siRNA in ER-stressed cells remained functional, showed longer retention, and prolonged silencing.

Conclusions:

ER stress impacts siRNA trafficking, increasing cellular accumulation while impairing gene silencing.
Autophagy plays a role in siRNA accumulation, particularly under ER stress conditions.
Understanding ER stress effects is crucial for optimizing siRNA delivery vehicles and dosing strategies.

Read full analysis

Keywords:

Autophagy Disease State ER stress SiRNA delivery

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