| Summary: | 碩士 === 國立臺灣大學 === 藥學研究所 === 100 === Many protein drugs need to overcome the cellular membrane barrier and be delivered intracellularly to exert their therapeutic action within the cell. Therefore, the development of a suitable strategy for intracellular protein delivery is a topic worth investigating. Active targeting delivery is one of the commonly used drug delivery strategy. Specific ligands are conjugated on the surface of the drug or carrier, and therefore enable the entire system to be recognized by a specific receptor expressed on the cellular surface of the target tissues and be taken into cells via receptor-mediated endocytosis. This strategy can further enhance the therapeutic efficacy of delivered drug or carrier, and at the same time reduce possible side effects on other tissues. Liver expresses the membrane protein ASGPR (asialoglycoprotein receptor) that can specifically recognize galactose residues. Therefore, we aim to modify protein with galactose ligand and then investigate the effect of galactose number on liver cellular uptake, and further apply this strategy to intracellular protein delivery.
First, we first used cytochrome C (cytC) as a model protein. Different numbers of galactose-CHO were conjugated to the amino groups on cytC via reductive amination to obtain cytC-galactose conjugates (cytC-gal). Hepatocellular carcinoma cell line, HepG2, and breast adenocarcinoma cell line, MCF7, were treated with cytC-gal conjugates to evaluate the intracellular delivery after galactose modification. CytC-gal were further labeled with FITC to detect cellular uptake via flow cytometry. We also performed MTT assay as the cell viability assay to investigate the effect of various cytC-gal conjugates on cell viability.
Our results showed that it took 16 hours overnight to conjugate 12 galactose residues to cytC, which greatly reduced the reaction time compared with traditional method. In terms of cellular uptake, cellular uptake of cytC-FITC-gal8 in HepG2 was significantly higher than cytC-FITC (p < 0.001), but similar observation was not seen on MCF7. According to literature, liver tissues express a type of lectin named asialoglycoprotein receptor (ASGPR) that specifically recognizes glycoproteins containing terminal galactose or N-acetylgalactosamine residues. To understand if the uptake was ASGPR-mediated, we co-treated cells with excess asialofetuin (ASF), a native ligand for ASGPR. In HepG2 cells, co-incubation of cytC-FITC-gal8 with only 1 fold of ASF can significantly reduce both the percent of FITC-positive cells and fluorescence intensity (p <0.001), suggesting that ASGPR is a major pathway for cytC-FITC-gal8 cellular uptake in HepG2 cells. On the other hand, in MCF7, co-incubation of cytC-FITC-gal8 with 30 fold of ASF still had no effect on cellular uptake, implying that the cellular uptake of cytC-FITC-gal8 was not ASGPR-mediated.
By adjusting the feed ratio of galactose-CHO to protein, we conjugated 5, 8 and 12 galactose residues. In cell viability experiments, we observed that a three cytC-gal conjugates can significantly reduce cell viability in both HepG2 and MCF7 cell lines in a dose-dependent manner. It indirectly indicated that galactose modification can enhance the intracellular delivery of cytC and induce cellular apoptosis. Cells were further treated with cytC-gal with identical enzyme activity to further investigate the effect of galactse residue numbers on intracellular delivery. However, we noticed that increasing galactose ligand from 5 to 8 and 12 did not further reduce cell viability in both cell lines, suggesting that the cellular uptake cannot be further enhanced either. We further treated cells with the endosomal escape reagent, chloroquine, to understand if the cellular uptake of cytC-gal was endosome-mediated. In HepG2 cells, it was found that chloroquine could further decrease the cell viability, suggesting that chloroquine might avoid further lysosomal degradation and facilitate the release of endosome-trapped cytC-gal into cytosol to exert therapeutic action. However, in the other cell line, MCF7, co-incubation of chloroquine had no further effect on cellular viability, suggesting that cytC was uptaken via endosome-independent pathway in MCF cells.
Although recombinant arginine deiminase (rADI) is a potent cell proliferation inhibitor towards most hepatocellular carcinoma and melanoma, rADI resistance is still observed in certain tumor cells, such as HepG2. Our lab previously showed intracellular delivery of rADI into resistant cells by cell-penetrating peptide restored sensitivity to rADI treatment. For this reason, we conjugated galactose-CHO to rADI in order to restore rADI sensitivity on HepG2 cells. We also performed cell viability assay and results shown that galactose modification could significantly inhibit cell proliferation, implying more rADI delivered into HepG2 cells.
To conclude, galactose-CHO could conjugate galactose ligand to both cytC and rADI protein and enhance anti-proliferation effect in HepG2 and MCF7 cells. The cellular uptake into HepG2 cells were via ASGPR; the cellular uptake for MCF7 cells was not observed, therefore the reason of anti-proliferation effect on MCF7 cells remained unclear.
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