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Virosomes of hepatitis B virus envelope L proteins containing doxorubicin: synergistic enhancement of human liver-specific antitumor growth activity by radiotherapy.

Liu Q, Jung J, Somiya M, Iijima M, Yoshimoto N, Niimi T, Maturana AD, Shin SH, Jeong SY, Choi EK, Kuroda S - Int J Nanomedicine (2015)

Bottom Line: The growth of target cell-derived tumors was retarded effectively and specifically.Next, the combination of high dose (10.0 mg/kg as DOX, once) with tumor-specific radiotherapy (3 Gy, once after 2 hours) exhibited the most effective antitumor growth activity in mice harboring target cell-derived tumors.These results demonstrated that the HBV-based virosomes containing DOX could be an effective antitumor nanomedicine specific to human hepatic tissues, especially in combination with radiotherapy.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan ; The Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Japan.

ABSTRACT
Bionanocapsules (BNCs) are hollow nanoparticles consisting of hepatitis B virus (HBV) envelope L proteins and have been shown to deliver drugs and genes specifically to human hepatic tissues by utilizing HBV-derived infection machinery. The complex of BNCs with liposomes (LPs), the BNC-LP complexes (a LP surrounded by BNCs in a rugged spherical form), could also become active targeting nanocarriers by the BNC function. In this study, under acidic conditions and high temperature, BNCs were found to fully fuse with LPs (smooth-surfaced spherical form), deploying L proteins with a membrane topology similar to that of BNCs (ie, virosomes displaying L proteins). Doxorubicin (DOX) was efficiently encapsulated via the remote loading method at 14.2%±1.0% of total lipid weight (mean ± SD, n=3), with a capsule size of 118.2±4.7 nm and a ζ-potential of -51.1±1.0 mV (mean ± SD, n=5). When mammalian cells were exposed to the virosomes, the virosomes showed strong cytotoxicity in human hepatic cells (target cells of BNCs), but not in human colon cancer cells (nontarget cells of BNCs), whereas LPs containing DOX and DOXOVES (structurally stabilized PEGylated LPs containing DOX) did not show strong cytotoxicity in either cell type. Furthermore, the virosomes preferentially delivered DOX to the nuclei of human hepatic cells. Xenograft mice harboring either target or nontarget cell-derived tumors were injected twice intravenously with the virosomes containing DOX at a low dose (2.3 mg/kg as DOX, 5 days interval). The growth of target cell-derived tumors was retarded effectively and specifically. Next, the combination of high dose (10.0 mg/kg as DOX, once) with tumor-specific radiotherapy (3 Gy, once after 2 hours) exhibited the most effective antitumor growth activity in mice harboring target cell-derived tumors. These results demonstrated that the HBV-based virosomes containing DOX could be an effective antitumor nanomedicine specific to human hepatic tissues, especially in combination with radiotherapy.

No MeSH data available.


Related in: MedlinePlus

CsCl isopycnic ultracentrifugation analyses of BNC–LP complexes.Notes: The mixtures of BNCs (as particle) and LPs (as lipids) at weight ratios of 1:10 (at a molar ratio of 1:8.0×104; A, B), 1:20 (at a molar ratio of 1:1.6×105; C, D), and 1:30 (at a molar ratio of 1:2.4×105; E, F) were incubated at 37°C and pH 3.0 (A, C, E) or 70°C and pH 3.0 (B, D, F) for 1 hour, and then analyzed using CsCl isopycnic ultracentrifugation (24,000 rpm, 25°C, 16 hours). Protein concentrations (red) were determined with a BCA protein assay kit. Lipid concentrations (blue) were estimated from cholesterol concentration, which was determined using a Cholesterol E-Test Wako kit. Density (green) was measured using an electronic balance. The yield (%) of BNCs in each BNC–LP complex preparation is indicated in the graphs.Abbreviations: BNC, bionanocapsule; LP, liposome; abs, absorbance; BCA, bicinchoninic acid.
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f1-ijn-10-4159: CsCl isopycnic ultracentrifugation analyses of BNC–LP complexes.Notes: The mixtures of BNCs (as particle) and LPs (as lipids) at weight ratios of 1:10 (at a molar ratio of 1:8.0×104; A, B), 1:20 (at a molar ratio of 1:1.6×105; C, D), and 1:30 (at a molar ratio of 1:2.4×105; E, F) were incubated at 37°C and pH 3.0 (A, C, E) or 70°C and pH 3.0 (B, D, F) for 1 hour, and then analyzed using CsCl isopycnic ultracentrifugation (24,000 rpm, 25°C, 16 hours). Protein concentrations (red) were determined with a BCA protein assay kit. Lipid concentrations (blue) were estimated from cholesterol concentration, which was determined using a Cholesterol E-Test Wako kit. Density (green) was measured using an electronic balance. The yield (%) of BNCs in each BNC–LP complex preparation is indicated in the graphs.Abbreviations: BNC, bionanocapsule; LP, liposome; abs, absorbance; BCA, bicinchoninic acid.

Mentions: Our previous studies17,18 demonstrated that the mixtures of BNCs and LPs at a weight ratio of 1:2 or 1:20 (at a molar ratio, BNC [as particle]:LP [as lipids] =1:1.6×104 or 1:1.6×105, respectively) formed BNC–LP complexes spontaneously at room temperature under neutral conditions. However, following CsCl isopycnic ultracentrifugation analyses, the free form of BNCs or LPs was always found in the complex preparations. Since these free BNCs were considered to suppress the human hepatic cell-specific delivery by BNC–LP complexes in a competitive inhibition manner, the weight ratio of BNC:LP was investigated from 1:10 (1:8.0×104 at molar ratio) to 1:30 (1:2.4×105 at molar ratio) to reduce the unexpected generation of free BNCs. Furthermore, since BNCs are stable up to 70°C,14 the phase transition temperature of LPs is estimated to be above 70°C, and BNC contains fusogenic activity in the translocation motifs of preS2 and/or S region.23,24 We then examined whether the BNC–LP complex formation was enhanced with incubation at 70°C under acidic conditions (pH 3.0). As shown in Figure 1, narrower peaks of BNC–LP complexes were observed at 70°C, suggesting that BNC–LP complexes became homogeneous. Among the BNC–LP complexes prepared at 70°C, the highest yield of BNCs was obtained at BNC:LP =1:20 (Figure 1D). Consequently, the BNC–LP complexes prepared at BNC:LP =1:20 (1:1.6×105 at molar ratio), 70°C, and pH 3.0 were promptly used for further experiments without any purification.


Virosomes of hepatitis B virus envelope L proteins containing doxorubicin: synergistic enhancement of human liver-specific antitumor growth activity by radiotherapy.

Liu Q, Jung J, Somiya M, Iijima M, Yoshimoto N, Niimi T, Maturana AD, Shin SH, Jeong SY, Choi EK, Kuroda S - Int J Nanomedicine (2015)

CsCl isopycnic ultracentrifugation analyses of BNC–LP complexes.Notes: The mixtures of BNCs (as particle) and LPs (as lipids) at weight ratios of 1:10 (at a molar ratio of 1:8.0×104; A, B), 1:20 (at a molar ratio of 1:1.6×105; C, D), and 1:30 (at a molar ratio of 1:2.4×105; E, F) were incubated at 37°C and pH 3.0 (A, C, E) or 70°C and pH 3.0 (B, D, F) for 1 hour, and then analyzed using CsCl isopycnic ultracentrifugation (24,000 rpm, 25°C, 16 hours). Protein concentrations (red) were determined with a BCA protein assay kit. Lipid concentrations (blue) were estimated from cholesterol concentration, which was determined using a Cholesterol E-Test Wako kit. Density (green) was measured using an electronic balance. The yield (%) of BNCs in each BNC–LP complex preparation is indicated in the graphs.Abbreviations: BNC, bionanocapsule; LP, liposome; abs, absorbance; BCA, bicinchoninic acid.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4487236&req=5

f1-ijn-10-4159: CsCl isopycnic ultracentrifugation analyses of BNC–LP complexes.Notes: The mixtures of BNCs (as particle) and LPs (as lipids) at weight ratios of 1:10 (at a molar ratio of 1:8.0×104; A, B), 1:20 (at a molar ratio of 1:1.6×105; C, D), and 1:30 (at a molar ratio of 1:2.4×105; E, F) were incubated at 37°C and pH 3.0 (A, C, E) or 70°C and pH 3.0 (B, D, F) for 1 hour, and then analyzed using CsCl isopycnic ultracentrifugation (24,000 rpm, 25°C, 16 hours). Protein concentrations (red) were determined with a BCA protein assay kit. Lipid concentrations (blue) were estimated from cholesterol concentration, which was determined using a Cholesterol E-Test Wako kit. Density (green) was measured using an electronic balance. The yield (%) of BNCs in each BNC–LP complex preparation is indicated in the graphs.Abbreviations: BNC, bionanocapsule; LP, liposome; abs, absorbance; BCA, bicinchoninic acid.
Mentions: Our previous studies17,18 demonstrated that the mixtures of BNCs and LPs at a weight ratio of 1:2 or 1:20 (at a molar ratio, BNC [as particle]:LP [as lipids] =1:1.6×104 or 1:1.6×105, respectively) formed BNC–LP complexes spontaneously at room temperature under neutral conditions. However, following CsCl isopycnic ultracentrifugation analyses, the free form of BNCs or LPs was always found in the complex preparations. Since these free BNCs were considered to suppress the human hepatic cell-specific delivery by BNC–LP complexes in a competitive inhibition manner, the weight ratio of BNC:LP was investigated from 1:10 (1:8.0×104 at molar ratio) to 1:30 (1:2.4×105 at molar ratio) to reduce the unexpected generation of free BNCs. Furthermore, since BNCs are stable up to 70°C,14 the phase transition temperature of LPs is estimated to be above 70°C, and BNC contains fusogenic activity in the translocation motifs of preS2 and/or S region.23,24 We then examined whether the BNC–LP complex formation was enhanced with incubation at 70°C under acidic conditions (pH 3.0). As shown in Figure 1, narrower peaks of BNC–LP complexes were observed at 70°C, suggesting that BNC–LP complexes became homogeneous. Among the BNC–LP complexes prepared at 70°C, the highest yield of BNCs was obtained at BNC:LP =1:20 (Figure 1D). Consequently, the BNC–LP complexes prepared at BNC:LP =1:20 (1:1.6×105 at molar ratio), 70°C, and pH 3.0 were promptly used for further experiments without any purification.

Bottom Line: The growth of target cell-derived tumors was retarded effectively and specifically.Next, the combination of high dose (10.0 mg/kg as DOX, once) with tumor-specific radiotherapy (3 Gy, once after 2 hours) exhibited the most effective antitumor growth activity in mice harboring target cell-derived tumors.These results demonstrated that the HBV-based virosomes containing DOX could be an effective antitumor nanomedicine specific to human hepatic tissues, especially in combination with radiotherapy.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan ; The Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Japan.

ABSTRACT
Bionanocapsules (BNCs) are hollow nanoparticles consisting of hepatitis B virus (HBV) envelope L proteins and have been shown to deliver drugs and genes specifically to human hepatic tissues by utilizing HBV-derived infection machinery. The complex of BNCs with liposomes (LPs), the BNC-LP complexes (a LP surrounded by BNCs in a rugged spherical form), could also become active targeting nanocarriers by the BNC function. In this study, under acidic conditions and high temperature, BNCs were found to fully fuse with LPs (smooth-surfaced spherical form), deploying L proteins with a membrane topology similar to that of BNCs (ie, virosomes displaying L proteins). Doxorubicin (DOX) was efficiently encapsulated via the remote loading method at 14.2%±1.0% of total lipid weight (mean ± SD, n=3), with a capsule size of 118.2±4.7 nm and a ζ-potential of -51.1±1.0 mV (mean ± SD, n=5). When mammalian cells were exposed to the virosomes, the virosomes showed strong cytotoxicity in human hepatic cells (target cells of BNCs), but not in human colon cancer cells (nontarget cells of BNCs), whereas LPs containing DOX and DOXOVES (structurally stabilized PEGylated LPs containing DOX) did not show strong cytotoxicity in either cell type. Furthermore, the virosomes preferentially delivered DOX to the nuclei of human hepatic cells. Xenograft mice harboring either target or nontarget cell-derived tumors were injected twice intravenously with the virosomes containing DOX at a low dose (2.3 mg/kg as DOX, 5 days interval). The growth of target cell-derived tumors was retarded effectively and specifically. Next, the combination of high dose (10.0 mg/kg as DOX, once) with tumor-specific radiotherapy (3 Gy, once after 2 hours) exhibited the most effective antitumor growth activity in mice harboring target cell-derived tumors. These results demonstrated that the HBV-based virosomes containing DOX could be an effective antitumor nanomedicine specific to human hepatic tissues, especially in combination with radiotherapy.

No MeSH data available.


Related in: MedlinePlus