Glioblastoma multiforme (GBM) the most aggressive (WHO grade IV) diffuse glioma, is also by far the most frequent one. After standard treatment, the 2-year overall survival of GBM patients is approximately only 25%. Although numerous experimental drugs have been tested in clinical trials, GBM patients have not yet profited of curative treatments. To overcome the big limitations regarding GBM treatment, we address the challenge of developing a drug delivery system based on highly biocompatible chitosan nanobubbles (NBs) conjugated with an anti-glypican1 (GPC1) antibody loaded with docetaxel as chemotherapeutic agent. This drug delivery approach has been proposed to counter major challenges as overcoming the BBB, allowing the therapeutic release exclusively to tumor cells, and minimizing the possible side effects in cancer patients. The GPC1 proteoglycan has been chosen as useful target for drug delivering with NBs, therefore GPC1 expression was characterized in-vitro, being found expressed in GBM cell lines (e.g., T98G, U87-MG) but not found expressed in non-GBM cell line. Consistently, we assessed the localization of GPC1 protein expression on the cell surface and in the cytoplasm of GBM cell lines whereas it was lacked in the negative control cells. Of note, in primary tumor sections of these 10 GBM cases, GPC1 was found overexpressed whereas in normal tissues was found not expressed. To obtain a specific anti-GPC1 antibody recognizing the last 70 amino acid of GPC1 protein and therefore the cell-surface form of GPC1, mouse immunization has been performed. Hybridomas have produced three different anti-GPC1 specific clones (A, B, C). By using the B and C clones, GPC1 expression was detected in GBM cells at levels comparable to the levels obtained by using the commercially available antibody by the B and C clones. On the contrary, the A clone was not capable to recognize GPC1. Therefore, we purified the B and C clones to obtain specific anti-GPC1 monoclonal Abs. Moreover, C and B appeared to be more efficient than the a-GPC1c for detection of GPC1 expression levels. According to the results of antibody testing in GBM cell lines and negative control cell lines, the B clone was chosen to be conjugated to the NBs to develop the active drug delivery strategy. To select the drug to be loaded in the NBs, the killing capability of temozolomide (TMZ), paclitaxel (PTX) and docetaxel (DTX) was evaluated in GBM cells. DTX have the highest killing capability compared to PTX and TMZ. Therefore, we used DTX for the NBs loading encapsulation. The in-vitro characterization of NBs showed the average diameter of about 350 nm and a positive charge and spherical morphology. In-vitro analysis of the treatment of NBs in GBM cells, showed the localization of NBs conjugated with B antibody in cell cytoplasm around the nucleus. In contrast, a lower mean fluorescence intensity was observed for the cells treated with unconjugated NBs. For the in-vitro cytotoxic effect of NBs, NB loaded with DTX, NBs loaded with DTX and conjugated with B antibody, showed a killing capability correlated with the concentration in each evaluated point, with cell viable levels comparable to those of free DTX for some concentrations. Blank NBs, NB conjugated with Cy 5.5, and NB conjugated with B antibody were not toxic at all tested concentrations. In-vivo and ex-vivo test of the biodistribution of anti-GPC1 NBs in xenograft GBM mouse models, showed that the presence of the conjugation with the B antibody seems to be allow a major accumulation of the injected NBs in the tumor as well as a higher retention time at least until the last time point of 96 h of treatment. In conclusion, the proposed active drug delivery approach using anti-GPC1 conjugated NBs loaded with DTX could be useful for the treatment of GBM.

Glioblastoma multiforme (GBM) the most aggressive (WHO grade IV) diffuse glioma, is also by far the most frequent one. After standard treatment, the 2-year overall survival of GBM patients is approximately only 25%. Although numerous experimental drugs have been tested in clinical trials, GBM patients have not yet profited of curative treatments. To overcome the big limitations regarding GBM treatment, we address the challenge of developing a drug delivery system based on highly biocompatible chitosan nanobubbles (NBs) conjugated with an anti-glypican1 (GPC1) antibody loaded with docetaxel as chemotherapeutic agent. This drug delivery approach has been proposed to counter major challenges as overcoming the BBB, allowing the therapeutic release exclusively to tumor cells, and minimizing the possible side effects in cancer patients. The GPC1 proteoglycan has been chosen as useful target for drug delivering with NBs, therefore GPC1 expression was characterized in-vitro, being found expressed in GBM cell lines (e.g., T98G, U87-MG) but not found expressed in non-GBM cell line. Consistently, we assessed the localization of GPC1 protein expression on the cell surface and in the cytoplasm of GBM cell lines whereas it was lacked in the negative control cells. Of note, in primary tumor sections of these 10 GBM cases, GPC1 was found overexpressed whereas in normal tissues was found not expressed. To obtain a specific anti-GPC1 antibody recognizing the last 70 amino acid of GPC1 protein and therefore the cell-surface form of GPC1, mouse immunization has been performed. Hybridomas have produced three different anti-GPC1 specific clones (A, B, C). By using the B and C clones, GPC1 expression was detected in GBM cells at levels comparable to the levels obtained by using the commercially available antibody by the B and C clones. On the contrary, the A clone was not capable to recognize GPC1. Therefore, we purified the B and C clones to obtain specific anti-GPC1 monoclonal Abs. Moreover, C and B appeared to be more efficient than the a-GPC1c for detection of GPC1 expression levels. According to the results of antibody testing in GBM cell lines and negative control cell lines, the B clone was chosen to be conjugated to the NBs to develop the active drug delivery strategy. To select the drug to be loaded in the NBs, the killing capability of temozolomide (TMZ), paclitaxel (PTX) and docetaxel (DTX) was evaluated in GBM cells. DTX have the highest killing capability compared to PTX and TMZ. Therefore, we used DTX for the NBs loading encapsulation. The in-vitro characterization of NBs showed the average diameter of about 350 nm and a positive charge and spherical morphology. In-vitro analysis of the treatment of NBs in GBM cells, showed the localization of NBs conjugated with B antibody in cell cytoplasm around the nucleus. In contrast, a lower mean fluorescence intensity was observed for the cells treated with unconjugated NBs. For the in-vitro cytotoxic effect of NBs, NB loaded with DTX, NBs loaded with DTX and conjugated with B antibody, showed a killing capability correlated with the concentration in each evaluated point, with cell viable levels comparable to those of free DTX for some concentrations. Blank NBs, NB conjugated with Cy 5.5, and NB conjugated with B antibody were not toxic at all tested concentrations. In-vivo and ex-vivo test of the biodistribution of anti-GPC1 NBs in xenograft GBM mouse models, showed that the presence of the conjugation with the B antibody seems to be allow a major accumulation of the injected NBs in the tumor as well as a higher retention time at least until the last time point of 96 h of treatment. In conclusion, the proposed active drug delivery approach using anti-GPC1 conjugated NBs loaded with DTX could be useful for the treatment of GBM.

Nanoparticles anti-GPC1 for glioblastoma multiforme treatment

DI CINTIO, FEDERICA
2022-03-11T00:00:00+01:00

Abstract

Glioblastoma multiforme (GBM) the most aggressive (WHO grade IV) diffuse glioma, is also by far the most frequent one. After standard treatment, the 2-year overall survival of GBM patients is approximately only 25%. Although numerous experimental drugs have been tested in clinical trials, GBM patients have not yet profited of curative treatments. To overcome the big limitations regarding GBM treatment, we address the challenge of developing a drug delivery system based on highly biocompatible chitosan nanobubbles (NBs) conjugated with an anti-glypican1 (GPC1) antibody loaded with docetaxel as chemotherapeutic agent. This drug delivery approach has been proposed to counter major challenges as overcoming the BBB, allowing the therapeutic release exclusively to tumor cells, and minimizing the possible side effects in cancer patients. The GPC1 proteoglycan has been chosen as useful target for drug delivering with NBs, therefore GPC1 expression was characterized in-vitro, being found expressed in GBM cell lines (e.g., T98G, U87-MG) but not found expressed in non-GBM cell line. Consistently, we assessed the localization of GPC1 protein expression on the cell surface and in the cytoplasm of GBM cell lines whereas it was lacked in the negative control cells. Of note, in primary tumor sections of these 10 GBM cases, GPC1 was found overexpressed whereas in normal tissues was found not expressed. To obtain a specific anti-GPC1 antibody recognizing the last 70 amino acid of GPC1 protein and therefore the cell-surface form of GPC1, mouse immunization has been performed. Hybridomas have produced three different anti-GPC1 specific clones (A, B, C). By using the B and C clones, GPC1 expression was detected in GBM cells at levels comparable to the levels obtained by using the commercially available antibody by the B and C clones. On the contrary, the A clone was not capable to recognize GPC1. Therefore, we purified the B and C clones to obtain specific anti-GPC1 monoclonal Abs. Moreover, C and B appeared to be more efficient than the a-GPC1c for detection of GPC1 expression levels. According to the results of antibody testing in GBM cell lines and negative control cell lines, the B clone was chosen to be conjugated to the NBs to develop the active drug delivery strategy. To select the drug to be loaded in the NBs, the killing capability of temozolomide (TMZ), paclitaxel (PTX) and docetaxel (DTX) was evaluated in GBM cells. DTX have the highest killing capability compared to PTX and TMZ. Therefore, we used DTX for the NBs loading encapsulation. The in-vitro characterization of NBs showed the average diameter of about 350 nm and a positive charge and spherical morphology. In-vitro analysis of the treatment of NBs in GBM cells, showed the localization of NBs conjugated with B antibody in cell cytoplasm around the nucleus. In contrast, a lower mean fluorescence intensity was observed for the cells treated with unconjugated NBs. For the in-vitro cytotoxic effect of NBs, NB loaded with DTX, NBs loaded with DTX and conjugated with B antibody, showed a killing capability correlated with the concentration in each evaluated point, with cell viable levels comparable to those of free DTX for some concentrations. Blank NBs, NB conjugated with Cy 5.5, and NB conjugated with B antibody were not toxic at all tested concentrations. In-vivo and ex-vivo test of the biodistribution of anti-GPC1 NBs in xenograft GBM mouse models, showed that the presence of the conjugation with the B antibody seems to be allow a major accumulation of the injected NBs in the tumor as well as a higher retention time at least until the last time point of 96 h of treatment. In conclusion, the proposed active drug delivery approach using anti-GPC1 conjugated NBs loaded with DTX could be useful for the treatment of GBM.
34
2020/2021
Settore BIO/14 - Farmacologia
Università degli Studi di Trieste
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11368/3015204
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