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. While we consistently observed a reduced cytokine response to lactosylated particles, this did not result in enhanced delivery or expression as compared to PEGylated formulations. We also document that repeat injection did not increase plasmid levels in the liver, lung, or spleen, but delivery to the tumor was enhanced under these conditions. In addition, we show that changes in neither blood nor tissue cytokines correlated strongly with reporter gene expression, and we observed relatively constant expression efficiencies (RLU/ng plasmid) across all tissues despite a considerably reduced cytokine response in the tumor. Together, these results indicate that both biodistribution and cytokine responses are dramatically altered by a repeat intravenous injection of lipoplexes, and that the mechanisms regulating reporter gene expression are not straightforward.

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. Our data indicate that lactose imparts similar effects as PEG in terms of reducing leukocyte uptake, extending circulation half-life, and enhancing delivery to the tumor and other organs. In addition, measurements of blood cytokine levels after repeat injection indicate that reduced levels of inflammatory cytokines (IL-6, IFN-γ, TNFα) are elicited in response to lipoplexes coated with lactose as compared to PEG. These data indicate that a lactose coating on lipoplexes results in slightly improved tumor accumulation as compared to PEGylated formulations while eliciting a reduced innate immune response.

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.

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. As an alternative strategy, some recent reports have clearly demonstrated that exosomes from cow milk are absorbed from the gastrointestinal tract in humans and could potentially be used for oral delivery of drugs that are traditionally administered intravenously. Our previous work has shown that antibodies are present in exosome preparations, and the current work with milk exosomes suggests that absorption from the gastrointestinal tract occurs via the "neonatal" Fc receptor, FcRn. Furthermore, our results demonstrate that milk exosomes are absorbed from the gut as intact particles that can be modified with ligands to promote retention in target tissues.

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. In agreement with previous studies, PEGylation dramatically extends circulation times and enhances tumor delivery. However, our studies suggest that this relationship is not straightforward, and that particle sequestration by leukocytes can significantly alter biodistribution, especially with non-PEGylated nanoparticle formulations. We conclude that leukocyte uptake should be considered in biodistribution studies, and that delivery to these circulating cells may present opportunities for treating viral infections and leukemia.

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. Our results indicate that initial tumor deposition (5 min) was slightly lower in nude mice but comparable after 24 h. In addition, some small differences in tissue deposition/accumulation and reporter gene expression were observed between the two mouse models. The results with this one tumor model suggest that delivery studies conducted in nude mice can provide comparable results to those in immunocompetent mouse models.

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. This study measured the effect of repeat administration of nontargeted lipoplexes on clearance, cytokine/chemokine response, plasmid distribution, reporter gene expression, and liver toxicity. We do not observe a refractory period or a statistically significant difference in blood clearance between the first administration and subsequent injections of this lipoplex formulation, consistent with the absence of a cytokine/chemokine response. However, we do see a significant effect on both plasmid accumulation and expression, an enhancement of 26-fold and 10-fold in tumor plasmid levels and expression, respectively, after 4 injections as compared to that after a single injection. In addition, in vivo imaging suggests that expression in other organs had diminished rapidly 72 h after each administration, in contrast to relatively constant expression in the tumor. Taken together, the findings indicate that gene delivery to tumors can be dramatically enhanced by employing repetitive administration.

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. CONCLUSION: Lipoplexes prepared at anionic charge ratios offer comparable tumor delivery and reduced liver toxicity despite shorter circulation times.

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. In this study, we compare the transfection efficiency of cationic lipoplexes to effects on viability of cultured cells in both the short and long term (7 days). Our results indicate that, while minimal toxicity is evident 24 h after exposure to DOTAP-based lipoplexes, cell viability continues to decline and ultimately compromises reporter gene expression at longer times. Substitution of a naturally occurring cationic amphiphile, sphingosine, for DOTAP greatly reduces toxicity and allows high expression to be maintained over prolonged periods.

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) to optimize formulations for transfection in cell culture and in vivo. The results of such experiments are often interpreted in the context of nuclease resistance and cell association, but effects on the protein corona are usually not considered. While many studies have demonstrated that lipoplex structure and function can be dramatically compromised in the presence of serum, little attention has been paid to the adsorption of specific proteins and how this might be affected by formulation parameters. In this study, we characterize changes in the protein corona that occur as DOTAP-based lipoplexes are formulated with different amounts of cholesterol and prepared at different charge ratios. Our results demonstrate a significant effect of lipid composition on both total protein adsorption as well as the individual proteins from fetal calf serum that are associated with lipoplexes. In addition, we show that PEGylation increases protein adsorption with our formulations; effects that depend on the type of PEG conjugate employed in the lipoplex. Attempts to identify a specific protein responsible for enhancing transfection were unsuccessful.

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. In addition, studies assessing transfection rates in the absence of serum demonstrate that the ability of domains to enhance transfection is not dependent on interactions with serum proteins. Consistent with this hypothesis, characterization of the adsorbed proteins composing the corona of these lipoplex formulations did not reveal a correlation between transfection and the adsorption of a specific protein. CONCLUSION: We have demonstrated that the interaction with serum proteins can promote domain formation in some formulations, and thereby result in enhanced transfection only after serum exposure.

Ligands located within a cholesterol domain enhance gene delivery to the target tissue.

Targeted gene delivery provides enormous potential for clinical treatment of many incurable diseases. Liposomes formulated with targeting ligands have been tested extensively both in vitro and in vivo, and many studies have strived to identify more efficacious ligands. However, the environment of the ligand within the delivery vehicle is generally not considered, and this study assesses the effect of ligand microenvironment by utilizing a lipoplex possessing a cholesterol domain. Our recent work has shown that the presence of the targeting ligand within the cholesterol domain promotes more productive transfection in cultured cells. In the present study, lipoplexes having the identical lipid composition were formulated with different conjugates of the folate ligand such that the ligand was included in, or excluded from, the cholesterol domain. The effect of locating the ligand within the cholesterol domain was then tested in a xenograft tumor model in mice. Lipoplexes that included the ligand within the cholesterol domain showed significantly higher luciferase expression and plasmid accumulation in tumors as compared to lipoplexes in which the ligand was excluded from the domain. These results demonstrate that the microenvironment of the ligand can affect gene delivery to tumors, and show that ligand-mediated delivery can be enhanced by locating targeting ligands within a cholesterol domain.

Application of a ultra performance liquid chromatography method in the determination of DNA quality and stability.

The development of plasmid DNA as a pharmaceutical requires that integrity (i.e., supercoil content) be monitored as part of quality control. The standard method of determining supercoil content is gel electrophoresis followed by staining and imaging, which is complicated by a variety of factors. Previously described chromatographic methods used to quantify supercoil content have had difficulty obtaining reliable separation of the different isoforms. Using ultra performance liquid chromatography, we have optimized buffer conditions, and utilized increased column temperatures in developing a method that allows accurate quantification of each isoform by ultraviolet detection. We found that increasing the column temperature to 55°C improved separation of the isoform peaks as well as increased the resolution of each peak. We demonstrate the utility of this method by quantifying supercoil content of samples subjected to sonication, acidification or lyophilization, and storage. Our results demonstrate that this method allows for a precise quantification of individual DNA isoforms within a heterogeneous sample.

Moisture content impacts the stability of DNA adsorbed onto gold microparticles.

Particle-mediated epidermal delivery (PMED) of small quantities of DNA (0.5-4.0 µg) has been reported to both induce an immune response and protect against disease in human subjects. In order for the PMED of DNA to be a viable technique for vaccination, the adsorbed DNA must be stable during shipping and storage. Here, we report that the storage stability of plasmid DNA adsorbed to 2-µm gold particles is strongly dependent on sample water content. Gold/DNA samples stored at 60°C and 6% relative humidity (RH) maintained supercoil content after 4-month storage, whereas storage at higher RHs facilitated degradation. Storage with desiccants had stabilizing effects on DNA similar to storage at 6% RH. However, storage with "indicating" Drierite and phosphorus pentoxide resulted in enhanced rates of DNA degradation.

Kemp elimination catalysts by computational enzyme design.

The design of new enzymes for reactions not catalysed by naturally occurring biocatalysts is a challenge for protein engineering and is a critical test of our understanding of enzyme catalysis. Here we describe the computational design of eight enzymes that use two different catalytic motifs to catalyse the Kemp elimination-a model reaction for proton transfer from carbon-with measured rate enhancements of up to 10(5) and multiple turnovers. Mutational analysis confirms that catalysis depends on the computationally designed active sites, and a high-resolution crystal structure suggests that the designs have close to atomic accuracy. Application of in vitro evolution to enhance the computational designs produced a >200-fold increase in k(cat)/K(m) (k(cat)/K(m) of 2,600 M(-1)s(-1) and k(cat)/k(uncat) of >10(6)). These results demonstrate the power of combining computational protein design with directed evolution for creating new enzymes, and we anticipate the creation of a wide range of useful new catalysts in the future.

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. Designs that used an explicit water molecule to mediate proton shuffling were significantly more successful, with rate accelerations of up to four orders of magnitude and multiple turnovers, than those involving charged side-chain networks. The atomic accuracy of the design process was confirmed by the x-ray crystal structure of active designs embedded in two protein scaffolds, both of which were nearly superimposable on the design model.