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Systemic delivery of protein nanocages bearing CTT peptides for enhanced imaging of MMP-2 expression in metastatic tumor models.

Kawano T, Murata M, Piao JS, Narahara S, Hamano N, Kang JH, Hashizume M - Int J Mol Sci (2014)

Bottom Line: Here, we describe a metastatic cancer cell-targeted protein nanocage.An MMP-2-binding peptide, termed CTT peptide (CTTHWGFTLC), was conjugated to the surface of a naturally occurring heat shock protein nanocage by genetic modification.The engineered protein nanocages showed a binding affinity for MMP-2 and selective uptake in cancer cells that highly expressed MMP-2 in vitro.

View Article: PubMed Central - PubMed

Affiliation: Innovation Center for Medical Redox Navigation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. t-kawano@dem.med.kyushu-u.ac.jp.

ABSTRACT
Matrix metalloproteinase 2 (MMP-2) in metastatic cancer tissue, which is associated with a poor prognosis, is a potential target for tumor imaging in vivo. Here, we describe a metastatic cancer cell-targeted protein nanocage. An MMP-2-binding peptide, termed CTT peptide (CTTHWGFTLC), was conjugated to the surface of a naturally occurring heat shock protein nanocage by genetic modification. The engineered protein nanocages showed a binding affinity for MMP-2 and selective uptake in cancer cells that highly expressed MMP-2 in vitro. In near-infrared fluorescence imaging, the nanocages showed specific and significant accumulation in tumor tissue after intravenous injection in vivo. These protein nanocages conjugated with CTT peptide could be potentially applied to a noninvasive near-infrared fluorescence detection method for imaging gelatinase activity in metastatic tumors in vivo.

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Cellular uptake of the protein nanocages in vitro. (a) Representative histograms of the fluorescence intensities of HT1080 and HT29 cells incubated with fluorophore-labeled nanocages in the presence of 10% fetal bovine serum for 3 h; (b) Cytotoxicity of HspG41C-CTT in HT1080 and HT29 cells. Data are presented as the mean ± standard error of the mean of three independent experiments.
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ijms-16-00148-f004: Cellular uptake of the protein nanocages in vitro. (a) Representative histograms of the fluorescence intensities of HT1080 and HT29 cells incubated with fluorophore-labeled nanocages in the presence of 10% fetal bovine serum for 3 h; (b) Cytotoxicity of HspG41C-CTT in HT1080 and HT29 cells. Data are presented as the mean ± standard error of the mean of three independent experiments.

Mentions: For cellular uptake experiments, Hsp nanocages labeled with a fluorophore (Alexa Fluor 488) were prepared by the Michael addition reaction of Alexa Fluor 488 maleimide and Cys41 (interior of Hsp nanocages). Based on the results of size exclusion chromatography, conjugation with a fluorophore inside of the nanocage did not significantly affect the structure or size of the nanocage. To determine whether HspG41C-CTT nanocages were selectively taken up by MMP-2 targeting, HT1080 and HT29 cells were used as positive and negative controls for MMP-2 expression, respectively. Cellular uptake was observed after 3 h of incubation using a flow cytometer (Figure 4a). Non-targeted HspG41C nanocages were taken up by both cell lines. However, HspG41C-CTT nanocages were taken up by HT1080 cells but not HT29 cells. Cell uptake of HspG41C displaying negatively charged linkers decreased by HT1080 cells due to reduce the non-specific binding. These results showed that the nanocages modified with CTT peptides selectively bound to HT1080 cells that highly expressed MMP-2. MMP-2 is known to localize to the cell surface and cytoplasmic compartment and release into the extracellular space, but is not known to internalize inhibitors upon binding [24,25]. The cellular uptakes of CTT-peptides conjugated imaging agents and liposome were energy dependent active receptor mediated endocytosis, as indicated by the observation of significantly reduced uptake at 4 °C [26,27]. Further studies are needed to clarify the role of MMP-2 in cellular internalization of HspG41C-CTT. Because cytotoxicity is an important factor in selecting materials for the development of imaging carriers, we characterized the effects of the nanocages on cell viability under the same conditions that were used to determine cellular uptake. The results showed no appreciable cytotoxic effects of HspG41C or HspG41C-CTT on HT1080 and HT29 cells (Figure 4b).


Systemic delivery of protein nanocages bearing CTT peptides for enhanced imaging of MMP-2 expression in metastatic tumor models.

Kawano T, Murata M, Piao JS, Narahara S, Hamano N, Kang JH, Hashizume M - Int J Mol Sci (2014)

Cellular uptake of the protein nanocages in vitro. (a) Representative histograms of the fluorescence intensities of HT1080 and HT29 cells incubated with fluorophore-labeled nanocages in the presence of 10% fetal bovine serum for 3 h; (b) Cytotoxicity of HspG41C-CTT in HT1080 and HT29 cells. Data are presented as the mean ± standard error of the mean of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4307240&req=5

ijms-16-00148-f004: Cellular uptake of the protein nanocages in vitro. (a) Representative histograms of the fluorescence intensities of HT1080 and HT29 cells incubated with fluorophore-labeled nanocages in the presence of 10% fetal bovine serum for 3 h; (b) Cytotoxicity of HspG41C-CTT in HT1080 and HT29 cells. Data are presented as the mean ± standard error of the mean of three independent experiments.
Mentions: For cellular uptake experiments, Hsp nanocages labeled with a fluorophore (Alexa Fluor 488) were prepared by the Michael addition reaction of Alexa Fluor 488 maleimide and Cys41 (interior of Hsp nanocages). Based on the results of size exclusion chromatography, conjugation with a fluorophore inside of the nanocage did not significantly affect the structure or size of the nanocage. To determine whether HspG41C-CTT nanocages were selectively taken up by MMP-2 targeting, HT1080 and HT29 cells were used as positive and negative controls for MMP-2 expression, respectively. Cellular uptake was observed after 3 h of incubation using a flow cytometer (Figure 4a). Non-targeted HspG41C nanocages were taken up by both cell lines. However, HspG41C-CTT nanocages were taken up by HT1080 cells but not HT29 cells. Cell uptake of HspG41C displaying negatively charged linkers decreased by HT1080 cells due to reduce the non-specific binding. These results showed that the nanocages modified with CTT peptides selectively bound to HT1080 cells that highly expressed MMP-2. MMP-2 is known to localize to the cell surface and cytoplasmic compartment and release into the extracellular space, but is not known to internalize inhibitors upon binding [24,25]. The cellular uptakes of CTT-peptides conjugated imaging agents and liposome were energy dependent active receptor mediated endocytosis, as indicated by the observation of significantly reduced uptake at 4 °C [26,27]. Further studies are needed to clarify the role of MMP-2 in cellular internalization of HspG41C-CTT. Because cytotoxicity is an important factor in selecting materials for the development of imaging carriers, we characterized the effects of the nanocages on cell viability under the same conditions that were used to determine cellular uptake. The results showed no appreciable cytotoxic effects of HspG41C or HspG41C-CTT on HT1080 and HT29 cells (Figure 4b).

Bottom Line: Here, we describe a metastatic cancer cell-targeted protein nanocage.An MMP-2-binding peptide, termed CTT peptide (CTTHWGFTLC), was conjugated to the surface of a naturally occurring heat shock protein nanocage by genetic modification.The engineered protein nanocages showed a binding affinity for MMP-2 and selective uptake in cancer cells that highly expressed MMP-2 in vitro.

View Article: PubMed Central - PubMed

Affiliation: Innovation Center for Medical Redox Navigation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. t-kawano@dem.med.kyushu-u.ac.jp.

ABSTRACT
Matrix metalloproteinase 2 (MMP-2) in metastatic cancer tissue, which is associated with a poor prognosis, is a potential target for tumor imaging in vivo. Here, we describe a metastatic cancer cell-targeted protein nanocage. An MMP-2-binding peptide, termed CTT peptide (CTTHWGFTLC), was conjugated to the surface of a naturally occurring heat shock protein nanocage by genetic modification. The engineered protein nanocages showed a binding affinity for MMP-2 and selective uptake in cancer cells that highly expressed MMP-2 in vitro. In near-infrared fluorescence imaging, the nanocages showed specific and significant accumulation in tumor tissue after intravenous injection in vivo. These protein nanocages conjugated with CTT peptide could be potentially applied to a noninvasive near-infrared fluorescence detection method for imaging gelatinase activity in metastatic tumors in vivo.

Show MeSH
Related in: MedlinePlus