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siRNA delivery targeting to the lung via agglutination-induced accumulation and clearance of cationic tetraamino fullerene.

Minami K, Okamoto K, Doi K, Harano K, Noiri E, Nakamura E - Sci Rep (2014)

Bottom Line: TPFE and siRNA form sub-micrometer-sized complexes in buffered solution and these complexes agglutinate further with plasma proteins in the bloodstream to form micrometer-sized particles.The agglutinate rapidly clogs the lung capillaries, releases the siRNA into lung cells to silence expression of target genes, and is then cleared rapidly from the lung after siRNA delivery.We applied our delivery system to an animal model of sepsis, indicating the potential of TPFE-based siRNA delivery for clinical applications.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Chemistry, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan [2].

ABSTRACT
The efficient treatment of lung diseases requires lung-selective delivery of agents to the lung. However, lung-selective delivery is difficult because the accumulation of micrometer-sized carriers in the lung often induces inflammation and embolization-related toxicity. Here we demonstrate a lung-selective delivery system of small interfering RNA (siRNA) by controlling the size of carrier vehicle in blood vessels. The carrier is made of tetra(piperazino)fullerene epoxide (TPFE), a water-soluble cationic tetraamino fullerene. TPFE and siRNA form sub-micrometer-sized complexes in buffered solution and these complexes agglutinate further with plasma proteins in the bloodstream to form micrometer-sized particles. The agglutinate rapidly clogs the lung capillaries, releases the siRNA into lung cells to silence expression of target genes, and is then cleared rapidly from the lung after siRNA delivery. We applied our delivery system to an animal model of sepsis, indicating the potential of TPFE-based siRNA delivery for clinical applications.

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Suppression of neutrophil sequestration in the lung by lung-specific delivery of siTLR4 by TPFE.(a–c) Neutrophils stained with Giemsa stain. TPFE–siGFP-treated C3H/HeJ mice (TLR4–/–, a) and C3H/HeN mice (TLR4+/+, b), and TPFE–siTLR4-treated C3H/HeN mice (TLR4+/+, c) treated by intratracheal LPS injection are shown. Scale bar is 20 μm. (d–e) Quantitative analysis of neutrophil sequestration into the lung and lung MPO activity, respectively (N = 4 for each group). Black and white bars show the intratracheal treatment of LPS and normal saline, respectively. (f) Knockdown of mRNA expression of TLR4 was detected by a real-time RT-PCR analysis (N = 4 for each group). All error bars are SEM. * P < 0.05. *** P < 0.001.
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f6: Suppression of neutrophil sequestration in the lung by lung-specific delivery of siTLR4 by TPFE.(a–c) Neutrophils stained with Giemsa stain. TPFE–siGFP-treated C3H/HeJ mice (TLR4–/–, a) and C3H/HeN mice (TLR4+/+, b), and TPFE–siTLR4-treated C3H/HeN mice (TLR4+/+, c) treated by intratracheal LPS injection are shown. Scale bar is 20 μm. (d–e) Quantitative analysis of neutrophil sequestration into the lung and lung MPO activity, respectively (N = 4 for each group). Black and white bars show the intratracheal treatment of LPS and normal saline, respectively. (f) Knockdown of mRNA expression of TLR4 was detected by a real-time RT-PCR analysis (N = 4 for each group). All error bars are SEM. * P < 0.05. *** P < 0.001.

Mentions: We evaluated the application of mouse toll-like receptor 4 (TLR4)-targeted siRNA delivery by TPFE to the suppression of neutrophil accumulation induced by LPS injection in the lung. siRNA targeting TLR4 (siTLR4) was injected into wild-type mice (C3H/HeN, TLR4+/+) and into mice harboring the TLR4 missense mutation (C3H/HeJ, TLR4–/–) as the negative control. We also carried out a control experiment using siGFP as non-targeting siRNA. LPS was injected intratracheally 24 h after intravenous injection of the TPFE–siTLR4 complex, and lung specimens were collected 6 h after the LPS injection. The accumulation of neutrophils was evaluated by lung pathology analysis and by measuring myeloperoxidase (MPO) activity (Fig. 6a–e). Both neutrophil sequestration and MPO activity in the lung of siTLR4-injected mice were significantly lower than the siGFP-injected mice, and were as low as the mice harboring the TLR4 missense mutation, C3H/HeJ (TLR4–/–). The expression of TLR4 mRNA was determined by real-time RT-PCR (Fig. 6f), which showed that the TLR4 mRNA was knocked down by siTLR4 in the lung. These results indicate that siTLR4 delivered by TPFE knocked down TLR4 and significantly suppressed the accumulation of the neutrophils by the LPS-TLR4 signaling pathway.


siRNA delivery targeting to the lung via agglutination-induced accumulation and clearance of cationic tetraamino fullerene.

Minami K, Okamoto K, Doi K, Harano K, Noiri E, Nakamura E - Sci Rep (2014)

Suppression of neutrophil sequestration in the lung by lung-specific delivery of siTLR4 by TPFE.(a–c) Neutrophils stained with Giemsa stain. TPFE–siGFP-treated C3H/HeJ mice (TLR4–/–, a) and C3H/HeN mice (TLR4+/+, b), and TPFE–siTLR4-treated C3H/HeN mice (TLR4+/+, c) treated by intratracheal LPS injection are shown. Scale bar is 20 μm. (d–e) Quantitative analysis of neutrophil sequestration into the lung and lung MPO activity, respectively (N = 4 for each group). Black and white bars show the intratracheal treatment of LPS and normal saline, respectively. (f) Knockdown of mRNA expression of TLR4 was detected by a real-time RT-PCR analysis (N = 4 for each group). All error bars are SEM. * P < 0.05. *** P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4017229&req=5

f6: Suppression of neutrophil sequestration in the lung by lung-specific delivery of siTLR4 by TPFE.(a–c) Neutrophils stained with Giemsa stain. TPFE–siGFP-treated C3H/HeJ mice (TLR4–/–, a) and C3H/HeN mice (TLR4+/+, b), and TPFE–siTLR4-treated C3H/HeN mice (TLR4+/+, c) treated by intratracheal LPS injection are shown. Scale bar is 20 μm. (d–e) Quantitative analysis of neutrophil sequestration into the lung and lung MPO activity, respectively (N = 4 for each group). Black and white bars show the intratracheal treatment of LPS and normal saline, respectively. (f) Knockdown of mRNA expression of TLR4 was detected by a real-time RT-PCR analysis (N = 4 for each group). All error bars are SEM. * P < 0.05. *** P < 0.001.
Mentions: We evaluated the application of mouse toll-like receptor 4 (TLR4)-targeted siRNA delivery by TPFE to the suppression of neutrophil accumulation induced by LPS injection in the lung. siRNA targeting TLR4 (siTLR4) was injected into wild-type mice (C3H/HeN, TLR4+/+) and into mice harboring the TLR4 missense mutation (C3H/HeJ, TLR4–/–) as the negative control. We also carried out a control experiment using siGFP as non-targeting siRNA. LPS was injected intratracheally 24 h after intravenous injection of the TPFE–siTLR4 complex, and lung specimens were collected 6 h after the LPS injection. The accumulation of neutrophils was evaluated by lung pathology analysis and by measuring myeloperoxidase (MPO) activity (Fig. 6a–e). Both neutrophil sequestration and MPO activity in the lung of siTLR4-injected mice were significantly lower than the siGFP-injected mice, and were as low as the mice harboring the TLR4 missense mutation, C3H/HeJ (TLR4–/–). The expression of TLR4 mRNA was determined by real-time RT-PCR (Fig. 6f), which showed that the TLR4 mRNA was knocked down by siTLR4 in the lung. These results indicate that siTLR4 delivered by TPFE knocked down TLR4 and significantly suppressed the accumulation of the neutrophils by the LPS-TLR4 signaling pathway.

Bottom Line: TPFE and siRNA form sub-micrometer-sized complexes in buffered solution and these complexes agglutinate further with plasma proteins in the bloodstream to form micrometer-sized particles.The agglutinate rapidly clogs the lung capillaries, releases the siRNA into lung cells to silence expression of target genes, and is then cleared rapidly from the lung after siRNA delivery.We applied our delivery system to an animal model of sepsis, indicating the potential of TPFE-based siRNA delivery for clinical applications.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Chemistry, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan [2].

ABSTRACT
The efficient treatment of lung diseases requires lung-selective delivery of agents to the lung. However, lung-selective delivery is difficult because the accumulation of micrometer-sized carriers in the lung often induces inflammation and embolization-related toxicity. Here we demonstrate a lung-selective delivery system of small interfering RNA (siRNA) by controlling the size of carrier vehicle in blood vessels. The carrier is made of tetra(piperazino)fullerene epoxide (TPFE), a water-soluble cationic tetraamino fullerene. TPFE and siRNA form sub-micrometer-sized complexes in buffered solution and these complexes agglutinate further with plasma proteins in the bloodstream to form micrometer-sized particles. The agglutinate rapidly clogs the lung capillaries, releases the siRNA into lung cells to silence expression of target genes, and is then cleared rapidly from the lung after siRNA delivery. We applied our delivery system to an animal model of sepsis, indicating the potential of TPFE-based siRNA delivery for clinical applications.

Show MeSH
Related in: MedlinePlus