Therapeutic effect for liver-metastasized tumor by sequential intravenous injection of anionic polymer and cationic lipoplex of siRNA.

Previously, we developed a novel siRNA transfer method to the liver by sequential intravenous injection of anionic polymer and cationic liposome/siRNA complex (cationic lipoplex). In this study, we investigated whether siRNA delivered by this sequential injection could significantly suppress mRNA expression of the targeted gene in liver metastasis and inhibit tumor growth. When cationic lipoplex was intravenously injected into mice bearing liver metastasis of human breast tumor MCF-7 at 1 min after intravenous injection of chondroitin sulfate C (CS) or poly-l-glutamic acid (PGA), siRNA was accumulated in tumor-metastasized liver. In terms of a gene silencing effect, sequential injections of CS or PGA plus cationic lipoplex of luciferase siRNA could reduce luciferase activity in liver MCF-7-Luc metastasis. Regarding the side effects, sequential injections of CS plus cationic lipoplex did not exhibit hepatic damage or induction of inflammatory cytokines in serum after repeated injections, but sequential injections of PGA plus cationic lipoplex did. Finally, sequential injections of CS plus cationic lipoplex of protein kinase N3 siRNA could suppress tumor growth in the mice bearing liver metastasis. From these findings, sequential injection of CS and cationic lipoplex of siRNA might be a novel systemic method of delivering siRNA to liver metastasis.

siRNA delivery into tumor cells by cationic cholesterol derivative-based nanoparticles and liposomes.

Previously, we reported that cationic nanoparticles (NP) composed of diamine-type cholesteryl-3-carboxamide (OH-Chol, N-(2-(2-hydroxyethylamino)ethyl)cholesteryl-3-carboxamide) and Tween 80 could deliver small interfering RNA (siRNA) with high transfection efficiency into tumor cells. In this study, we synthesized new diamine-type cationic cholesteryl carbamate (OH-C-Chol, cholesteryl (2-((2-hydroxyethyl)amino)ethyl)carbamate) and triamine-type carbamate (OH-NC-Chol, cholesteryl (2-((2-((2-hydroxyethyl)amino)ethyl)amino)ethyl)carbamate), and prepared cationic nanoparticles composed of OH-C-Chol or OH-NC-Chol with Tween 80 (NP-C and NP-NC, respectively), as well as cationic liposomes composed of OH-C-Chol or OH-NC-Chol with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (LP-C and LP-NC, respectively) for evaluation of their possible use as siRNA delivery vectors. LP-C and LP-NC/siRNA complexes (lipoplexes) exhibited larger gene silencing effects than NP-C and NP-NC/siRNA complexes (nanoplexes), respectively, in human breast tumor MCF-7 cells, although the NP-C nanoplex showed high association with the cells. In particular, LP-NC lipoplex could induce strong gene suppression, even at a concentration of 5 nM siRNA. From these results, cationic liposomes composed of OH-NC-Chol and DOPE may have potential as gene vectors for siRNA transfection to tumor cells.

siRNA delivery to lung-metastasized tumor by systemic injection with cationic liposomes.

CONTEXT: Cationic liposomes can efficiently deliver siRNA to the lung by intravenous injection of cationic liposome/siRNA complexes (lipoplexes). OBJECTIVE: The aim of this study was to examine a formulation of cationic liposomes for siRNA delivery to lung metastasis of breast tumor. MATERIALS AND METHODS: For the preparation of cationic liposomes, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or dimethyldioctadecylammonium bromide (DDAB) as a cationic lipid and cholesterol (Chol) or 1,2-dioleoyl-L-α-glycero-3-phosphatidylethanolamine (DOPE) as a neutral lipid were used. In vitro and in vivo gene silencing effects by cationic lipoplexes were evaluated after transfection into stably luciferase-expressing human breast tumor MCF-7-Luc cells and after intravenous injection into mice with lung MCF-7-Luc metastasis, respectively. Intracellular localization of siRNA after transfection into MCF-7 cells by cationic lipoplexes and biodistribution of siRNA after intravenous injection of cationic lipoplexes into the mice with lung metastasis were examined by confocal and fluorescent microscopy analyses, respectively. RESULTS: In in vitro transfection, DOTAP/DOPE and DDAB/DOPE lipoplexes of luciferase siRNA strongly suppressed luciferase activity in MCF-7-Luc cells, but DOTAP/Chol and DDAB/Chol lipoplexes did not, although DOTAP/Chol and DDAB/Chol lipoplexes exhibited higher cellular uptake than DOTAP/DOPE and DDAB/DOPE lipoplexes. When their cationic lipoplexes were intravenously injected into mice with lung MCF-7-Luc metastasis, siRNAs were mainly accumulated in the lungs; however, the reduced luciferase activities in the lung-metastasized tumors were observed only by injections of DOTAP/Chol and DOTAP/DOPE lipoplexes, but not by DDAB/Chol and DDAB/DOPE lipoplexes. CONCLUSIONS: DOTAP-based liposomes might be useful as an in vivo siRNA delivery carrier that can induce gene silencing in lung-metastasized tumors.

Sequential intravenous injection of anionic polymer and cationic lipoplex of siRNA could effectively deliver siRNA to the liver.

In this study, we developed novel siRNA transfer method to the liver by sequential intravenous injection of anionic polymer and cationic liposome/cholesterol-modified siRNA complex (cationic lipoplex). When cationic lipoplex was intravenously injected into mice, the accumulation of siRNA was mainly observed in the lungs. In contrast, when cationic lipoplex was intravenously injected at 1 min after intravenous injection of poly-L-glutamic acid (PGA) or chondroitin sulfate C (CS), siRNA was accumulated in the liver. In terms of suppression of gene expression in vivo, apolipoprotein B (ApoB) mRNA in the liver and low-density-lipoprotein (LDL) and very low-density-lipoprotein (VLDL) cholesterol level in serum were reduced at 48 h after single sequential injection of PGA or CS plus cationic lipoplex of cholesterol-modified ApoB siRNA. Furthermore, sequential injections of PGA plus cationic lipoplex of cholesterol-modified luciferase siRNA could reduce luciferase activity in tumor xenografts bearing liver metastasis of human breast tumor MCF-7-Luc. From these findings, sequential injection of anionic polymer and cationic lipoplex of siRNA might produce a systemic vector of siRNA to the liver.

In vivo siRNA delivery system for targeting to the liver by poly-l-glutamic acid-coated lipoplex.

In this study, we developed anionic polymer-coated liposome/siRNA complexes (lipoplexes) with chondroitin sulfate C (CS), poly-l-glutamic acid (PGA) and poly-aspartic acid (PAA) for siRNA delivery by intravenous injection, and evaluated the biodistribution and gene silencing effect in mice. The sizes of CS-, PGA- and PAA-coated lipoplexes were about 200?nm and their ?-potentials were negative. CS-, PGA- and PAA-coated lipoplexes did not induce agglutination after mixing with erythrocytes. In terms of biodistribution, siRNAs after intravenous administration of cationic lipoplexes were largely observed in the lungs, but those of CS-, PGA- and PAA-coated lipoplexes were in both the liver and the kidneys, indicating that siRNA might be partially released from the anionic polymer-coated lipoplexes in the blood circulation and accumulate in the kidney, although the lipoplexes can prevent the agglutination with blood components. To increase the association between siRNA and cationic liposome, we used cholesterol-modified siRNA (siRNA-Chol) for preparation of the lipoplexes. When CS-, PGA- and PAA-coated lipoplexes of siRNA-Chol were injected into mice, siRNA-Chol was mainly observed in the liver, not in the kidneys. In terms of the suppression of gene expression in vivo, apolipoprotein B (ApoB) mRNA in the liver was significantly reduced 48?h after single intravenous injection of PGA-coated lipoplex of ApoB siRNA-Chol (2.5?mg?siRNA/kg), but not cationic, CS- and PAA-coated lipoplexes. In terms of toxicity after intravenous injection, CS-, PGA- and PAA-coated lipoplexes did not increase GOT and GPT concentrations in blood. From these findings, PGA coatings for cationic lipoplex of siRNA-Chol might produce a systemic vector of siRNA to the liver.

Simultaneous determination of free capsorubin and capsanthin in red pepper powder using u-HPLC.

A simultaneous ultra-HPLC (u-HPLC) method for the determination of free capsorubin and capsanthin in red pepper powder was validated in terms of its precision, accuracy, and linearity. The u-HPLC separation was performed on an RP C18 column (particle size 2 pm, id 2 mm length 100 mm) and with a photodiode-array detector. The recoveries of capsorubin were greater than 83.8 +/- 1.7%; the LOD and LOQ of the u-HPLC analyses were 0.043 and 0.129 mg/kg, respectively. The intraday and interday precisions for capsorubin were less than 9.01%. The recoveries of capsanthin were greater than 87.7 +/- 1.5%, and the LOD and LOQ were 0.101 and 0.306 mglkg, respectively. The intraday and interday precisions for capsanthin were less than 12.66%. All calibration curves for capsorubin and capsanthin exhibited good linearity (r2 = 0.99) within the tested ranges.

Transdermal Delivery of Small Interfering RNA with Elastic Cationic Liposomes in Mice.

We developed elastic cationic liposomal vectors for transdermal siRNA delivery. These liposomes were prepared with 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) as a cationic lipid and sodium cholate (NaChol) or Tween 80 as an edge activator. When NaChol or Tween 80 was included at 5, 10, and 15% (w/w) into DOTAP liposomal formulations (C5-, C10-, and C15-liposomes and T5-, T10-, and T15-liposomes), C15- and T10-liposomes showed 2.4- and 2.7-fold-higher elasticities than DOTAP liposome, respectively. Although the sizes of all elastic liposomes prepared in this study were about 80-90 nm, the sizes of C5-, C10- and C15-liposome/siRNA complexes (lipoplexes) were about 1,700-1,800 nm, and those of T5-, T10-, and T15-lipoplexes were about 550-780 nm. Their elastic lipoplexes showed strong gene suppression by siRNA without cytotoxicity when transfected into human cervical carcinoma SiHa cells. Following skin application of the fluorescence-labeled lipoplexes in mice, among the elastic lipoplexes, C15- and T5-lipoplexes showed effective penetration of siRNA into skin, compared with DOTAP lipoplex and free siRNA solution. These data suggest that elastic cationic liposomes containing an appropriate amount of NaChol or Tween 80 as an edge activator could deliver siRNA transdermally.