Differential Uptake of Antisense Oligonucleotides in Mouse Hepatocytes and Macrophages Revealed by Simultaneous Two-Photon Excited Fluorescence and Coherent Raman Imaging

Prabuddha Mukherjee, Edita Aksamitiene, Aneesh Alex, Jindou Shi, Kajari Bera, Chi Zhang, Darold R. Spillman, Marina Marjanovic, Michael Fazio, Punit P. Seth, Kendall Frazier, Steve R. Hood, Stephen A. Boppart

Research output: Contribution to journalArticlepeer-review

Abstract

Antisense oligonucleotides (ASOs), a novel paradigm in modern therapeutics, modulate cellular gene expression by binding to complementary messenger RNA (mRNA) sequences. While advances in ASO medicinal chemistry have greatly improved the efficiency of cellular uptake, selective uptake by specific cell types has been difficult to achieve. For more efficient and selective uptake, ASOs are often conjugated with molecules with high binding affinity for transmembrane receptors. Triantennary N-Acetyl-galactosamine conjugated phosphorothioate ASOs (GalNAc-PS-ASOs) were developed to enhance targeted ASO delivery into liver through the hepatocyte-specific asialoglycoprotein receptor (ASGR). We assessed the kinetics of uptake and subsequent intracellular distribution of AlexaFluor 488 (AF488)-labeled PS-ASOs and GalNAc-PS-ASOs in J774A.1 mouse macrophages and primary mouse or rat hepatocytes using simultaneous coherent anti-Stokes Raman scattering (CARS) and two-photon fluorescence (2PF) imaging. The CARS modality captured the dynamic lipid distributions and overall morphology of the cells; two-photon fluorescence (2PF) measured the time-and dose-dependent localization of ASOs delivered by a modified treatment of suspension cells. Our results show that in macrophages, the uptake rate of PS-ASOs did not significantly differ from that of GalNAc-PS-ASOs. However, in hepatocytes, GalNAc-PS-ASOs exhibited a peripheral uptake distribution compared to a polar uptake distribution observed in macrophages. The peripheral distribution correlated with a significantly larger amount of internalized GalNAc-PS-ASOs compared to the PS-ASOs. This work demonstrates the relevance of multimodal imaging for elucidating the uptake mechanism, accumulation, and fate of different ASOs in liver cells that can be used further in complex in vitro models and liver tissues to evaluate ASO distribution and activity.

Original languageEnglish (US)
Pages (from-to)163-176
Number of pages14
JournalNucleic Acid Therapeutics
Volume32
Issue number3
DOIs
StatePublished - Jun 1 2022

Keywords

  • GalNAc
  • Hepatocytes
  • Macrophages
  • Multimodal imaging
  • PS-ASO

ASJC Scopus subject areas

  • Drug Discovery
  • Genetics
  • Molecular Medicine
  • Molecular Biology
  • Biochemistry

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