Glycan structure determinants for cation-independent mannose 6-phosphate receptor binding and cellular uptake of a recombinant protein.

The cation-independent mannose 6-phosphate receptor (CI-MPR) plays a critical role in intracellular transport of lysosomal enzymes as well as the uptake of recombinant proteins. To define the minimal glycan structure determinants necessary for receptor binding and cellular uptake, we synthesized a series of glycans containing mono-, di-, tri-, tetra-, and hexamannoses terminated with either one or two phosphates for conjugating to a model protein, recombinant human acid α-glucosidase. A high affinity interaction with the CI-MPR can be achieved for the enzyme conjugated to a dimannose glycan with a single phosphate. However, tightest binding to a CI-MPR affinity column was observed with a hexamannose structure containing two phosphates. Moreover, maximal cellular uptake and a 5-fold improvement in in vivo potency were achieved when the bisphosphorylated hexamannose glycan is conjugated to the protein by a ß linker. Nevertheless, even a monophosphorylated dimannose glycan conjugate showed stronger binding to the receptor affinity column, higher cellular uptake, and significantly greater in vivo efficacy compared to the unconjugated protein which contains a low level of high affinity glycan structure. These results demonstrate that the phosphorylated dimannose moiety appears to be the minimal structure determinant for enhanced CI-MPR binding and that the orientation of the glycan is critical for maximum receptor interaction. In summary, we have improved the understanding of the mechanism of CI-MPR binding and developed a simple alternative for CI-MPR targeting.

Assessment of in vivo degradation profiles of hyaluronic acid hydrogels using temporal evolution of chemical exchange saturation transfer (CEST) MRI.

Hyaluronic acid (HA) hydrogels have found a wide range of applications in biomedicine: regenerative medicine to drug delivery applications. In vivo quantitative assessment of these hydrogels using magnetic resonance imaging (MRI) provides an effective, accurate, safe, and non-invasive translational approach to assess the biodegradability of HA hydrogels. Chemical exchange saturation transfer (CEST) is an MRI contrast enhancement technique that overcomes the concentration limitation of other techniques like magnetic resonance spectroscopy (MRS) by detecting metabolites at up to two orders of magnitude or higher. In this study, HA hydrogels were synthesized based on different crosslinking agents and assessed using CEST MRI to investigate the in vivo degradation profiles of these gels in a mouse subcutaneous injection model over a three-month period. Nature of crosslinking agents was found to influence their degradation profiles. Since CEST MRI provides a unique chemical signature to visualize HA hydrogels, our studies proved that this technique could be used as a guide in the hydrogel optimization process for drug delivery and regenerative medicine applications.