Hyaluronan is a Key Component in Cryoprotection and Formulation of Targeted Unilamellar Liposomes

Lyophilized unilamellar liposomes (ULV), the dosage form of choice for shelf-life, revert upon reconstitution to the larger multilamellar liposomes (MLV), which is detrimental to the many carrier-mediated therapies that require small particles. High doses of sugars such as trehalose, sucrose and others, included in the original formulations for cryoprotection, were shown to prevent the conversion to MLV. In this study we set out to test whether hyaluronan (HA), the surface-bound ligand in our previously developed targeted bioadhesive liposomes (BAL), can also act as a cryoprotectant. The studies included structural and physicochemical characterization of original and reconstituted hyaluronan-ULV (HA-ULV). For each HA-ULV, similar regular ULV (RL-ULV) served as controls. Four properties were tested: particle size, zeta potential, encapsulation efficiency and half-life of drug release (tau(1/2)), for three drugs-chloramphenicol (CAM), vinblastine (VIN) and mitomycin C (MMC). Encapsulation efficiencies of the original systems were quite alike for similar RL-ULV and HA-ULV ranging from 25% to 70%. All systems acted as sustained-release drug depots, tau(1/2) ranging from 1.3 to 5.3 days. Drug species and lipid composition were the major determinants of encapsulation and release magnitudes. By all tests, as anticipated, lyophilization generated significant changes in the reconstituted RL-ULV: 17-fold increase in diameter; tripling of zeta potential; 25-60% drop in encapsulation efficiencies; 25-30% decrease in tau(1/2). In contrast, the reconstituted HA-ULV retained the same dimensions, zeta potentials, encapsulation efficiencies and tau(1/2) of the original systems. These data clearly show HA to be a cryoprotectant, adding another clinically relevant advantage to HA-BAL. We propose that, like the sugars, HA cryoprotects by providing substitute structure-stabilizing H-bonds.