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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

Characterization of BNC–LP complexes.Notes: (A) Transmission electron microscopy (TEM) photographs of (a) the BNC–LP complexes prepared at room temperature under neutral conditions, (b) the interface between BNCs and LP in the boxed area of (a), White arrowheads indicate the interface between BNCs and LP. (c) The BNC–LP complexes prepared at 70°C under acid conditions (namely virosomes), and (d) LP. (B) The virosomes treated with trypsin were separated by SDS-PAGE, and then immunoblotted with anti-preS1 antibody (upper panel) and anti-S antibody (lower panel). Asterisks indicate the bands of anti-preS1 immunoreactive proteins (~48 and ~43 kDa) and anti-S immunoreactive proteins (~16, ~19, ~26, and ~29 kDa). The sizes (C) and ζ-potentials (D) of virosomes (green), virosomes containing DOX (red), and LPs (blue) were measured by DLS. Scale bars represent 100 nm.Abbreviations: BNC, bionanocapsule; LP, liposome; Try, trypsin; Ab, antibody; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; DOX, doxorubicin; DLS, dynamic light scattering.
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f2-ijn-10-4159: Characterization of BNC–LP complexes.Notes: (A) Transmission electron microscopy (TEM) photographs of (a) the BNC–LP complexes prepared at room temperature under neutral conditions, (b) the interface between BNCs and LP in the boxed area of (a), White arrowheads indicate the interface between BNCs and LP. (c) The BNC–LP complexes prepared at 70°C under acid conditions (namely virosomes), and (d) LP. (B) The virosomes treated with trypsin were separated by SDS-PAGE, and then immunoblotted with anti-preS1 antibody (upper panel) and anti-S antibody (lower panel). Asterisks indicate the bands of anti-preS1 immunoreactive proteins (~48 and ~43 kDa) and anti-S immunoreactive proteins (~16, ~19, ~26, and ~29 kDa). The sizes (C) and ζ-potentials (D) of virosomes (green), virosomes containing DOX (red), and LPs (blue) were measured by DLS. Scale bars represent 100 nm.Abbreviations: BNC, bionanocapsule; LP, liposome; Try, trypsin; Ab, antibody; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; DOX, doxorubicin; DLS, dynamic light scattering.

Mentions: On TEM observation, the BNC–LP complexes prepared at room temperature under neutral conditions (pH 7.0) (Figure 2A, panel a) exhibited a rugged spherical structure of 112.5±19.7 nm (mean ± SD, n=10) in diameter, which agreed well with a previous report.17 The interface between BNCs and LP became obscure (Figure 2A, panel b), suggesting the occurrence of membrane fusion. When prepared at 70°C and pH 3.0, BNC–LP complexes exhibited a smooth-surfaced spherical structure of 111.4±19.6 nm (mean ± SD, n=10) in diameter (Figure 2A, panel c), which is similar to that of LPs (Figure 2A, panel d). When BNCs and BNC–LP complexes were digested with trypsin, anti-preS1 immunoreactive ~48 and ~43 kDa peptides (full-length N,O-glycosylated and N-glycosylated L protein, respectively) disappeared (Figure 2B, upper panel). Following trypsinization of BNCs, the anti-S immunoreactive peptides with molecular weights of ~16, ~19, ~26, and ~29 kDa were protected (Figure 2B, lower panel), these peptides corresponded to nonglycosylated and O-glycosylated forms of the ~16 kDa peptide (from Gly-19 in the preS2 region to Arg-129 in the S region) and ~26 kDa peptide (from Gly-19 in the preS2 region to the C-terminal in the S region), respectively.12 Similar peptides were found in the trypsinized BNC–LP complexes that strongly suggests that the membrane topology of Hepatitis B virus surface antigen (HBsAg) L protein was well conserved and the preS region was presented outside of the BNC–LP complexes. Based on the elimination of rugged structure in the BNC–LP complexes by incubation at 70°C in acidic conditions, it was postulated that HBsAg L proteins in BNCs were transferred to LPs, presumably by membrane fusion. Compared with the TEM images of previous BNC–LP complexes (Figure 2A, panel a),17 the shape was dramatically changed from a rugged spherical structure to a smooth spherical structure (Figure 2A, panel c). Since BNC contains fusogenic activity in the translocation motifs of the preS2 and/or S region,23,24 both high temperature and acidic conditions might enhance the membrane fusion between BNCs and LPs. In addition, the proteinase protection assay showed that the membrane topology of HBsAg L protein was well conserved after the fusion between BNCs and LPs. It was strongly suggested that HBsAg L proteins in BNCs were disassembled into micelles and then spontaneously integrated into LPs (Figure 3), indicating the formation of virosomes.19 While the composition of BNCs is ~10% (w/w) lipids and ~90% L proteins,25 that of virosomes is estimated to be ~96% lipids and ~4% L proteins. It was expected that virosomes can incorporate DOX by remote loading at comparable levels of LPs alone. Collectively, virosomes could be regarded as LPs transplanted with the targeting ability and fusogenic activity of HBsAg L protein, namely LPs armed with HBV-derived infection machineries.


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)

Characterization of BNC–LP complexes.Notes: (A) Transmission electron microscopy (TEM) photographs of (a) the BNC–LP complexes prepared at room temperature under neutral conditions, (b) the interface between BNCs and LP in the boxed area of (a), White arrowheads indicate the interface between BNCs and LP. (c) The BNC–LP complexes prepared at 70°C under acid conditions (namely virosomes), and (d) LP. (B) The virosomes treated with trypsin were separated by SDS-PAGE, and then immunoblotted with anti-preS1 antibody (upper panel) and anti-S antibody (lower panel). Asterisks indicate the bands of anti-preS1 immunoreactive proteins (~48 and ~43 kDa) and anti-S immunoreactive proteins (~16, ~19, ~26, and ~29 kDa). The sizes (C) and ζ-potentials (D) of virosomes (green), virosomes containing DOX (red), and LPs (blue) were measured by DLS. Scale bars represent 100 nm.Abbreviations: BNC, bionanocapsule; LP, liposome; Try, trypsin; Ab, antibody; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; DOX, doxorubicin; DLS, dynamic light scattering.
© Copyright Policy
Related In: Results  -  Collection

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

f2-ijn-10-4159: Characterization of BNC–LP complexes.Notes: (A) Transmission electron microscopy (TEM) photographs of (a) the BNC–LP complexes prepared at room temperature under neutral conditions, (b) the interface between BNCs and LP in the boxed area of (a), White arrowheads indicate the interface between BNCs and LP. (c) The BNC–LP complexes prepared at 70°C under acid conditions (namely virosomes), and (d) LP. (B) The virosomes treated with trypsin were separated by SDS-PAGE, and then immunoblotted with anti-preS1 antibody (upper panel) and anti-S antibody (lower panel). Asterisks indicate the bands of anti-preS1 immunoreactive proteins (~48 and ~43 kDa) and anti-S immunoreactive proteins (~16, ~19, ~26, and ~29 kDa). The sizes (C) and ζ-potentials (D) of virosomes (green), virosomes containing DOX (red), and LPs (blue) were measured by DLS. Scale bars represent 100 nm.Abbreviations: BNC, bionanocapsule; LP, liposome; Try, trypsin; Ab, antibody; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; DOX, doxorubicin; DLS, dynamic light scattering.
Mentions: On TEM observation, the BNC–LP complexes prepared at room temperature under neutral conditions (pH 7.0) (Figure 2A, panel a) exhibited a rugged spherical structure of 112.5±19.7 nm (mean ± SD, n=10) in diameter, which agreed well with a previous report.17 The interface between BNCs and LP became obscure (Figure 2A, panel b), suggesting the occurrence of membrane fusion. When prepared at 70°C and pH 3.0, BNC–LP complexes exhibited a smooth-surfaced spherical structure of 111.4±19.6 nm (mean ± SD, n=10) in diameter (Figure 2A, panel c), which is similar to that of LPs (Figure 2A, panel d). When BNCs and BNC–LP complexes were digested with trypsin, anti-preS1 immunoreactive ~48 and ~43 kDa peptides (full-length N,O-glycosylated and N-glycosylated L protein, respectively) disappeared (Figure 2B, upper panel). Following trypsinization of BNCs, the anti-S immunoreactive peptides with molecular weights of ~16, ~19, ~26, and ~29 kDa were protected (Figure 2B, lower panel), these peptides corresponded to nonglycosylated and O-glycosylated forms of the ~16 kDa peptide (from Gly-19 in the preS2 region to Arg-129 in the S region) and ~26 kDa peptide (from Gly-19 in the preS2 region to the C-terminal in the S region), respectively.12 Similar peptides were found in the trypsinized BNC–LP complexes that strongly suggests that the membrane topology of Hepatitis B virus surface antigen (HBsAg) L protein was well conserved and the preS region was presented outside of the BNC–LP complexes. Based on the elimination of rugged structure in the BNC–LP complexes by incubation at 70°C in acidic conditions, it was postulated that HBsAg L proteins in BNCs were transferred to LPs, presumably by membrane fusion. Compared with the TEM images of previous BNC–LP complexes (Figure 2A, panel a),17 the shape was dramatically changed from a rugged spherical structure to a smooth spherical structure (Figure 2A, panel c). Since BNC contains fusogenic activity in the translocation motifs of the preS2 and/or S region,23,24 both high temperature and acidic conditions might enhance the membrane fusion between BNCs and LPs. In addition, the proteinase protection assay showed that the membrane topology of HBsAg L protein was well conserved after the fusion between BNCs and LPs. It was strongly suggested that HBsAg L proteins in BNCs were disassembled into micelles and then spontaneously integrated into LPs (Figure 3), indicating the formation of virosomes.19 While the composition of BNCs is ~10% (w/w) lipids and ~90% L proteins,25 that of virosomes is estimated to be ~96% lipids and ~4% L proteins. It was expected that virosomes can incorporate DOX by remote loading at comparable levels of LPs alone. Collectively, virosomes could be regarded as LPs transplanted with the targeting ability and fusogenic activity of HBsAg L protein, namely LPs armed with HBV-derived infection machineries.

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