New Techniques to Assess In Vitro Release of siRNA from Nanoscale Polyplexes

Krieg, Bettina; Hirsch, Markus; Scholz, Erik; Nuhn, Lutz; Tabujew, Ilja; Bauer, Horst; Decker, Sandra; Khobta, Andriy; Schmidt, Manfred; Tremel, Wolfgang; Zentel, Rudolf; Peneva, Kalina; Koynov, Kaloian; Mason, A. James; Helm, Mark

doi:10.1007/s11095-014-1589-7

Cited by 18 publications

(16 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Although a dye at the 5′ of the antisense strand increases the IC 50 somewhat, the resulting siRNAs still show IC 50 values of 700 pM, which is excellent activity and clearly validating the FRET-labeled siRNA as biologically active in the entire RNAi pathway. Knockdown was confirmed in different cell lines and with different dye labels ( 38 , 39 ) after 24 h, delineating a time window in which decisive intracellular delivery and distribution events must take place. Several other groups have employed a number of different dye pairs in the cuvette, in fixed cells and in live cell imaging ( 32 , 34 , 40 – 42 ).…”

Section: Introductionmentioning

confidence: 91%

Live cell imaging of duplex siRNA intracellular trafficking

Hirsch

Helm

2015

Nucleic Acids Research

Self Cite

View full text Add to dashboard Cite

Intracellular distribution of siRNA after in vitro transfection typically depends on lipopolyplexes, which must release the siRNA into the cytosol. Here, the fate of siRNAs was monitored by FRET-based live cell imaging. Subsequent to in situ observation of uptake and release processes, this approach allowed the observation of a number of hitherto uncharacterized intracellular distribution and degradation processes, commencing with a burst of endosomal releases, followed, in some cases, by fast siRNA influx into the nucleus. The continued observation of intact siRNA against a background of free fluorophores resulting from advanced degradation was possible by a specifically developed imaging algorithm, which identified populations of intact siRNA in pixels based on FRET. This proved to be essential in the end point definition of siRNA distribution, which typically featured partially degraded siRNA pools in perinuclear structures. Our results depict the initial 4 h as a critical time window, characterized by fast initial burst release into the cytosol, which lay the foundations for subsequent intracellular distribution of siRNA. Combination with a subsequent slower, but sustained release from endosomal reservoirs may contribute to the efficiency and duration of RNAi, and explain the success of lipopolyplexes in RNAi experiments in cell culture.

show abstract

Section: Introductionmentioning

confidence: 91%

Live cell imaging of duplex siRNA intracellular trafficking

Hirsch

Helm

2015

Nucleic Acids Research

Self Cite

View full text Add to dashboard Cite

show abstract

“…Instead of addressing this receptor by nanobodies, we took advantage of the macrophage mannose receptor's intrinsic binding affinity to mannose and developed a densely α‐mannosyl‐functionalized siRNA carrier system for effective targeting of M2 macrophages. To protect the sensitive cargo from nuclease degradation and promote cellular uptake, the polyanionic siRNA has to be loaded into a cationic nanohydrogel particle system via electrostatic interactions . Such systems can be derived from amphiphilic reactive ester block copolymers which self‐assemble into micellar structures.…”

Section: Introductionmentioning

confidence: 99%

α‐Mannosyl‐Functionalized Cationic Nanohydrogel Particles for Targeted Gene Knockdown in Immunosuppressive Macrophages

Leber

Kaps

Yang

et al. 2019

Macromolecular Bioscience

Self Cite

View full text Add to dashboard Cite

Immunosuppressive M2 macrophages govern the immunophathogenic micromilieu in many severe diseases including cancer or fibrosis, thus, their re‐polarization through RNA interference is a promising concept to support combinatorial therapies. For targeted siRNA delivery, however, safe and stable carriers are required that manage cell specific transport to M2 macrophages. Here, siRNA‐loaded cationic nanogels are reported with α‐mannosyl decorated surfaces that target and modify M2 macrophages selectively. Via amphiphilic precursor block copolymers bearing one single α‐mannosyl moiety at their chain end mannosylated cationic nanohydrogel particles (ManNP) were obtained of 20 nm diameter determined by dynamic light scattering and cryogenic electron transmission microscopy. α‐Mannosyl surface modification is confirmed by agglutination with concanavalin A. SiRNA‐loaded ManNP preferentially targets the overexpressed mannose receptor CD206 on M2 macrophages, as shown by in vitro cell uptake studies in M2 polarized primary macrophages. This specificity is confirmed, since ManNP uptake could be reduced by blocking of CD206 with mannan. Effective ManNP‐guided siRNA delivery is confirmed by sequence‐specific gene knockdown of CSF‐1R in M2‐type macrophages exclusively, while the expression levels in M1‐polarized macrophages is not affected. In conclusion, α‐mannosyl‐functionalized ManNPs are promising universal siRNA carriers for targeted immunomodulatory treatment of immunosuppressive macrophages.

show abstract

“…In 2004, Merkle et al [68] applied FCS to study the formation of lipoplexes of a 40 bp oligonucleotide with the cationic lipid, DOTAP. In the last two decades FCS was used extensively to investigate various types of polyplexes [66,[69][70][71][72][73][74][75][76][77][78][79] and lipoplexes. [68,[80][81][82]…”

Section: Monitoring Ncs Formationmentioning

confidence: 99%

Shining Light on Polymeric Drug Nanocarriers with Fluorescence Correlation Spectroscopy

Schmitt

Nuhn

Barz

et al. 2022

Macromol. Rapid Commun.

Self Cite

View full text Add to dashboard Cite

The use of nanoparticles as carriers is an extremely promising way for administration of therapeutic agents, such as drug molecules, proteins, and nucleic acids. Such nanocarriers (NCs) can increase the solubility of hydrophobic compounds, protect their cargo from the environment, and if properly functionalized, deliver it to specific target cells and tissues. Polymer‐based NCs are especially promising, because they offer high degree of versatility and tunability. However, in order to get a full advantage of this therapeutic approach and develop efficient delivery systems, a careful characterization of the NCs is needed. This review highlights the fluorescence correlation spectroscopy (FCS) technique as a powerful and versatile tool for NCs characterization at all stages of the drug delivery process. In particular, FCS can monitor and quantify the size of the NCs and the drug loading efficiency after preparation, the NCs stability and possible interactions with, e.g., plasma proteins in the blood stream and the kinetic of drug release in the cytoplasm of the target cells.

show abstract

New Techniques to Assess In Vitro Release of siRNA from Nanoscale Polyplexes

Cited by 18 publications

References 43 publications

Live cell imaging of duplex siRNA intracellular trafficking

Live cell imaging of duplex siRNA intracellular trafficking

α‐Mannosyl‐Functionalized Cationic Nanohydrogel Particles for Targeted Gene Knockdown in Immunosuppressive Macrophages

Shining Light on Polymeric Drug Nanocarriers with Fluorescence Correlation Spectroscopy

Contact Info

Product

Resources

About