The Effect of Repeat Administration of Lipoplexes on Gene Delivery, Biodistribution, and Cytokine Response in Immunocompetent Tumor-Bearing Mice.

It is becoming increasingly clear that the intravenous administration of nanoparticles elicits an immune response that compromises delivery efficiency and can be life threatening. This study investigated both the systemic and tissue-level cytokine response to repeat administration of lipoplexes coated with either lactose or PEG. We report that blood cytokine levels differ significantly from that observed in individual tissues.

The Use of Lactose as an Alternative Coating for Nanoparticles.

Nanoparticle-mediated drug delivery has long utilized PEGylation as a mechanism for reducing uptake by the reticuloendothelial system and extending circulation lifetimes. However, studies over the past 2 decades have established that immune responses to PEG can promote clearance on repeat injection and elicit life-threatening anaphylactic reactions in some patients. As a potential alternative to PEGylation, we explored the ability of utilizing lactose, a naturally occurring sugar that is common on the surface of blood cells, as a coating for lipoplexes.

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.

The Potential of Exosomes From Cow Milk for Oral Delivery.

Many pharmaceuticals must be administered intravenously due to their poor oral bioavailability. In addition to issues associated with sterility and inconvenience, the cost of repeated infusion over a 6-week course of therapy costs the health care system tens of billions of dollars per year. Attempts to improve oral bioavailability have traditionally focused on enhancing drug solubility and membrane permeability, and the use of synthetic nanoparticles has also been investigated.

Nanoparticle uptake by circulating leukocytes: A major barrier to tumor delivery.

Decades of research into improving drug delivery to tumors has documented uptake of particulate delivery systems by resident macrophages in the lung, liver, and spleen, and correlated short circulation times with reduced tumor accumulation. An implicit assumption in these studies is that nanoparticles present in the blood are available for distribution to the tumor. This study documents significant levels of lipoplex uptake by circulating leukocytes, and its effect on distribution to the tumor and other organs.

Nonadditive Effects of Repetitive Administration of Lipoplexes in Immunocompetent Mice.

Repetitive administration is routinely used to maintain therapeutic drug levels, but previous studies have documented an accelerated blood clearance of some lipid-based delivery systems under these conditions. To assess the effect of repetitive administration, non-PEGylated lipoplexes (+/-0.5) were administered 4 times via tail vein injection at 3-day intervals to immunocompetent BALB/c mice bearing 4T1 tumors.

Assessing the effect of a nude mouse model on nanoparticle-mediated gene delivery.

The relevance of using nude mouse models for evaluating drug delivery to human tumors has recently been questioned by numerous researchers. While the immune response is known to play a critical role in cancer, this study assesses the effect of using immunocompromised "nude" mice on drug delivery. By inoculating both nude and immunocompetent mice with a mouse mammary carcinoma cell line (4T1), differences in the "first pass effect", distribution, and reporter gene expression due to the use of the nude mouse model could be elucidated.

Effect of charge ratio on lipoplex-mediated gene delivery and liver toxicity.

Background: The vast majority of studies investigating gene delivery have utilized cationic delivery vehicles, but anionic nanoparticles can also possess high transfection activity, and offer significant benefits in terms of ease of preparation and reduced toxicity.

Results: Our study on lipoplexes possessing cholesterol nanodomains demonstrates that in vitro transfection after exposure to serum can be high at anionic charge ratios, and that this effect is also evident in studies assessing delivery to tumors in vivo, despite reduced circulation times. In addition, accumulation in the liver and lungs is reduced as compared with lipoplexes formulated at cationic charge ratios.

Relating toxicity to transfection: using sphingosine to maintain prolonged expression in vitro.

Cationic reagents are commonly used to facilitate DNA delivery, and transfection experiments are typically initiated in cell culture where the optimal charge ratio is determined. While transfection rates are often enhanced at higher +/- charge ratios, the cellular toxicity associated with the greater amounts of cationic components at elevated charge ratios is often not considered. In addition, the prolonged effects of cationic lipid uptake on cell viability are not evident in a typical 24-48 h transfection experiment.

The effects of lipoplex formulation variables on the protein corona and comparisons with in vitro transfection efficiency.

The use of lipoplexes for the intracellular delivery of nucleic acids typically involves the optimization of several parameters that are known to affect delivery. Researchers commonly vary charge ratio, and often incorporate different amounts of helper lipids (e.g.

Cholesterol domains enhance transfection.

Background: The formation of cholesterol domains in lipoplexes has been associated with enhanced serum stability and transfection rates both in cell culture and in vivo.

Results: This study utilizes the ability of saturated phosphatidylcholines to promote the formation of cholesterol domains at much lower cholesterol contents than have been utilized in previous work. The results demonstrate that lipoplexes with identical cholesterol and cationic lipid contents exhibit significantly improved transfection efficiencies when a domain is present, consistent with previous work.

De novo computational design of retro-aldol enzymes.

The creation of enzymes capable of catalyzing any desired chemical reaction is a grand challenge for computational protein design. Using new algorithms that rely on hashing techniques to construct active sites for multistep reactions, we designed retro-aldolases that use four different catalytic motifs to catalyze the breaking of a carbon-carbon bond in a nonnatural substrate. Of the 72 designs that were experimentally characterized, 32, spanning a range of protein folds, had detectable retro-aldolase activity.