Sulfation patterns determine cellular internalization of heparin-like polysaccharides

Abstract

Heparin is a highly sulfated polysaccharide that serves biologically relevant roles as an anticoagulant and anticancer agent. While it is well-known that modification of heparin's sulfation pattern can drastically influence its ability to bind growth factors and other extracellular molecules, very little is known about the cellular uptake of heparin and the role sulfation patterns serve in affecting its internalization. In this study, we chemically synthesized several fluorescently labeled heparins consisting of a variety of sulfation patterns. These polysaccharides were thoroughly characterized using anion exchange chromatography and size exclusion chromatography. Subsequently, we utilized flow cytometry and confocal imaging to show that sulfation patterns differentially affect the amount of heparin uptake in multiple cell types. This study provides the first comprehensive analysis of the effect of sulfation pattern on the cellular internalization of heparin or heparan sulfate like polysaccharides. The results of this study expand current knowledge regarding heparin internalization and provide insights into developing more effective heparin-based drug conjugates for applications in intracellular drug delivery.

Figure 1

Structures of modified heparins prepared in this study.

Figure 2

Sulfation patterns determine the total amount of cellular uptake of M. Heps at 6 hours. The different panels indicate M. Hep uptake in a) BLMVEC, b) K1 CHO, c) BXPC-3, d) HT-29, and e) U87 Mg cells. Values are normalized against heparin and show that M. Heps such as NA, NS, and CDSNS show enhanced cell uptake relative to heparin. Internalization of M. Heps was determined by fluorescence assisted cell sorting analysis as described in the experimental section. Cellular auto fluorescence at the settings used was minimal.

Figure 3

Sulfation patterns determine the cellular localization of M. Heps in U87 Mg cells. Panels in this image are fluorescence from Rhodamine Phalloidin (actin), DAPI (nucleus), Fluoresceinamine (M. Heps), and an overlay of all fluorophores. Representative substrates are: A) NA, B) NS, C) CDSNS, D) 2ODS, E) CDSHep, F) Heparin. It is evident that NA polymers colocalize with DAPI in the nucleus of U87 Mg cells.

Figure 4

Sulfation patterns determine the cellular localization of M. Heps, however no nuclear localization is visible for any substrate in HT-29 cells. Panels in this image are fluorescence from Rhodamine Phalloidin (actin), DAPI (nucleus), Fluoresceinamine (M. Heps), and an overlay of all fluorophores. Representative substrates are: A) NA, B) NS, C) CDSNS, D) 2ODS, E) CDSHep, F) Heparin.

Figure 5

Sulfation patterns determine rate of entry of M. Heps into HT-29 colon cancer cells. Values are normalized to the 30 hour time points for each substrate. Representative panels indicate the time-dependent internalization of: A) NA, B) NS, C) CDSNS, D) 2ODS, E) CDSHep, and F) Heparin

Figure 6

Sulfation pattern has little effect on the concentration dependence of internalization. Representative bars indicate the relative fluorescence measured by FACS when 20 µg (■), 100 µg (), or 200 µg (□) of M.Heps were incubated with cells for 24 hours. Values are normalized to the total substrate internalized after 24 hours when 200 ▪g of the respective substrate are added.