The anti-glypican 1 AT101 antibody as targeting agent to effectively deliver chitosan nanobubbles to glioblastoma cells

Abstract

Background: Recently, we developed AT101, an IgM-class mouse monoclonal antibody directed against glypican-1 (GPC1), a proteoglycan that can be considered as useful target for glioblastoma multiforme (GBM) treatment being specifically and highly expressed on GBM cell surface. Here, we proposed the use of AT101 as targeting agent in a drug delivery nanoplatfom to effectively deliver chitosan nanobubbles (NBs) for GBM treatment.

Methods: Chitosan NBs were prepared and conjugated with AT101 or left unconjugated as control.

Results: The ability of AT101 to bind the GPC1 protein was demonstrated by flow cytometry and immunofluorescence analysis in the "GBM-like" GPC1-expressing cell lines U-87 MG and T98G. AT101 was shown to bind GPC1-expressing GBM tumor samples by immunofluorescence. In-vivo experiments in the U-87 MG xenograft model showed that AT101 was able to bind GPC1 on cell surface and accumulate in U-87 MG tumor masses (p = 0.0002 respect to control). Moreover, in-vivo experiments showed that AT101 is able to target GPC1 when conjugated to chitosan NBs, thus increasing their specific deliver to GPC1-expressing cells of U-87 MG tumor, as compared to chitosan NBs not conjugated to AT101 (p = 0.02).

Conclusions: AT101 is an useful targeting agent for the development of drug delivery nanoplatforms for GBM treatment.

Keywords: GBM; GPC1; chitosan nanobubbles; monoclonal antibody; targeted therapy.

Figure 1.

Characterization of GPC1 expression. (a) Evaluation of GPC1 expression using α-GPC1c in 2 representative cases of frozen primary GBM tumor specimens and of healthy tissues (e.g., liver, lung). Negative controls refer to the use of the secondary antibody only. Cell nuclei are shown in blue and GPC1 is shown in red. Scale bar = 25 μm. (b) Evaluation of GPC1 expression by western blot in GBM cell line models using the α-GPC1c. The representative western blot shows GPC1 protein levels of U-87 MG and T98G cell lines, and Jurkat cell line as negative control. Vinculin was used as a housekeeping protein. (c) Evaluation of GPC1 expression by flow cytometry in GBM cell line models, and in the Jurkat cell line, employed as negative control. Histogram showing GPC1 expression in U-87 MG, T98G, and Jurkat cells. (* p < 0.05, p was calculated by using paired t-test). (d) Evaluation of GPC1 expression by immunofluorescence in GBM cell line models, and in the Jurkat cell line, employed as negative control. Representative immunofluorescence of GPC1 protein localization of U-87 MG, T98G, and Jurkat. The corresponding negative control is represented by the use of the secondary antibody only. Scale bar = 25 μm.

Figure 2.

Characterization of AT101. (a) Flow cytometry analysis to evaluate GPC1 expression in T98G, U-87 MG, and Jurkat cells using AT101. (* p<0.05, p was calculated using paired t-test). (b) Evaluation of GPC1 expression by immunofluorescence using AT101 in GBM cell line models, and in the Jurkat cell line. The corresponding negative control is represented by the use of the secondary antibody only. Scale bar = 25 μm.

Figure 3.

Evaluation of GPC1 expression in GBM tumor specimens by using AT101. evaluation of GPC1 expression using AT101 in 2 representative cases of frozen primary GBM tumor specimens and of healthy tissues (e.g., liver, lung). Negative controls refer to the use of the secondary antibody only. Cell nuclei are shown in blue and GPC1 is shown in red. Scale bar = 25 μm.

Figure 4.

Characterization of AT101 biodistribution. (a) In-vivo biodistribution of AT101 in the U-87 MG cell-bearing mouse model. A whole body scan of a representative mouse is shown of the in-vivo biodistribution, using VIVOVISION IVIS®Lumina, of AT101 (1 nanomole of Cy5.5) compared with the negative control (saline solution). Average fluorescent efficiency images were acquired at the indicated pre-injection and at different time points (24 hours (h), 48 hours, 72 hours, 96 hours). (b) dot-and-line plot of the in-vivo biodistribution, using VIVOVISION IVIS®Lumina, of AT101 (1 nanomole of Cy5.5) compared with the negative control (saline solution). Data are reported as average fluorescent efficiency mean ± sd (n = 3) (* p<0.05, p was calculated by using paired t-test). (c) Ex-vivo biodistribution, using VIVOVISION IVIS®Lumina, of AT101 (1 nanomole of Cy5.5) in comparison with the negative control (saline solution). Ex-vivo optical imaging of organs (tumor, brain, heart, kidneys, liver, spleen, lungs) explanted 96 hours after treatment with AT101 or administration of saline, as control, is shown. (d) bar chart showing average fluorescent efficiency for tumor, brain, kidneys, liver, lungs, spleen, and heart tissues explanted 96 h after administration of AT101 antibody and saline. Data are reported as average fluorescent efficiency mean ± SD (n = 3). (* p< 0.05, p was calculated using paired t-test). (e) Ex-vivo evaluation of AT101 deposition in the subcutaneous mouse model of U-87 MG analyzed 96 hours after administration by immunofluorescence. Upper panel: evaluation of IgM deposition in a mouse treated with AT101 and in a mouse treated with saline solution as negative control. Cell nuclei are shown in blue and IgM in green. Scale bar = 25 μm. Lower panel: histological evaluation of tumor tissue by hematoxylin-eosin in a mouse treated with AT101 and in a mouse treated with saline solution. In purple nuclei, in pink the cytoplasm.

Figure 5.

Characterization of AT101 contribute in the in-vivo biodistribution of chitosan NBs. (a) in-vivo biodistribution of Cy5.5-AT101-NB and Cy5.5-nb in the U-87 MG cell-bearing mouse model. Upper panel: a the whole body scan of a representative mouse is shown of the in-vivo biodistribution, using VIVOVISION IVIS®Lumina, of Cy5.5-AT101-nb (1 nanomole of Cy5.5) in comparison with the Cy5.5-nb (1 nanomole of Cy5.5). Average fluorescent efficiency images were acquired at pre-injection and at different time points (24 hours (h), 48 hours, 72 hours, 96 hours). Data are reported as average fluorescent efficiency mean ± SD (n = 3). (b) dot-and-line plot of the in-vivo biodistribution, using VIVOVISION IVIS®Lumina, of Cy5.5-AT101-nb (1 nanomole of Cy5.5) in comparison with Cy5.5-nb (1 nanomole of Cy5.5). Data are reported as average fluorescent efficiency mean ± SD (n = 3). (* p < 0.05, p was calculated using paired t-test). (c) Ex-vivo biodistribution, using VIVOVISION IVIS®Lumina, of Cy5.5-AT101-nb (1 nanomole of Cy5.5) compared with the Cy5.5-nb (1 nanomole of Cy5.5). Ex-vivo optical imaging of organs (tumor, brain, heart, kidneys, liver, spleen, lungs) explanted 96 hours after treatment with Cy5.5-AT101-nb or administration of Cy5.5-nb, is shown. (d) bar chart showing average fluorescence efficiency for tumor, brain, kidneys, liver, lungs, spleen, and heart tissues explanted 96 hours after administration of Cy5.5-AT101-nb or Cy5.5-nb. Data are reported as average fluorescent efficiency mean ± SD (n = 3). * p < 0.05, p was calculated using paired t-test.