Conjugates of gold nanoparticles and antifreeze protein III for cryopreservation of cells and tissues

Abstract

The development of non-toxic cryoprotectants is crucial for advancing fields such as regenerative medicine, cell therapy and tissue engineering, where the preservation of the viability of cells, tissues and organs during cryopreservation is essential. This interdisciplinary effort involves areas such as cryobiology, nanotechnology, biochemistry and material science to create more efficient and safer cryoprotective solutions. This study explores the development and application of gold nanoparticles (AuNPs) conjugated with antifreeze protein III (AFPIII) for improving the cryopreservation of bone marrow stem cells (bMSCs) encapsulated in alginate macrospheres (AMSs). Two types of AuNPs, stabilized with citrate (CitAuNPs) and BSPP (BSPPAuNPs), were functionalized with AFPIII using both covalent and non-covalent conjugation methods and were characterized for their size, surface charge and protein layer thickness. The cytotoxicity assays indicated that both types of AuNPs and their AFPIII conjugates had no adverse effects on bMSC viability and proliferation over 48 h, demonstrating their non-toxicity. Furthermore, the cryopreservation of bMSC-contained AMSs revealed that the covalent BSPPAuNPs-AFPIII conjugate provided superior preservation of cell viability and metabolic activity, outperforming both non-covalent conjugates and individual components. Cryomicroscopic analysis revealed that AFPIII altered ice crystal formation, promoting smaller, multidirectional crystals, which minimized cellular damage during freezing. The covalent BSPPAuNPs-AFPIII conjugate exhibited superior cryoprotective effects, preserving cell viability and function better than the non-covalent CitAuNPs-AFPIII conjugate. These findings suggest that AuNPs-AFPIII conjugates, particularly the covalent BSPPAuNPs-AFPIII complex, hold great promise for improving cell and tissue cryopreservation protocols.