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Synthetic Integrin-Targeting Dextran-Fc Hybrids Efficiently Inhibit Tumor Proliferation In Vitro.

ABSTRACT: Herein, we present the design, synthesis, and biological evaluation of novel integrin-targeting molecular hybrids combining RGD peptides and a potent cytotoxin presented on dextran polysaccharides. Based on an aglycosylated Fc as a centerpiece, endosomal-cleavable cytotoxic agent monomethyl auristatin E (MMAE) and dextran as multimerization site were covalently connected by two bioorthogonal enzyme-mediated reactions site-specifically. Decoration of dextran with cyclic RGD peptides, introduced by copper "click" reaction, resulted in the final constructs with the potential to kill integrin-overexpressing tumor cells. We found that these modifications had little impact on the stability of the Fc scaffold and the RGD-bearing construct showed good binding properties of αvβ3-expressing U87MG cells. Furthermore, the construct showed a remarkable antiproliferative activity. These results demonstrate the general capability of our design to provoke receptor-mediated endocytosis upon binding to the cellular surface, followed by endosomal cleavage of the linkage between Fc-dextran and MMAE and its subsequent release. Our approach opens new avenues to transcribe small molecule binders into tailor-made multimeric molecular hybrids with antitumor potential.

SUBMITTER: Schneider H

PROVIDER: S-EPMC8339797 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:PurposeTo synthesize a ternary cationic copolymer called CS-g-(PEI-b-mPEG) and characterize its features as a non-viral siRNA carrier; in turn, to investigate the influence of small interfering RNA (siRNA) targeting IkappaB kinase subunit beta (IKKbeta) on the proliferation of human Tenon's capsule fibroblasts (HTFs) in vitro.MethodsFirst, a novel cationic copolymer composed of low molecular weight, linear poly(ethyleneimine) [PEI] blocked with polyethylene glycol (PEG) and grafted onto a chitosan (CS) molecule was synthesized. CS-g-(PEI-b-mPEG) was then compacted with 21nt siRNA at various copolymer/siRNA charge (N/P) ratios, and the resulting complexes were characterized by dynamic light scattering, gel electrophoresis, and serum incubation. Cell Titer 96 AQ(ueous) One Solution cell proliferation assay was used to investigate the cytotoxicity of this cationic copolymer. Second, siRNAs targeting IKKbeta (IKKBeta-siRNAs) were delivered into the HTFs using CS-g-(PEI-b-mPEG) as the vehicle. Real-time reverse transcription polymerase chain reaction (RT-PCR) subsequently assessed the mRNA level of IKKbeta, and western blot assay was used to determine protein expression. After IKKB-siRNA transfection, Cell Titer 96 AQ(ueous) One Solution cell proliferation assay was used to evaluate the proliferation of HTFs.ResultsThe diameter of the CS-g-(PEI-b-mPEG)/siRNA complexes tended to decrease whereas their zeta potential tended to increase as the N/P ratio increased. The CS-g-(PEI-b-mPEG) copolymer showed good siRNA binding ability and high siRNA protection capacity. Furthermore, the copolymer presented remarkable transfection efficiency and showed much less cytotoxicity than 25 kDa PEI. IKKB-siRNAs were successfully delivered into HTFs using CS-g-(PEI-b-mPEG) as a vector. As a result, the expression of IKKbeta was downregulated at both the mRNA and protein levels, and the activation of nuclear factor-kappaB (NF-kappaB) in the HTFs was subsequently inhibited. Most impressively, the proliferation of HTFs was also effectively suppressed through the blocking of the NF-kappaB pathway.ConclusionsAll the results demonstrate that CS-g-(PEI-b-mPEG) is a promising candidate for siRNA delivery, featuring excellent biocompatibility, biodegradability, and transfection efficiency. The RNA interference (RNAi) strategy using cationic copolymers as siRNA carriers will be a safe and efficient anti-scarring method following glaucoma filtration surgery.

Project description:Fucosylation is one of the most prevalent modifications on N- and O-glycans of glycoproteins, and it plays an important role in various cellular processes and diseases. Small molecule inhibitors of fucosylation have shown promise as therapeutic agents for sickle cell disease, arthritis, and cancer. We describe here the design and synthesis of a panel of fluorinated l-fucose analogs bearing fluorine atoms at the C2 and/or C6 positions of l-fucose as metabolic fucosylation inhibitors. Preliminary study of their effects on cell proliferation revealed that the 6,6-difluoro-l-fucose (3) and 6,6,6-trifluoro-l-fucose (6) showed significant inhibitory activity against proliferation of human colon cancer cells and human umbilical vein endothelial cells. In contrast, the previously reported 2-deoxy-2-fluoro-l-fucose (1) had no apparent effects on proliferations of all the cell lines tested. To understand the mechanism of cell proliferation inhibition by the fluorinated l-fucose analogs, we performed chemoenzymatic synthesis of the corresponding GDP-fluorinated l-fucose analogs and tested their inhibitory activities against the mammalian α1,6-fucosyltransferase (FUT8). Interestingly, the corresponding GDP derivatives of 6,6-difluoro-l-fucose (3) and 6,6,6-trifluoro-l-fucose (6), which are the stronger proliferation inhibitors, showed much weaker inhibitory activity against FUT8 than that of the 2-deoxy-2-fluoro-l-fucose (1). These results suggest that FUT8 is not the major target of the 6-fluorinated fucose analogs (3 and 6). Instead, other factors, such as the key enzymes involved in the de novo GDP-fucose biosynthetic pathway and/or other fucosyltransferases involved in the biosynthesis of tumor-associated glyco-epitopes are most likely the targets of the fluorinated l-fucose analogs to achieve cell proliferation inhibition. To our knowledge, this is the first comparative study of various fluorinated l-fucose analogs for suppressing the proliferation of human cancer and primary endothelial cells required for angiogenesis.

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Synthetic Integrin-Targeting Dextran-Fc Hybrids Efficiently Inhibit Tumor Proliferation In Vitro.

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