Enhancing Endosomal Escape and Gene Regulation Activity for Spherical Nucleic Acids

Jungsoo Park; Michael Evangelopoulos; Matthew Kuo Vasher; Sergej Kudruk; Namrata Ramani; Vinzenz Mayer; Alexander Carlos Solivan; Andrew Lee; Chad Alexander Mirkin

doi:10.1002/smll.202306902

Enhancing Endosomal Escape and Gene Regulation Activity for Spherical Nucleic Acids

Jungsoo Park, Michael Evangelopoulos, Matthew Kuo Vasher, Sergej Kudruk, Namrata Ramani, Vinzenz Mayer, Alexander Carlos Solivan, Andrew Lee, Chad Alexander Mirkin^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

2 Scopus citations

Abstract

The therapeutic potential of small interfering RNAs (siRNAs) is limited by their poor stability and low cellular uptake. When formulated as spherical nucleic acids (SNAs), siRNAs are resistant to nuclease degradation and enter cells without transfection agents with enhanced activity compared to their linear counterparts; however, the gene silencing activity of SNAs is limited by endosomal entrapment, a problem that impacts many siRNA-based nanoparticle constructs. To increase cytosolic delivery, SNAs are formulated using calcium chloride (CaCl₂) instead of the conventionally used sodium chloride (NaCl). The divalent calcium (Ca²⁺) ions remain associated with the multivalent SNA and have a higher affinity for SNAs compared to their linear counterparts. Importantly, confocal microscopy studies show a 22% decrease in the accumulation of CaCl₂-salted SNAs within the late endosomes compared to NaCl-salted SNAs, indicating increased cytosolic delivery. Consistent with this finding, CaCl₂-salted SNAs comprised of siRNA and antisense DNA all exhibit enhanced gene silencing activity (up to 20-fold), compared to NaCl-salted SNAs regardless of sequence or cell line (U87-MG and SK-OV-3) studied. Moreover, CaCl₂-salted SNA-based forced intercalation probes show improved cytosolic mRNA detection.

Original language	English (US)
Article number	2306902
Journal	Small
Volume	20
Issue number	11
DOIs	https://doi.org/10.1002/smll.202306902
State	Published - Mar 15 2024

Funding

Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under Award Numbers P50CA221747, R01CA257926, and R01CA275430. The content was solely the responsibility of the authors and did not necessarily represent the official views of the National Institutes of Health. This work made use of the IMSERC MS facility at Northwestern University, which had received support from the Soft and Hybrid Nanotechnology Experimental (SHyNE) Resource (NSF ECCS‐2025633), the State of Illinois, and the International Institute for Nanotechnology (IIN). J.P. was supported in part by the Chicago Cancer Baseball Charities and the H Foundation at the Lurie Cancer Center of Northwestern University. M.E. was supported in part by the Dr. John N. Nicholson Fellowship and the Alexander S. Onassis Public Benefit Foundation.

Keywords

PLGA nanoparticles
calcium chloride
gene regulation
siRNAs (small interfering RNAs)
spherical nucleic acids

ASJC Scopus subject areas

General Chemistry
Engineering (miscellaneous)
Biotechnology
General Materials Science
Biomaterials

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10.1002/smll.202306902

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title = "Enhancing Endosomal Escape and Gene Regulation Activity for Spherical Nucleic Acids",

abstract = "The therapeutic potential of small interfering RNAs (siRNAs) is limited by their poor stability and low cellular uptake. When formulated as spherical nucleic acids (SNAs), siRNAs are resistant to nuclease degradation and enter cells without transfection agents with enhanced activity compared to their linear counterparts; however, the gene silencing activity of SNAs is limited by endosomal entrapment, a problem that impacts many siRNA-based nanoparticle constructs. To increase cytosolic delivery, SNAs are formulated using calcium chloride (CaCl2) instead of the conventionally used sodium chloride (NaCl). The divalent calcium (Ca2+) ions remain associated with the multivalent SNA and have a higher affinity for SNAs compared to their linear counterparts. Importantly, confocal microscopy studies show a 22% decrease in the accumulation of CaCl2-salted SNAs within the late endosomes compared to NaCl-salted SNAs, indicating increased cytosolic delivery. Consistent with this finding, CaCl2-salted SNAs comprised of siRNA and antisense DNA all exhibit enhanced gene silencing activity (up to 20-fold), compared to NaCl-salted SNAs regardless of sequence or cell line (U87-MG and SK-OV-3) studied. Moreover, CaCl2-salted SNA-based forced intercalation probes show improved cytosolic mRNA detection.",

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Enhancing Endosomal Escape and Gene Regulation Activity for Spherical Nucleic Acids

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