Hyaluronan is a key component in cryoprotection and formulation of targeted unilamellar liposomes

Abstract

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.

Supramolecular Organization and siRNA Binding of Hyaluronic Acid-Coated Lipoplexes for Targeted Delivery to the CD44 Receptor

“…(17,18) Surface modification of cationic liposomes with high-molecularweight HA can improve their efficacy by mediating active CD44 targeting in tumors and can also increase their circulation time because of a possible dysopsonization effect due to the hydrophilic coating effect of HA. (19)(20)(21)(22) In this study, we describe the design and physicochemical characterization of novel HA-lipoplexes entrapping siRNA. The lipoplex composition is based on cationic lipid 2-(2,3-didodecyloxypropyl)hydroxyethyl] ammonium bromide (DE), which has shown promising transfection efficiency in different cell lines compared to the cationic lipids currently available on the market.…”

Systemic Leukocyte-Directed siRNA Delivery Revealing Cyclin D1 as an Anti-Inflammatory Target

“…Hyaluronan was then attached to the outer surface of the liposomes, through covalent linkage to dipalmitoylphosphatidylethanolamine (DPPE), thereby stabilizing the particles both during subsequent siRNA entrapment ( Fig. 1) and during systemic circulation in vivo (11). The resulting stabilized nano-particles (sNPs) were successfully equipped with a targeting capacity by covalently attaching a monoclonal antibody against the integrins to hyaluronan (fig.…”

RNA interference-based nanosystems for inflammatory bowel disease therapy

“…The two considerations for introducing hyaluronan into nanoscale liposomes are as follows: 1) as a bridge linker, the FIB504 antibody subsequently attached to the hyaluronan and 2) hyaluronan was used to stabilize the particles during the cycle of lyophilization and rehydration. In another research report by Peer et al, 31 the surface-binding ligand of hyaluronan on liposomes acted as a cryoprotectant during lyophilization, which may derive from providing a substitute structure to form stabilizing H bonds. It was found that with hyaluronan bound to antibodies, the change in the mean particle size during lyophilization and rehydration was 107±14 to 123±24 nm, whereas a typically growing aggregation of 110±21 to 1,330±750 nm was detected without hyaluronan.…”