6-Amino-6-deoxy-curdlan is a promising nucleic acid carrier that efficiently delivers plasmid DNA as well as short interfering RNA (siRNA) to various cell lines. The highly reactive C6-NH groups of 6-amino-6-deoxy-curdlan prompt conjugation of various side groups including tissue-targeting ligands to enhance cell-type-specific nucleic acid delivery to specific cell lines. Herein, to test the primary-cell-targeting efficiency of the curdlan derivative, we chemically conjugated a macrophage-targeting ligand, mannose, to 6-amino-6-deoxy-curdlan. The resulting curdlan derivative (denoted CMI) readily complexed with siRNA and formed nanoparticles with a diameter of 50-80 nm. The CMI nanoparticles successfully delivered a dye-labeled siRNA to mouse peritoneal macrophages. The delivery efficiency was blocked by mannan, a natural ligand for a macrophage surface mannose receptor (CD206), but not by zymosan, a ligand for the dectin-1 receptor, which is also present on the surface of macrophages. Moreover, CMI nanoparticles were internalized by macrophages only at 37 °C, suggesting that the cellular uptake of CMI nanoparticles was energy-dependent. Furthermore, CMI nanoparticle efficiently delivered siRNA against tumor necrosis factor α (TNFα) to lipopolysaccharide-stimulated primary mouse peritoneal macrophages. In vivo experiments demonstrated that CMI nanoparticles successfully delivered siTNFα to mouse peritoneal macrophages, liver, and lung and induced significant knockdown of the TNFα expression at both messenger RNA and protein levels. Therefore, our design of CMI may be a promising siRNA carrier for targeting CD206-expressing primary cells such as macrophage and dendritic cells.
Microglia polarization
plays an important role in poststroke recovery.
Inhibition of proinflammatory (M1) polarization and promotion of anti-inflammatory
(M2) polarization of microglia are potential therapeutic strategies
for inflammation reduction and neuronal recovery after stroke. Here,
we evaluated the central nervous system (CNS)-targeted
short interfering RNA (siRNA) delivery ability of functionalized curdlan
nanoparticles (CMI) and investigated the nuclear factor-κB (NF-κB)
p65 silencing efficiency of CMI-mediated siRNA in microglia, as well
as the resulting neuroprotective effect of microglia polarization
and neuroprotection in vitro and in vivo. The systemic delivery of
NF-κB p65 siRNA (sip65) complexed to CMI nanoparticles in the
mouse model of transient middle cerebral artery occlusion (tMCAO)
resulted in the distribution of siRNA in microglia and significant
silencing in NF-κB p65 in the peri-infarct region. Knockdown
of NF-κB p65 resulted in M1 to M2 phenotypic transition of microglia,
evidenced by the change in the expression pattern of signature cytokines
as well as inducible nitric oxide synthase and CD206. Moreover, the
CMI-mediated silencing of p65 increased the density of neurons and
decreased pyknosis and edema in the peri-infarct region. Assessment
of the neurological deficit score on the Bederson scale revealed a
significantly reduced score in the mouse model of tMCAO treated with
the sip65/CMI complex. Collectively, our data suggest that CMI nanoparticles
are a promising CNS-targeting siRNA delivery system, and NF-κB
p65 may be a potential therapeutic target for inflammation reduction
and poststroke recovery.
RNA interference (RNAi) down-regulates gene expression post-transcriptionally, which is a therapeutically significant phenomenon that could potentially reduce the level of disease related proteins that are undruggable by conventional small molecular approaches. However, clinical application of small interference RNAs (siRNAs) requires design of potent siRNA sequences and development of safe and efficient delivery systems. To create a biocompatible siRNA delivery agent, we chemically modified natural polysaccharide curdlan in a regioselective manner to introduce amino group in the glucose units. The resulting 6-amino-curdlan (6AC) is water soluble and forms nanoparticles upon complexing with siRNAs. The novel curdlan-based nanoparticles efficiently delivered siRNAs to human cancer cells and mouse primary cells, and reduced 70–90% of target mRNA level. Moreover, 6AC nanoparticles delivered siRNA targeting eGFP to mouse embryonic stem (mES) cells stably expressing eGFP, and produced substantial reductions of eGFP protein level. The novel curdlan-based nanoparticle is a promising vehicle for delivery of short RNAs to knock down endogenous mRNAs.
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