Minoo J. Moghaddam

Delivery of nucleic acids

Nucleic acids have revolutionized biomedical research and have become indispensable research tools. In pharmaceutical development, nucleic acids are at present mostly used as diagnostic tools and for target validation (1– 3). Applications of microarrays and PCR, treatment with antisense oligonucleotides or small interfering RNA and breeding of knock-in/knock-out-models can be used for accurate diagnosis and biomarker detection, can improve insight into disease processes and can pinpoint pathways where treatments may interfere. Although the application of nucleic acids as therapeutics promises to be even more exciting, their role as clinically applied drugs is still modest. At present, two nucleic acid-based drugs (Vitravenei and Macugeni) are on the market (4). Both drugs are oligonucleotides. Macugeni is an extracellularly acting aptamer that functions as a growth factor decoy and Vitravenei is an intracellularly acting antisense molecule that inhibits a viral gene. Both oligonucleotides contain chemically modified backbones and are injected at the site of the pathology in the vitreous of the eye. This exemplifies the difficulties associated with the use of nucleic acids for therapeutic intervention, both regarding their physicochemical as well as their biological properties. The physicochemical properties of nucleic acids, with molecular weights ranging from 7 kDa for antisense oligonucleotides to over 1 MDa for plasmid DNA, and strong negative charge do not favor membrane passage. Only one class of nucleic acids, aptamers, can act extracellularly, which circumvents the need for cell membrane translocation. Conversely, all other classes need to interact with intracellular targets to be active. The problem is most prominent for plasmid DNA, which has the largest size of all proposed nucleic acid therapeutics and also needs to arrive inside the cell nucleus to be effective. Nuclear localization would in principle require passage through the nuclear pore for which the DNA-molecule is too large (5). These qualities at least partly explain why the marketed drugs are an aptamer and an antisense oligonucleotide. The biological properties also do not support their application as therapeutics. Nucleic acids are susceptible to the action of nucleases. Therefore the two marked oligonucleotides bear chemically modified backbones. In addition, nucleic acids are rapidly cleared from the body, either via glomerular filtration by the kidneys and excretion into the urine or by (scavenger) receptor uptake and intracellular degradation. Therefore, local injection at the site of the pathology is the preferred administration route for the clinically applied oligonucleotides. Despite these difficulties, nucleic acids still capture the mind of many pharmaceutical scientists as possible therapeutics. One of the most appealing properties is that a change in a disease target would in principle only require a change in the nucleic acid sequence to obtain a new drug. As the physicochemical properties like size and charge of the molecules remain the same, the same principles can be applied during the drug formulation steps for this new sequence. After successful formulation of the first nucleic acid drug it can be expected that subsequent formulations will follow more easily. In contrast, for small molecular weight drugs, lead compound identification requires high throughput screening, and drug formulation is dependent on the physicochemical and biological characteristics of the compound. Nevertheless, the difficult biopharmaceutical characteristics of nucleic acids put a lot of demands on the delivery systems that should compensate for these qualities by increasing stability against the action of nucleases, reducing excretion and uptake by non-target tissues and promoting target tissue interaction, target cell association, membrane translocation, and correct intracellular trafficking (6). The articles in this theme issue address this difficult drug formulation process. The group of Klibanov approached the problem of identifying suitable vectors for plasmid DNA delivery using a high-throughput-synthesis coupled to combinatorial chemistry approach. Their study is based on the cationic polymer poly(ethylene imine) (PEI). Experimental observations of their group and others indicate that PEI molecular weight is positively correlated with degree of transfection but also with severity of toxicity (7, 8). These observations provided the input for synthesizing small molecular weight PEI-derivatives that were cross-linked with oligo-acrylate esters. As many of the factors that contribute to degree of transfection and toxicity as well as the relative contribution of each factor to the overall transfection efficiency are unknown, the highthroughput synthesis approach likely provides a higher chance of finding successful polymers. Indeed, their results show that superior PEI-derivatives could be identified as compared to the presently used Fgolden standard_ 22 kD PEI both with respect to degree of transfection as well as toxicity both in vitro and in vivo. Most cationic polymers exhibit a molecular weight distribution. De Wolf et al. investigated the effects of fractionation of the biodegradable polymer poly(2-dimethylamino ethylamino)phosphazene (p(DMAEA)-ppz) into four different molecular weight fractions on in vitro/in vivo transfection of plasmid DNA and polymer-DNA-complex-

A transfection compound series based on a versatile Tris linkage

The family of cationic lipid transfection reagents described here demonstrates a modular design that offers potential for the ready synthesis of a wide variety of molecular variants. The key feature of these new molecules is the use of Tris as a linker for joining the hydrophobic domain to a cationic head group. The molecular design offers the opportunity to conveniently synthesise compounds differing in charge, the number and nature of hydrophobic groups in the hydrophobic domain and the characteristics of the spacer between the cationic and hydrophobic moieties. We show that prototype reagents of this design can deliver reporter genes into cultured cells with efficiencies rivaling those of established cationic lipid transfection reagents. A feature of these reagents is that they are not dependent on formulation with a neutral lipid for activity.

Ordered Nanostructured Amphiphile Self-Assembly Materials from Endogenous Nonionic Unsaturated Monoethanolamide Lipids in Water

The self-assembly, solid state and lyotropic liquid crystalline phase behavior of a series of endogenous n-acylethanolamides (NAEs) with differing degrees of unsaturation, viz., oleoyl monoethanolamide, linoleoyl monoethanolamide, and linolenoyl monoethanolamide, have been examined. The studied molecules are known to possess inherent biological function. Both the monoethanolamide headgroup and the unsaturated hydrophobe are found to be important in dictating the self-assembly behavior of these molecules. In addition, all three molecules form lyotropic liquid crystalline phases in water, including the inverse bicontinuous cubic diamond (Q(II)(D)) and gyroid (Q(II)(G)) phases. The ability of the NAEs to form inverse cubic phases and to be dispersed into ordered nanostructured colloidal particles, cubosomes, in excess water, combined with their endogenous nature and natural medicinal properties, makes this new class of soft mesoporous amphiphile self-assembly materials suitable candidates for investigation in a variety of advanced multifunctional applications, including encapsulation and controlled release of therapeutic agents and incorporation of medical imaging agents.

High-throughput development of amphiphile self-assembly materials: fast-tracking synthesis, characterization, formulation, application, and understanding.

Amphiphile self-assembly materials, which contain both a hydrophilic and a hydrophobic domain, have great potential in high-throughput and combinatorial approaches to discovery and development. However, the materials chemistry community has not embraced these ideas to anywhere near the extent that the medicinal chemistry community has. While this situation is beginning to change, extracting the full potential of high-throughput approaches in the development of self-assembling materials will require further development in the synthesis, characterization, formulation, and application domains. One of the key factors that make small molecule amphiphiles prospective building blocks for next generation multifunctional materials is their ability to self-assemble into complex nanostructures through low-energy transformations. Scientists can potentially tune, control, and functionalize these structures, but only after establishing their inherent properties. Because both robotic materials handling and customized rapid characterization equipment are increasingly available, high-throughput solutions are now attainable. These address traditional development bottlenecks associated with self-assembling amphiphile materials, such as their structural characterization and the assessment of end-use functional performance. A high-throughput methodology can help streamline materials development workflows, in accord with existing high-throughput discovery pipelines such as those used by the pharmaceutical industry in drug discovery. Chemists have identified several areas that are amenable to a high-throughput approach for amphiphile self-assembly materials development. These allow an exploration of not only a large potential chemical, compositional, and structural space, but also material properties, formulation, and application variables. These areas of development include materials synthesis and preparation, formulation, characterization, and screening performance for the desired end application. High-throughput data analysis is crucial at all stages to keep pace with data collection. In this Account, we describe high-throughput advances in the field of amphiphile self-assembly, focusing on nanostructured lyotropic liquid crystalline materials, which form when amphiphiles are added to a polar solvent. We outline recent progress in the automated preparation of amphiphile molecules and their nanostructured self-assembly systems both in the bulk phase and in dispersed colloidal particulate systems. Once prepared, we can structurally characterize these systems by establishing phase behavior in a high-throughput manner with both laboratory (infrared and light polarization microscopy) and synchrotron facilities (small-angle X-ray scattering). Additionally, we provide three case studies to demonstrate how chemists can use high-throughput approaches to evaluate the functional performance of amphiphile self-assembly materials. The high-throughput methodology for the set-up and characterization of large matrix in meso membrane protein crystallization trials can illustrate an application of bulk phase self-assembling amphiphiles. For dispersed colloidal systems, two nanomedicine examples highlight advances in high-throughput preparation, characterization, and evaluation: drug delivery and magnetic resonance imaging agents.

Enhanced uptake of an integral membrane protein, the dopamine D2L receptor, by cubic nanostructured lipid nanoparticles doped with Ni(II) chelated EDTA amphiphiles

Intrinsic difficulties in characterizing the structure of combined membrane protein–lyotropic liquid crystalline lipidic cubic systems have hampered the development of techniques such as membrane protein (MP) crystallization, which remain largely empirical with consequently low success rates. Here we have incorporated an integral membrane protein and important neurological drug target, the dopamine D2L receptor, within nanostructured nanoparticles of lipidic cubic phase, known as Cubosomes. We show that MPs are incorporated within Cubosomes and that they exert a structural effect which is qualitatively similar to that seen in bulk cubic phase for some systems, exemplifying the potential of Cubosomes to characterize MP incorporation. In addition we have shown, for Cubosomes doped with Ni(II) chelated EDTA amphiphiles, that the strong affinity interaction between the bio-engineered histidine(His)-tag on the protein and the Ni(II) chelated EDTA headgroup of the doped amphiphile leads to enhanced interaction between the membrane protein and the nanostructured cubic nanoparticle. This indicates that protein loading within a cubic phase can be increased as required either to facilitate crystal growth within cubic mesophases or for drug loading. In addition it exemplifies the potential of Cubosome nanostructured nanoparticles to be targeted to specific sites in the body.

A novel lyotropic liquid crystal formed by triphilic star-polyphiles: hydrophilic/oleophilic/fluorophilic rods arranged in a 12.6.4. tiling

Triphilic star-polyphiles are short-chain oligomeric molecules with a radial arrangement of hydrophilic, hydrocarbon and fluorocarbon chains linked to a common centre. They form a number of liquid crystalline structures when mixed with water. In this contribution we focus on a hexagonal liquid crystalline mesophase found in star-polyphiles as compared to the corresponding double-chain surfactant to determine whether the hydrocarbon and fluorocarbon chains are in fact demixed in these star-polyphile systems, or whether both hydrocarbon and fluorocarbon chains are miscible, leading to a single hydrophobic domain, making the star-polyphile effectively amphiphilic. We report SANS contrast variation data that are compatible only with the presence of three distinct immiscible domains within this hexagonal mesophase, confirming that these star-polyphile liquid crystals are indeed hydrophilic/oleophilic/fluorophilic 3-phase systems. Quantitative comparison with scattering simulations shows that the experimental data are in very good agreement with an underlying 2D columnar (12.6.4) tiling. As in a conventional amphiphilic hexagonal mesophase, the hexagonally packed water channels (dodecagonal prismatic domains) are embedded in a hydrophobic matrix, but that matrix is split into oleophilic hexagonal prismatic domains and fluorophilic quadrangular prismatic domains.

Endogenous nonionic saturated monoethanolamide lipids: solid state, lyotropic liquid crystalline, and solid lipid nanoparticle dispersion behavior.

The n-acylethanolamides (NAEs) are a family of naturally occurring monoethanolamide containing lipids that display a variety of interesting biological properties. In this study, some physicochemical properties of a series of saturated monoethanolamide lipids with increasing hydrocarbon chain length (lauroyl, myristoyl, palmitoyl, and stearoyl) have been investigated. Temperature induced phase transitions for these NAEs indicate that both the monoethanolamide headgroups and the unsaturated hydrophobic tails play a role in the melting behavior of these lipids. All four lipids examined demonstrate the presence of at least three different polymorphic crystal forms. Transitions in crystal structure can be induced via heating and visualized with polarized optical microscopy. At room and physiological temperature, the four NAEs are solid lamellar crystalline materials. All four molecules form lyotropic liquid crystalline phases in water, albeit at relatively high temperatures, including the lamellar liquid crystalline phase and at least two isotropic phases. Lamellar crystalline palmitoyl monoethanolamide was dispersed as solid lipid nanoparticles (SLNs). The cytotoxicity of these SLNs toward human mammary epithelial cells (HMEpiC) and the MCF7 breast cancer cell line was assessed at physiological temperature. The palmitoyl monoethanolamide SLNs showed little to no toxicity to the HMEpiC even at a concentration of 30 microM. At concentrations above 3 microM, the HMEpiC population was reduced by less than 15%, while the MCF7 population was reduced by approximately 20-30%. The endogenous nature and natural medicinal properties make this series of lipids ideal candidates for further investigation as solid lipid nanoparticle drug delivery systems.

Targeting fibroblast-like synovial cells at sites of inflammation with peptide targeted liposomes results in inhibition of experimental arthritis.

In this study we examined a synovium-specific targeted liposomal drug delivery system for its ability to localize and release its drug cargo to inflamed joints. Targeted liposomes were tested in vitro for binding to synovial fibroblast like (FLS) and endothelial cells using flow cytometry and in vivo for localization to joints using a rat model of adjuvant induced arthritis (AIA). Targeted liposomes were then loaded with anti-arthritic medications and examined for clinical efficacy in AIA. Targeted liposomes specifically bound to rabbit FLS and human FLS and showed a 7-10 fold increase in vivo localization in affected joints compared to unaffected joints. Histological sections from rats treated with prednisone and a new immunosuppressive peptide CP showed minimal inflammation. This report substantiates the ability of the novel FLS sequence to target liposomal drug delivery and offers an alternative therapeutic approach for the treatment of arthritis.

Nanostructured self-assembly materials from neat and aqueous solutions of C18 lipid pro-drug analogues of Capecitabine—a chemotherapy agent. Focus on nanoparticulate cubosomes™ of the oleyl analogue

A series of prodrug analogues based on the established chemotherapy agent, 5-fluorouracil, have been prepared and characterized. C18 alkyl and alkenyl chains with increasing degree of unsaturation were attached to the N4 position of the 5-fluorocytosine (5-FC) base via a carbamate bond. Physicochemical characterization of the prodrug analogues was carried out using a combination of differential scanning calorimetry, cross-polarized optical microscopy, X-ray diffraction and small-angle X-ray scattering. The presence of a monounsaturated oleyl chain was found to promote lyotropic liquid crystalline phase formation in excess water with a fluid lamellar phase observed at room temperature and one or more bicontinuous cubic phases at 37 °C. The bulk phase was successfully dispersed into liposomes or cubosomes at room and physiological temperature respectively. In vitro toxicity of the nanoparticulate 5-FCOle dispersions was evaluated against several normal and cancer cell types over a 48 h period and exhibited an IC50 of ∼100 μM against all cell types. The in vivo efficacy of 5-FCOle cubosomes was assessed against the highly aggressive mouse 4T1 breast cancer model and compared to Capecitabine (a water-soluble commercially available 5-FU prodrug) delivered at the same dosages. After 21 days of treatment, the 0.5 mmol 5-FCOle treatment group exhibited a significantly smaller average tumour volume than all other treatment groups including Capecitabine at similar dosage. These results exemplify the potential of self-assembled amphiphile prodrugs for delivery of bioactives in vivo.

Chelating DTPA amphiphiles: ion-tunable self-assembly structures and gadolinium complexes

A series of chelating amphiphiles and their gadolinium (Gd(III)) metal complexes have been synthesized and studied with respect to their neat and lyotropic liquid crystalline phase behavior. These amphiphiles have the ability to form ion-tunable self-assembly nanostructures and their associated Gd(III) complexes have potential as magnetic resonance imaging (MRI) contrast enhancement agents. The amphiphiles are composed of diethylenetriaminepentaacetic acid (DTPA) chelates conjugated to one or two oleyl chain(s) (DTPA-MO and DTPA-BO), or isoprenoid-type chain(s) of phytanyl (DTPA-MP and DTPA-BP). The thermal phase behavior of the neat amphiphiles was examined by differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and cross polarizing optical microscopy (POM). Self-assembly of neat amphiphiles and their associated Gd complexes, as well as their lyotropic phase behavior in water and sodium acetate solutions of different ionic strengths, were examined by POM and small and wide angle X-ray scattering (SWAXS). All neat amphiphiles exhibited lamellar structures. The non-complexed amphiphiles showed a variety of lyotropic phases depending on the number and nature of the hydrophobic chain in addition to the ionic state of the hydration. Upon hydration with increased Na-acetate concentration and the subtle changes in the effective headgroup size, the interfacial curvature of the amphiphile increased, altering the lyotropic liquid crystalline structures towards higher order mesophases such as the gyroid (Ia3d) bicontinuous cubic phase. The chelation of Gd with the DTPA amphiphiles resulted in lamellar crystalline structures for all the neat amphiphiles. Upon hydration with water, the Gd-complexed mono-conjugates formed micellar or vesicular self-assemblies, whilst the bis-conjugates transformed only partially into lyotropic liquid crystalline mesophases.