Neutron reflectivity studies of the interaction of cubic-phase nanoparticles with phospholipid bilayers of different coverage.

Liquid-crystalline cubic-phase nanoparticles (CPNPs) (known as Cubosome particles), based on the lipid glycerol monooleate and stabilized by the nonionic block copolymer Pluronic F-127, interact with supported model membranes consisting of dioleoylphosphatidylcholine (DOPC) in a complex and dynamic fashion. Neutron reflectivity measurements on the interaction of CPNPs with bilayers of different coverage have increased our understanding of an interfacial exchange mechanism that is relevant to delivery applications. To access the composition of the adsorption layer, the method of isotopic contrast between the components was exploited by using DOPC with perdeuterated acyl chains, which are distinguishable (high scattering contrast) from the hydrogenous components of the CPNPs. The exchange of material between CPNPs and the bilayer takes place regardless of the initial bilayer coverage. However, this parameter has a strong influence on the physical nature of the layer formed upon interaction. For a bilayer of "high coverage" (80%), extensive exchange takes place between the CPNP components and the bilayer, and at steady state the surface layer comprises 72% glycerol monooleate and 8% DOPC, with no change in the solvent content. An analogous experiment involving pure glycerol monooleate liquid crystals shows that lipid exchange occurs even in the absence of the stabilizing polymer. For bilayers of "low coverage" (55%), the exchange mechanism involves an initial adsorption of material from the CPNPs to fill in the bilayer defects. However, most of the bilayer breaks up and only 15% coverage remains after 30 h. The evolution of a Bragg diffraction peak was monitored in this case to show that the bound nanoparticles occupy >7% surface coverage and have a periodicity in the density of the internal lipid structure that decreases with time. This progression is attributed to the incorporation of d-DOPC molecules within the internal cubic structure of the nanoparticles. The broadening of the diffraction peak with time, together with a final mean position that is closely related to the periodicity of the lamellar phase organization of GMO, shows that the lipid-exchange process results in either a contraction of the unit cell of the cubic-phase nanoparticles or a progression of the lipid arrangement to the lamellar phase.

Interaction between lamellar (vesicles) and nonlamellar lipid liquid-crystalline nanoparticles as studied by time-resolved small-angle X-ray diffraction.

The kinetics of structure change when dispersions of two different types of lipid-based liquid-crystalline phases, one lamellar and one reversed, are mixed has been investigated using synchrotron small-angle X-ray diffraction and ellipsometry. The systems studied were (i) cubic-phase nanoparticles (CPNPs) based on glycerol monooleate (GMO) stabilized with a nonionic block copolymer, Pluronic F-127; (ii) CPNPs based on phytantriol (PtOH) stabilized with D-alpha-Tocopheryl polyethylene glycol 1000 succinate (Vitamin E TPGS); and (iii) hexagonal-phase nanoparticles (HPNPs) based on a lipid mixture of diglycerol monooleate/glycerol dioleate, stabilized by Pluronic F-127. Time-resolved small-angle X-ray diffraction was used to track structural changes within nonlamellar nanoparticles when they interact with uni- and multilamellar vesicles of dioleoylphosphatidylcholine and dipalmitoylphatidylcholine. The results are very dependent on the type of nanoparticles under investigation. For GMO-based CPNPs, a strong interaction is observed on mixing with vesicular dispersions that leads to large changes in unit size dimensions as well as a later transition from cubic to lamellar structure. These results are in good agreement with previous studies on the interaction of GMO-based CPNPs with planar bilayers using neutron reflectivity, where the diffraction peak shifted with time upon mixing. The structural changes are much less prominent for the PtOH-based CPNPs and the HPNPs upon mixing with phospholipid vesicles. These results are correlated with those from measurement studying interactions between the liquid-crystalline nanoparticles and supported phospholipid bilayers by ellipsometry. Also, here the GMO-based CPNPs show more pronounced and rapid adsorption and interaction with the supported bilayer surface than do the other types of nonlamellar nanoparticles. The interaction also depends on the bilayer properties, where significantly slower lipid mixing is observed for a bilayer in the gel state compared to a bilayer in the liquid-crystalline phase. This study is not only relevant for drug-delivery applications but also shows the potential of synchrotron small-angle X-ray diffraction in studying time-dependent structural changes as a consequence of the interaction between different lipid self-assembled aggregates in complex systems.

A combined in vitro and in vivo study on the interactions between somatostatin and lipid-based liquid crystalline drug carriers and bilayers.

Somatostatin (SST) is a peptide hormone active in the regulation of the endocrine system via different somatostatin receptors subtypes. It inhibits the release of multiple secondary peptide hormones, affecting neurotransmission and cell proliferation. SST has a high therapeutic potential in the treatment of disease, such as acromegali, acute pancreatitis and gastroenteropathic endocrine tumors. However, its practical use is hampered by a short in vivo half-life of only a few minutes in man. For this reason more long-lived SST analogues, including octreotide and lanreotide, have been developed. Here we have used native SST as a model compound for a different approach of extending plasma half-lives of in vivo labile biomolecules. Through association of the peptide hormone with lipid-based liquid crystalline nanoparticle (LCNP) carriers, the terminal half-life of SST injected intravenously in rats is shown to be significantly extended from less than 10min to more than 1h. The effect on the in vivo circulation behavior depends on the mode of peptide association to the lipid particles and related physicochemical properties are discussed on the basis of in vitro light scattering, z-potential and adsorption measurements. It is concluded that application of the LCNP delivery system represents an interesting alternative to chemical modifications of in vivo sensitive therapeutically interesting peptides.

Neutron reflectometry to investigate the delivery of lipids and DNA to interfaces (Review).

The application of scattering methods in the study of biological and biomedical problems is a field of research that is currently experiencing fast growth. In particular, neutron reflectometry (NR) is a technique that is becoming progressively more widespread, as indicated by the current commissioning of several new reflectometers worldwide. NR is valuable for the characterization of biomolecules at interfaces due to its capability to provide quantitative structural and compositional information on relevant molecular length scales. Recent years have seen an increasing number of applications of NR to problems related to drug and gene delivery. We start our review by summarizing the experimental methodology of the technique with reference to the description of biological liquid interfaces. Various methods for the interpretation of data are then discussed, including a new approach based on the lattice mean-field theory to help characterize stimulus-responsive surfaces relevant to drug delivery function. Recent progress in the subject area is reviewed in terms of NR studies relevant to the delivery of lipids and DNA to surfaces. Lastly, we discuss two case studies to exemplify practical features of NR that are exploited in combination with complementary techniques. The first case concerns the interactions of lipid-based cubic phase nanoparticles with model membranes (a drug delivery application), and the second case concerns DNA compaction at surfaces and in the bulk solution (a gene delivery application).

Interfacial behavior of cubic liquid crystalline nanoparticles at hydrophilic and hydrophobic surfaces.

The adsorption behavior of self-assembled lipid liquid crystalline nanoparticles at different model surfaces was investigated in situ by use of ellipsometry. The technique allows time-resolved monitoring of the adsorbed amount and layer thickness under transient and steady-state conditions. The system under study was cubic-phase nanoparticle (CPNP) dispersions of glycerol monooleate stabilized by a nonionic block copolymer, Pluronic F-127. Depending on the surface properties and presence of electrolytes, different adsorption scenarios were discerned: At hydrophilic silica thick surface layers of CPNPs are generated by particle adsorption from dispersions containing added electrolyte, but no adsorption is observed in pure water. Adsorption at the hydrophobic surface involves extensive structural relaxation and formation, which is not electrolyte sensitive, of a classic monolayer structure. The different observations are rationalized in terms of differences in interactions among the CPNP aggregates, their unimer constituents, and the surface and show a strong influence of interfacial interactions on structure formation. Surface self-assembly structures with properties similar to those of the corresponding bulk aggregates appear exclusively in the weak interaction limit. This observation is in agreement with observations for surfactant self-assembly systems, and our findings indicate that this behavior is applicable also to complex self-assembly structures such as the CPNP structures discussed herein.