Interaction between CD44 and highly condensed hyaluronic acid through crosslinking with proteins.

Proteins and nucleic acids derived from bioresources have become a novel medical modality. However, the intrinsic features of proteins, such as their high molecular weight and polarity, impede their passage through the cell membrane. In this study, to deliver proteins to cancer cells, we prepared conjugates consisting of bovine serum albumin (BSA) and hyaluronic acid (HA). These conjugates contained multiple BSA and HA molecules and adopted a highly condensed structure by crosslinking through BSA. When the interaction between the HA-BSA conjugates and recombinant CD44 (rCD44) was examined using a quartz crystal microbalance, the conjugates induced a larger decrease of frequency change than HA. CT26 cells treated with FITC-labeled HA-BSA conjugates showed high fluorescence intensity. The uptake of the conjugates decreased upon adding excess HA. Therefore, the conjugates and nanoparticles with densely packed HA structures could be a potent and effective platform for delivering proteins to cancer tissues.

Modification of the antigenicity of cancer cells by conjugates consisting of hyaluronic acid and foreign antigens.

Tumor-specific cytotoxic T-lymphocytes (CTLs) recognize tumor-associated antigens presented on major histocompatibility complex (MHC) class I molecules. However, it is difficult to induce potent CTLs by vaccination because the antigenicity is not so high, compared with that of foreign antigens derived from viruses and microbes. The affinity of binding to MHC class I molecules is proportional to the antigenicity of the antigen that they are presenting. Here, we prepared several conjugates consisting of hyaluronic acid (HA) as a carrier to cancer cells and ovalbumin (OVA) as a foreign protein and changed the antigens on cancer cells from intrinsic antigens to OVA fragments. The conjugate containing multiple HA and OVA molecules (100k4HA-3OVA) adopted a highly condensed structure and was well recognized by recombinant CD44 molecules in quartz crystal microbalance analysis and incorporated into cancer cells (CT26 cells). A mixture of CT26 cells treated with 100k4HA-3OVA and splenocytes including OVA-specific CTLs induced abundant secretion of IFN-γ into the supernatant. At 48 h after mixing with the CTLs, almost all CT26 cells had died. These results indicate that 100k4HA-3OVA is actively internalized into the cells through interaction between HA and CD44. Subsequently, CT26 cells present not only self-antigens, but also OVA fragments on MHC class I molecules and are recognized by OVA-specific CTLs. We thus succeeded in modifying the antigenicity from self- to non-self-antigens on cancer cells. Therefore, this foreign-antigen delivery using HA to cancer cells, followed by antigen replacement, could be used as a novel strategy for treating cancers.