The role of lipid charge density in the serum stability of cationic lipid/DNA complexes

AbstractTo evaluate the role of lipid charge density in the serum stability of DOTAP-Chol/DNA complexes (lipoplexes), lipid–DNA interactions, extent of aggregation, supercoil content, and in vitro transfection efficiency of lipoplexes were investigated. In general, higher serum concentration destabilized, and increasing molar charge ratio of DOTAP to negatively charged phosphates in the DNA (DOTAP+/DNA−) stabilized lipoplexes in serum as assessed by the criteria used in this study. The increase of cholesterol content led to increased serum stability, and DOTAP:Chol (mol/mol 1:4)/DNA lipoplex with DOTAP+/DNA− ratio 4 was the most serum stable formulation of all the formulations examined, and maintained lipid–DNA interactions, did not aggregate and exhibited high in vitro transfection efficiency in 50% (v/v) serum. The increased stability of this formulation could not be explained by the decreased charge density of the lipid component. Furthermore, no single parameter examined in the study could be used to consistently predict the in vitro transfection efficiency of lipoplexes in serum. Surprisingly, no correlation between the maintenance of supercoiled DNA content and in vitro transfection efficiency was found in the study

Metal contaminants promote degradation of lipid/DNA complexes during lyophilization

AbstractOxidation reactions represent an important degradation pathway of nucleic acid-based pharmaceuticals. To evaluate the role of metal contamination and chelating agents in the formation of reactive oxygen species (ROS) during lyophilization, ROS generation and the stability of lipid/DNA complexes were investigated. Trehalose-containing formulations were lyophilized with different levels of transition metals. ROS generation was examined by adding proxyl fluorescamine to the formulations prior to freeze-drying. Results show that ROS were generated during lyophilization, and both supercoil content and transfection rates decreased as the levels of metal-induced ROS increased. The experiments incorporating chelators demonstrated that some of these agents (e.g., DTPA, desferal) clearly suppress ROS generation, while others (e.g., EDTA) enhance ROS. Surprisingly, there was not a strong correlation of ROS generated in the presence of chelators with the maintenance of supercoil content. In this study, we demonstrated the adverse effects of the presence of metals (especially Fe2+) in nonviral vector formulations. While some chelators attenuate ROS generation and preserve DNA integrity, the effects of these additives on vector stability during lyophilization are difficult to predict. Further study is needed to develop potent formulation strategies that inhibit ROS generation and DNA degradation during lyophilization and storage

The use of fluorescence resonance energy transfer to monitor dynamic changes of lipid–DNA interactions during lipoplex formation

AbstractFluorescence resonance energy transfer (FRET) was used to monitor interactions between Cy3-labeled plasmid DNA and NBD-labeled cationic liposomes. FRET data show that binding of cationic liposomes to DNA occurs immediately upon mixing (within 1 min), but FRET efficiencies do not stabilize for 1–5 h. The time allowed for complex formation has effects on in vitro luciferase transfection efficiencies of DOPE-based lipoplexes; i.e., lipoplexes prepared with a 1-h incubation have much higher transfection efficiencies than samples with 1-min or 5-h incubations. The molar charge ratio of DOTAP to negatively charged phosphates in the DNA (DOTAP+/DNA−) also affected the interaction between liposomes and plasmid DNA, and interactions stabilized more rapidly at higher charge ratios. Lipoplexes formulated with DOPE were more resistant to high ionic strength than complexes formulated with cholesterol. Taken together, our data demonstrate that lipid–DNA interactions and in vitro transfection efficiencies are strongly affected by the time allowed for complex formation. This effect is especially evident in DOPE-based lipoplexes, and suggests that the time allowed for lipoplex formation is a parameter that should be carefully controlled in future studies

An initial animal proof-of-concept study for central administration of clozapine to schizophrenia patients

While clozapine is the acknowledged superior pharmacotherapeutic for the treatment of schizophrenia, the side effect profile, which includes potentially fatal complications, limits its usefulness. Central administration of clozapine directly into the brain could circumvent many of the side effect issues due to the dramatic reduction in dose and the limitation of the drug primarily to the CNS. The present study demonstrates that clozapine can be formulated as a stable solution at physiological pH, which does not have in vitro neurotoxic effects at concentrations which may be effective at treating symptoms. Acute central administration improved auditory gating deficits in a mouse model of schizophrenia-like deficits. Assessment of behavioral alterations in rats receiving chronic central infusions of clozapine via osmotic minipump was performed with the open field and elevated plus mazes. Neither paradigm revealed any detrimental effects of the infusion. While these data represent only an initial investigation, they none-the-less suggest that central administration of clozapine may be a viable alternate therapeutic approach for schizophrenia patients which may be effective in symptom reduction without causing behavioral or neurotoxic effects

Contribution of Hydrophobicity to Thermodynamics of Ligand-DNA Binding and DNA Collapse

The importance of understanding the dynamics of DNA condensation is inherent in the biological significance of DNA packaging in cell nuclei, as well as for gene therapy applications. Specifically, the role of ligand hydrophobicity in DNA condensation has received little attention. Considering that only multivalent cations can induce true DNA condensation, previous studies exploring monovalent lipids have been unable to address this question. In this study we have elucidated the contribution of the hydrophobic effect to multivalent cation- and cationic lipid-DNA binding and DNA collapse by studying the thermodynamics of cobalt hexammine-, spermine-, and lipospermine-plasmid DNA binding at different temperatures. Comparable molar heat capacity changes (ΔC(p)) associated with cobalt hexammine- and spermine-DNA binding (−23.39 cal/mol K and −17.98 cal/mol K, respectively) suggest that upon binding to DNA, there are insignificant changes in the hydration state of the methylene groups in spermine. In contrast, the acyl chain contribution to the ΔC(p) of lipospermine-DNA binding (ΔC(p φ) = ΔC(p lipospermine) − ΔC(p spermine)) is significant (−220.94 cal/mol K). Although lipopermine induces DNA ordering into “tubular” suprastructures, such structures do not assume toroidal dimensions as observed for spermine-DNA complexes. We postulate that a steric barrier posed by the acyl chains in lipospermine precludes packaging of DNA into dimensions comparable to those found in nature

Potential induction of anti-PEG antibodies and complement activation toward PEGylated therapeutics

Conjugation of polyethylene glycol (PEG) to therapeutics has proven to be an effective approach to increase the serum half-life. However, the increased use of PEGylated therapeutics has resulted in unexpected immune-mediated side-effects. There are claims that these are caused by anti-PEG antibodies inducing rapid clearance. These claims are however hampered by the lack of standardized and well-validated antibody assays. PEGylation has also been associated with the activation of the complement system causing severe hypersensitivity reactions. Here, we critically review the clinical and analytical tools used. In addition, we propose an explanation of the immune-mediated side-effects of PEGylated products based on the haptogenic properties of PEG, responsible for complement activation and the induction of anti-PEG antibodies

Potential induction of anti-PEG antibodies and complement activation toward PEGylated therapeutics

Conjugation of polyethylene glycol (PEG) to therapeutics has proven to be an effective approach to increase the serum half-life. However, the increased use of PEGylated therapeutics has resulted in unexpected immune-mediated side-effects. There are claims that these are caused by anti-PEG antibodies inducing rapid clearance. These claims are however hampered by the lack of standardized and well-validated antibody assays. PEGylation has also been associated with the activation of the complement system causing severe hypersensitivity reactions. Here, we critically review the clinical and analytical tools used. In addition, we propose an explanation of the immune-mediated side-effects of PEGylated products based on the haptogenic properties of PEG, responsible for complement activation and the induction of anti-PEG antibodies

A novel method for conjugating the terminal amine of peptide ligands to cholesterol: Synthesis iRGD-cholesterol

Aim: Conventional conjugation reactions often involve the use of activated PEG as a linker, but concerns about PEG-mediated reduction in intracellular delivery and enhanced immunogenicity have generated interest in developing methods that eliminate the need for a PEG linker. Materials & methods: Reaction conditions were identified that specifically couples the terminal amine of a cyclic iRGD peptide (CRGDRGPDC) to the hydroxyl moiety of cholesterol through a short carbamate linker. Results & conclusion: Using this method for synthesizing iRGD-cholesterol, peptide ligands can be incorporated into lipid-based delivery systems, thereby eliminating concerns about adverse reactions to PEG. Toxicity and stability data indicate low toxicity and adequate serum stability at low ligand levels