Nhiem Tran

Nanostructure and cytotoxicity of self-assembled monoolein–capric acid lyotropic liquid crystalline nanoparticles

Monoolein forms self-assembled nanoparticles with various internally ordered nanostructures, including the lyotropic liquid crystalline inverse hexagonal and inverse bicontinuous cubic phases. This study investigated the influence of a saturated fatty acid, capric acid (decanoic acid), on the formation of different lyotropic liquid crystalline phases in monoolein-based systems. The nanoparticles were characterized by synchrotron small angle X-ray scattering (SAXS), cryogenic transmission electron microscopy (cryo-TEM), dynamic light scattering, and zeta potential measurements. The addition of capric acid to monoolein triggered concentration dependent phase changes with the sequence evolving from an inverse primitive cubic phase to inverse double-diamond cubic, inverse hexagonal (HII), and emulsified microemulsions. SAXS and cryo-TEM revealed the formation of both single phase and mixed phases within a nanoparticle. To understand the cytotoxicity effects of the different nanoparticles, cellular cytotoxicity and hemolysis assays were performed. Nanoparticles in emulsion and hexagonal phases were found to be less toxic than cubic phase nanoparticles. The hemolysis assays followed the same trend with cubic phase dispersions causing the highest level of hemoglobin release. In summary, this study showed that the internal lyotropic liquid crystal mesophase structure of self-assembled nanoparticles needs careful consideration in the design of drug delivery vehicles.

Lipid-PEG conjugates sterically stabilize and reduce the toxicity of phytantriol-based lyotropic liquid crystalline nanoparticles.

Lyotropic liquid crystalline nanoparticle dispersions are of interest as delivery vectors for biomedicine. Aqueous dispersions of liposomes, cubosomes, and hexosomes are commonly stabilized by nonionic amphiphilic block copolymers to prevent flocculation and phase separation. Pluronic stabilizers such as F127 are commonly used; however, there is increasing interest in using chemically reactive stabilizers for enhanced functionalization and specificity in therapeutic delivery applications. This study has explored the ability of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine conjugated with poly(ethylene glycol) (DSPE-PEGMW) (2000 Da ≤ MW ≤ 5000 Da) to engineer and stabilize phytantriol-based lyotropic liquid crystalline dispersions. The poly(ethylene glycol) (PEG) moiety provides a tunable handle to the headgroup hydrophilicity/hydrophobicity to allow access to a range of nanoarchitectures in these systems. Specifically, it was observed that increasing PEG molecular weight promotes greater interfacial curvature of the dispersions, with liposomes (Lα) present at lower PEG molecular weight (MW 2000 Da), and a propensity for cubosomes (QII(P) or QII(D) phase) at MW 3400 Da or 5000 Da. In comparison to Pluronic F127-stabilized cubosomes, those made using DSPE-PEG3400 or DSPE-PEG5000 had enlarged internal water channels. The toxicity of these cubosomes was assessed in vitro using A549 and CHO cell lines, with cubosomes prepared using DSPE-PEG5000 having reduced cytotoxicity relative to their Pluronic F127-stabilized analogues.

First Direct Observation of Stable Internally Ordered Janus Nanoparticles Created by Lipid Self-Assembly

We present the first observation of Janus nanoparticles consisting of stable, coexisting ordered mesophases in discrete particles created by lipid self-assembly. Cryo-TEM images provided visual identification of the multicompartment Janus nanoparticles and, combined with SAXS data, confirmed the presence of mixed cubic phases and mixed cubic/hexagonal phases within individual nanoparticles. We further investigated computer visualization models to interpret the potential interface between the interconnected coexisting nanostructured domains within a single nanoparticle.

In vivo caprine model for osteomyelitis and evaluation of biofilm-resistant intramedullary nails.

Bone infection remains a formidable challenge to the medical field. The goal of the current study is to evaluate antibacterial coatings in vitro and to develop a large animal model to assess coated bone implants. A novel coating consisting of titanium oxide and siloxane polymer doped with silver was created by metal-organic methods. The coating was tested in vitro using rapid screening techniques to determine compositions which inhibited Staphylococcus aureus growth, while not affecting osteoblast viability. The coating was then applied to intramedullary nails and evaluated in vivo in a caprine model. In this pilot study, a fracture was created in the tibia of the goat, and Staphylococcus aureus was inoculated directly into the bone canal. The fractures were fixed by either coated (treated) or non-coated intramedullary nails (control) for 5 weeks. Clinical observations as well as microbiology, mechanical, radiology, and histology testing were used to compare the animals. The treated goat was able to walk using all four limbs after 5 weeks, while the control was unwilling to bear weight on the fixed leg. These results suggest the antimicrobial potential of the hybrid coating and the feasibility of the goat model for antimicrobial coated intramedullary implant evaluation.

Dual-modality NIRF-MRI cubosomes and hexosomes: High throughput formulation and in vivo biodistribution

Engineered nanoparticles with multiple complementary imaging modalities are of great benefit to the rapid treatment and diagnosis of disease in various organs. Herein, we report the formulation of cubosomes and hexosomes that carry multiple amphiphilic imaging contrast agents in their self-assembled lipid bilayers. This is the first report of the use of both near infrared fluorescent (NIRF) imaging and gadolinium lipid based magnetic resonance (MR) imaging modalities in cubosomes and hexosomes. High-throughput screening was used to rapidly optimize formulations with desirable nano-architectures and low in vitro cytotoxicity. The dual-modal imaging nanoparticles in vivo biodistribution and organ specific contrast enhancement were then studied. The NIRF in vivo imaging results indicated accumulation of both cubosomes and hexosomes in the liver and spleen of mice up to 20h post-injection. Remarkably, the biodistribution of the nanoparticle formulations was affected by the mesophase (i.e. cubic or hexagonal), a finding of significant importance for the future use of these compounds, with hexosomes showing higher accumulation in the spleen than the liver compared to cubosomes. Furthermore, in vivo MRI data of animals injected with either type of lyotropic liquid crystal nanoparticle displayed enhanced contrast in the liver and spleen.

High-Throughput Screening of Saturated Fatty Acid Influence on Nanostructure of Lyotropic Liquid Crystalline Lipid Nanoparticles

Self-assembled lyotropic liquid crystalline lipid nanoparticles have been developed for a wide range of biomedical applications with an emerging focus for use as delivery vehicles for drugs, genes, and in vivo imaging agents. In this study, we report the generation of lipid nanoparticle libraries with information regarding mesophase and lattice parameter, which can aid the selection of formulation for a particular end-use application. In this study we elucidate the phase composition parameters that influence the internal structure of lipid nanoparticles produced from monoolein, monopalmitolein and phytantriol incorporating a variety of saturated fatty acids (FA) with different chain lengths at varying concentrations and temperatures. The material libraries were established using high throughput formulation and screening techniques, including synchrotron small-angle X-ray scattering. The results demonstrate the rich polymorphism of lipid nanoparticles with nonlamellar mesophases in the presence of saturated FAs. The inclusion of saturated FAs within the lipid nanoparticles promotes a gradual phase transition at all temperatures studied toward structures with higher negative surface curvatures (e.g., from inverse bicontinuous cubic phase to hexagonal phase and then emulsified microemulsion). The three partial phase diagrams produced are discussed in terms of the influence of FA chain length and concentration on nanoparticle internal mesophase structure and lattice parameters. The study also highlights a compositionally dependent coexistence of multiple mesophases, which may indicate the presence of multicompartment nanoparticles containing cubic/cubic and cubic/hexagonal mesophases.

Amphiphilic brush polymers produced using the RAFT polymerisation method stabilise and reduce the cell cytotoxicity of lipid lyotropic liquid crystalline nanoparticles

Self-assembled lipid lyotropic liquid crystalline nanoparticles such as hexosomes and cubosomes contain internal anisotropic and isotropic nanostructures, respectively. Despite the remarkable potential of such nanoparticles in various biomedical applications, the stabilisers used in formulating the nanoparticles are often limited to commercially available polymers such as the Pluronic block copolymers. This study explored the potential of using Reversible Addition-Fragmentation chain Transfer (RAFT) technology to design amphiphilic brush-type polymers for the purpose of stabilising phytantriol and monoolein-based lipid dispersions. The synthesised brush-type polymers consisted of a hydrophobic C12 short chain and a hydrophilic poly(ethylene glycol)methyl ether acrylate (PEGA) long chain with multiple 9-unit poly(ethylene oxide) (PEO) brushes with various molecular weights. It was observed that increasing the PEO brush density and thus the length of the hydrophilic component improved the stabilisation effectiveness for phytantriol and monoolein-based cubosomes. Synchrotron small-angle X-ray scattering (SAXS) experiments confirmed that the RAFT polymer-stabilised cubosomes had an internal double-diamond cubic phase with tunable water channel sizes. These properties were dependent on the molecular weight of the polymers, which were considered in some cases to be anisotropically distributed within the cubosomes. The in vitro toxicity of the cubosomes was assessed by cell viability of two human adenocarcinoma cell lines and haemolytic activities to mouse erythrocytes. The results showed that phytantriol cubosomes stabilised by the RAFT polymers were less toxic compared to their Pluronic F127-stabilised analogues. This study provides valuable insight into designing non-linear amphiphilic polymers for the effective stabilisation and cellular toxicity improvement of self-assembled lipid lyotropic liquid crystalline nanoparticles.

Self-assembled Lyotropic Liquid Crystalline Phase Behavior of Monoolein–Capric Acid–Phospholipid Nanoparticulate Systems

We report here the lyotropic liquid crystalline phase behavior of two lipid nanoparticulate systems containing mixtures of monoolein, capric acid, and saturated diacyl phosphatidylcholines dispersed by the Pluronic F127 block copolymer. Synchrotron small-angle X-ray scattering (SAXS) was used to screen the phase behavior of a library of lipid nanoparticles in a high-throughput manner. It was found that adding capric acid and phosphatidylcholines had opposing effects on the spontaneous membrane curvature of the monoolein lipid layer and hence the internal mesophase of the final nanoparticles. By varying the relative concentration of the three lipid components, we were able to establish a library of nanoparticles with a wide range of mesophases including at least the inverse bicontinuous primitive and double diamond cubic phases, the inverse hexagonal phase, the fluid lamellar phase, and possibly other phases. Furthermore, the in vitro cytotoxicity assay showed that the endogenous phospholipid-containing nanoparticles were less toxic to cultured cell lines compared to monoolein-based counterparts, improving the potential of the nonlamellar lipid nanoparticles for biomedical applications.

Manipulating the Ordered Nanostructure of Self-Assembled Monoolein and Phytantriol Nanoparticles with Unsaturated Fatty Acids

Mesophase structures of self-assembled lyotropic liquid crystalline nanoparticles are important factors that directly influence their ability to encapsulate and release drugs and their biological activities. However, it is difficult to predict and precisely control the mesophase behavior of these materials, especially in complex systems with several components. In this study, we report the controlled manipulation of mesophase structures of monoolein (MO) and phytantriol (PHYT) nanoparticles by adding unsaturated fatty acids (FAs). By using high throughput formulation and small-angle X-ray scattering characterization methods, the effects of FAs chain length, cis-trans isomerism, double bond location, and level of chain unsaturation on self-assembled systems are determined. Additionally, the influence of temperature on the phase behavior of these nanoparticles is analyzed. We found that in general, the addition of unsaturated FAs to MO and PHYT induces the formation of mesophases with higher Gaussian surface curvatures. As a result, a rich variety of lipid polymorphs are found to correspond with the increasing amounts of FAs. These phases include inverse bicontinuous cubic, inverse hexagonal, and discrete micellar cubic phases and microemulsion. However, there are substantial differences between the phase behavior of nanoparticles with trans FA, cis FAs with one double bond, and cis FAs with multiple double bonds. Therefore, the material library produced in this study will assist the selection and development of nanoparticle-based drug delivery systems with desired mesophase.

Inverse hexagonal and cubic micellar lyotropic liquid crystalline phase behaviour of novel double chain sugar-based amphiphiles

The lyotropic phase behaviour of a library of sugar-based amphiphiles was investigated using high-throughput small-angle X-ray scattering (SAXS). Double unsaturated-chain monosaccharide amphiphiles formed inverse hexagonal and cubic micellar (Fd3m) lyotropic phases under excess water conditions. A galactose-oleyl amphiphile from the library was subsequently formulated into hexosome nanoparticles, which have potential uses as drug delivery vehicles. The nanoparticles were shown to be stable at elevated temperatures and non-cytotoxic up to at least 200μgmL-1.