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Published on: February 27, 2019
Folate receptor-directed orthogonal click-functionalization of siRNA lipopolyplexes for tumor cell killing in vivo
Philipp Michael Klein1, Sarah Kern1, Dian-Jang Lee1
1Ludwig-Maximilians-University (LMU) Munich, Department of Pharmacy, Butenandtstr. 5-13, 81377 Munich, Germany.
Abstract:
The delivery of small interfering RNA (siRNA) and its therapeutic usage as an anti-cancer agent requires a carrier system for selective internalization into the cytosol of tumor cells. We prepared folate-bearing formulations by first complexing siRNA with the novel azido-functionalized sequence-defined cationizable lipo-oligomer 1106 (containing two cholanic acids attached to an oligoaminoamide backbone in T-shape configuration) into spherical, ∼100-200 nm sized lipopolyplexes, followed by surface-functionalization with various folate-conjugated DBCO-PEG agents. Both the lipo-oligomer and the different defined shielding and targeting agents with mono- and bis-DBCO and varying PEG length were generated by solid phase supported synthesis. A bivalent DBCO surface agent with a PEG24 spacer was identified as the optimal formulation in terms of nanoparticle size, folate receptor (FR) targeting, cellular uptake and gene silencing in vitro. Notably, near-infrared fluorescence bioimaging studies showed that double-click incorporation of bivalent DBCO-PEG24 resulted in siRNA/1106/DBCO2-ss2-PEG24-FolA lipopolyplexes with extended biodistribution and intratumoral delivery in a subcutaneous FR-positive leukemia mouse model. Intravenous administration of analogous therapeutic siRNA lipopolyplexes (directed against the kinesin spindle motor protein EG5) mediated tumoral EG5 mRNA knockdown by ∼60% and, in combination with the novel antitubulin drug pretubulysin, significantly prolonged survival of aggressive leukemia bearing mice without noticeable side effects.
Insights
Researchers developed novel folate-bearing lipopolyplexes for targeted delivery of small interfering RNA (siRNA) cancer therapy. The optimal formulation enhanced tumor delivery and gene silencing, significantly improving survival in leukemia models.
Area of Science:
- Nanotechnology for drug delivery
- Cancer therapeutics
- RNA interference (RNAi)
Background:
- Effective delivery of small interfering RNA (siRNA) to cancer cells requires advanced carrier systems.
- Targeting tumor cells selectively is crucial for enhancing therapeutic efficacy and minimizing side effects.
- Folate receptor (FR) is often overexpressed on various cancer cells, making it an attractive target for drug delivery.
Purpose of the Study:
- To develop and optimize folate-functionalized lipopolyplexes for targeted siRNA delivery to cancer cells.
- To evaluate the in vitro and in vivo performance of these novel nanocarriers for cancer gene therapy.
- To assess the therapeutic potential of FR-targeted siRNA in combination with chemotherapy.
Main Methods:
- Preparation of spherical lipopolyplexes (∼100-200 nm) by complexing siRNA with a novel azido-functionalized lipo-oligomer (1106).
- Surface functionalization of lipopolyplexes with folate-conjugated DBCO-PEG agents via click chemistry.
- Solid-phase synthesis of the lipo-oligomer and targeting agents; evaluation of nanoparticle characteristics, FR targeting, cellular uptake, gene silencing, biodistribution, and therapeutic efficacy in a leukemia mouse model.
Main Results:
- A bivalent DBCO surface agent with a PEG24 spacer (siRNA/1106/DBCO2-ss2-PEG24-FolA) demonstrated optimal nanoparticle properties, FR targeting, cellular uptake, and in vitro gene silencing.
- Near-infrared fluorescence bioimaging confirmed enhanced biodistribution and intratumoral delivery in a subcutaneous FR-positive leukemia mouse model.
- Therapeutic siRNA targeting EG5 mRNA achieved ~60% tumoral knockdown and, combined with pretubulysin, significantly prolonged survival in aggressive leukemia-bearing mice with no observable side effects.
Conclusions:
- Novel folate-bearing lipopolyplexes represent a promising platform for targeted siRNA delivery in cancer therapy.
- The optimized formulation facilitates efficient tumor targeting, cellular internalization, and potent gene silencing.
- Combination therapy using FR-targeted siRNA and pretubulysin shows significant therapeutic potential for aggressive leukemia.
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