Freezing-mediated formation of supraproteins using depletion forces.

Hypothesis Long-acting formulations such as microparticles, injectable depots and implantable devices can realize spatiotemporally controlled delivery of protein drugs to extend their therapeutic in vivo half-lives. To efficiently encapsulate the protein drugs into such drug delivery systems, (sub)micron-sized protein particles are needed. The formation of micronized supraproteins can be induced through the synergistic combination of attractive depletion forces and freezing. The size of the supraproteins can be fine-tuned from submicron to several microns by adjusting the ice crystallization rate through the freeze-quench depth, which is set by the target temperature. Methods Supraprotein micron structures were prepared from protein solutions under various conditions in the presence and absence of nonadsorbing polyethylene glycol. Scanning electron microscopy and dynamic light scattering were employed to determine the sizes of the supraproteins and real-time total internal reflection fluorescent microscopy was used to follow the supraprotein formation during freezing. The protein secondary structure was measured before and after micronization by circular dichroism. A phase diagram of a protein-polyethylene glycol mixture was theoretically predicted to investigate whether the depletion interaction can elucidate the phase behavior. Findings Micronized protein supraparticles could be prepared in a controlled manner by rapid freeze-drying of aqueous mixtures of bovine serum albumin, horseradish peroxidase and lysozyme mixed with polyethylene glycol. Upon freezing, the temperature quench initiates a phase separation process which is reminiscent of spinodal decomposition. This demixing is subsequently arrested during droplet phase separation to form protein-rich microstructures. The final size of the generated protein microparticles is determined by a competition between phase separation and cooling rate, which can be controlled by target temperature. The experimental phase diagram of the aqueous protein-polyethylene glycol dispersion aligns with predictions from depletion theory for charged colloids and nonadsorbing polymers.

Design of block-copolymer-based micelles for active and passive targeting.

A self-consistent field study is presented on the design of active and passive targeting block-copolymeric micelles. These micelles form in water by self-assembly of triblock copolymers with a hydrophilic middle block and two hydrophobic outer blocks. A minority amount of diblock copolymers with the same chemistry is taken to coassemble into these micelles. At the end of the hydrophilic block of the diblock copolymers, a targeting moiety (TM) is present. Assuming that the rotation of the micelle towards the target is sufficiently fast, we can elaborate a single gradient cell model, wherein the micelle is in the center and the receptor (R) substrate exists on the outer plane of the spherical coordinate system. The distribution function of the targeting moiety corresponds to a Landau free energy with local minima and corresponding maxima. The lowest minimum, which is the ground state, shifts from within the micelle to the adsorbing state upon bringing the substrate closer to the micelle, implying a jumplike translocation of the targeting moiety. Equally deep minima represent the binodal of the phase transition, which is, due to the finite chain length, first-order like. The maximum in-between the two relevant minima implies that there is an activation barrier for the targeting moiety to reach the receptor surface. We localize the parameter space wherein the targeting moiety is (when the micelle is far from the target) preferably hidden in the stealthy hydrophilic corona of the micelle, which is desirable to avoid undesired immune responses, and still can jump out of the corona to reach the target quick enough, that is, when the barrier height is sufficiently low. The latter requirement may be identified by a spinodal condition. We found that such hidden TMs can still establish a TM-R contact at distances up to twice the corona size. The translocation transition will work best when the affinity of the TM for the core is avoided and when hydrophilic TMs are selected.

Formulation and antifungal performance of natamycin-loaded liposomal suspensions: the benefits of sterol-enrichment.

The aim of this study is to develop and evaluate food-grade liposomal delivery systems for the antifungal compound natamycin. Liposomes made of various soybean lecithins are prepared by solvent injection, leading to small unilamellar vesicles (<130 nm) with controlled polydispersity, able to encapsulate natamycin without significant modification of their size characteristics. Presence of charged phospholipids and reduced content of phosphatidylcholine in the lecithin mixture are found to be beneficial for natamycin encapsulation, indicating electrostatic interactions of the preservative with the polar head of the phospholipids. The chemical instability of natamycin upon storage in these formulations is however significant and proves that uncontrolled leakage out of the liposomes occurs. Efficient prevention of natamycin degradation is obtained by incorporation of sterols (cholesterol, ergosterol) in the lipid mixture and is linked to higher entrapment levels and reduced permeability of the phospholipid membrane provided by the ordering effect of sterols. Comparable action of ergosterol is observed at concentrations 2.5-fold lower than cholesterol and attributed to a preferential interaction of natamycin-ergosterol as well as a higher control of membrane permeability. Fine-tuning of sterol concentration allows preparation of liposomal suspensions presenting modulated in vitro release kinetics rates and enhanced antifungal activity against the model yeast Saccharomyces cerevisiae.

Controlled block copolymer micelle formation for encapsulation of hydrophobic ingredients.

We report on the formation of polymeric micelles in water using triblock copolymers with a polyethylene glycol middle block and various hydrophobic outer blocks prepared with the precipitation method. We form micelles in a reproducible manner with a narrow size distribution. This suggests that during the formation of the micelles the system had time to form micelles under close-to-thermodynamic control. This may explain why it is possible to use an equilibrium self-consistent field theory to predict the hydrodynamic size and the loading capacity of the micelles in accordance with experimental finding. Yet, the micelles are structurally quenched as concluded from the observation of size stability in time. We demonstrate that our approach enables to prepare rather hydrophobic block copolymer micelles with tunable size and loading.