Utilizing Cholesterol Nanodomains for Nucleic Acid Delivery - ACS

Chapter 3

Downloaded by UNIV OF FLORIDA on January 22, 2018 | http://pubs.acs.org Publication Date (Web): November 15, 2017 | doi: 10.1021/bk-2017-1271.ch003

Utilizing Cholesterol Nanodomains for Nucleic Acid Delivery Jamie L. Betker,1 Long Xu,2 Ye Zhang,3 and Thomas J. Anchordoquy*,1 1Skaggs

School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, 12850 E. Montview Blvd., Aurora, Colorado 80045, United States 2Janssen (China) R&D Center, 4560 Jinke Rd., Shanghai 201210, China 3The Food and Drug Administration (FDA), 10903 New Hampshire Avenue, Silver Spring, Maryland 20993, United States *E-mail: [email protected].

The ability of cholesterol to impart stability and serumresistance to lipid nanoparticles has been well-known for several decades. Thus, the use of cholesterol offers an alternative to utilizing the increasingly problematic “stealth” technology. More recently, the ability of high amounts of cholesterol to form nanodomains within lipid nanoparticles has been firmly established. The dense molecular packing and lack of ionic character of these nanodomains cause them to largely avoid the adsorption of proteins upon intravenous injection that can mask targeting moieties on the particle surface. In addition, formulations can be altered such that nanodomains form at low cholesterol concentrations with only naturally-occurring components, thereby minimizing the toxicity of the delivery vehicle and allowing the recipient cell to express therapeutic genes for prolonged periods. Furthermore, recent results suggest that repeated injection of some formulations elicits minimal, if any, cytokine response, thereby allowing delivery to tumors to be enhanced by multiple dosing. The chapter will review the evidence for cholesterol domains and their role in enhancing gene delivery both in vitro and in vivo, and provide a history of the development of these systems. Although this strategy has mostly focused on gene delivery, the approaches © 2017 American Chemical Society Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


could be employed for the delivery of other agents (e.g., small molecules, proteins/vaccines) with lipid-based systems.

The inability to effectively treat hereditary diseases has frustrated clinicians ever since the genetic underpinnings of diseases were appreciated. As scientists have gained a greater understanding of genes and molecular biology, the potential to modify and/or replace problematic sequences gave hope that hereditary diseases may someday be curable. While early laboratory studies utilized calcium phosphate precipitation to introduce genetic material into living cells, this method was much too inefficient for in vivo applications. However, such an approach offers the potential for altering cells in culture, and then transplanting these cells into a patient (i.e., “ex vivo” or cell-based therapy). Not long after the discovery of liposomes in the 1960s by Bangham and colleagues (1–4), attempts were made to use lipid vesicles to more efficiently introduce genes into mammalian cells, with potential in vivo applications (5–9). Early attempts to incorporate DNA and RNA into liposomes primarily utilized lipids extracted from natural sources, but this approach led to inefficient incorporation of nucleic acids into the liposomes (7–9). It is important to recognize that naturally-occurring lipids are predominantly anionic (e.g., phosphatidylserine, phosphatidylglycerol) or zwitterionic (e.g., phosphatidylcholine, phosphatidylethanolamine), and thus the incorporation of nucleic acids into lipid formulations was hindered by electrostatic repulsion between the negatively-charged phosphates of the nucleic acid and the anionic lipids. Initial attempts to utilize cationic amphiphiles (e.g., stearyl amine) to improve incorporation were not successful at achieving efficient transfection (7–10). However, a breakthrough publication by Phil Felgner and colleagues in 1987 demonstrated that cationic lipids could be used to efficiently introduce genetic material into mammalian cells (6, 11). This compelling demonstration ushered an era of investigating cationic lipids and polymers in an attempt to identify reagents capable of efficiently transfecting mammalian cells. Indeed, some early efforts laboriously screened large numbers of cationic lipids and “lipidoids” in an attempt to discover and patent reagents that might be used to cure a wide range of hereditary diseases. In addition to the ultimate goal of utilizing cationic lipids to deliver genes in vivo, there was significant interest in developing reagents that could be used to transfect cells in culture. It became evident in early experiments that transfection was most efficient if conducted in the absence of serum, and thus it became routine for research laboratories to perform experiments in serum-free media. A typical protocol involved cells being maintained in media containing 10% serum, but the media would be replaced with serum-free media in which the lipid/DNA complexes were administered. After several hours, serum would be re-introduced to the media, and reporter gene expression would be measured after 24 - 48 h. Although this general protocol works very well for introducing exogenous genes and monitoring their effects on cell processes in vitro, the overwhelming adoption of this approach led to the selection of cationic reagents that performed 72 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


well in the absence of serum. Beginning in the late nineties, a series of papers was published on the inhibitory effects of serum on transfection, and it became clear that this represented a major barrier to intravenous administration in vivo (12–17). In addition to merely the presence of serum, the source (i.e., species) as well as the concentration of serum were shown to be critical parameters (12, 17). Given this clear documentation regarding the devastating effects of serum, it is disappointing that the majority of studies claiming to be focused on identifying clinical formulations for systemic administration continue to utilize serum-free or 10% serum in their in vitro experiments. Because analogous studies with liposomes had shown that PEGylation could impart resistance to the destabilizing effects of serum, the field of non-viral gene delivery quickly adopted this technology (18–20). However, it was clear in early studies that the use of PEGylated components dramatically reduced transfection rates (21). More recent studies have demonstrated other adverse effects of utilizing PEGylated formulations including accelerated blood clearance, promotion of tumor angiogenesis and growth, and life-threatening immune responses in clinical trials (22–31). An alternative approach to imparting resistance to serum-induced perturbations was to incorporate cholesterol; a strategy that had been demonstrated for liposomes (32). Similar effects had been reported for lipoplexes, and our early experiments investigated the effects of cholesterol content on serum stability (17, 33). Our results were consistent with previous studies showing that serum concentration, charge ratio, and cholesterol content all contribute to lipoplex stability in terms of aggregation, lipid-DNA interactions, DNA stability, and transfection. The most striking finding was that increasing cholesterol contents to 66% by weight (80% mol/mol) endowed lipoplexes with superb serum stability. More specifically, minimal changes in particle size or transfection rates were observed when lipoplexes formulated at these cholesterol contents were incubated in 50% serum for 24 h (17)! Furthermore, although increasing cholesterol generally resulted in progressive increases in stability, there was a distinct enhancement as cholesterol content was increased from approximately 50 to 66 weight percent (66 to 80% mol/mol). Previous investigations on the biophysical properties of liposomes had demonstrated polymorphic behavior when high cholesterol contents were achieved, suggesting that lipid mixing between cholesterol and other acyl lipids (e.g., dioleoylphosphatidylcholine, DOPC) was not entirely homogeneous (34). This seemed especially relevant to our findings because our lipid formulation combined cholesterol with a lipid possessing identical hydrocarbon chains (i.e., 18 carbons with a single unsaturation), dioleoyl-trimethylammonium-propane (DOTAP). Considering that mixing with cholesterol would be predominantly governed by packing within the hydrocarbon chain region of the bilayer, the potential for incomplete lipid mixing within our lipoplexes seemed like a distinct possibility. Furthermore, X-ray diffraction studies by Huang et al. (35) had shown a solubility limit of cholesterol in other diacyl lipids at approximately 50 weight percent (66% mol/mol); the same concentration range at which we had observed a distinct enhancement of both serum stability and transfection rates in our previous work (17)! Therefore, we conducted studies to determine whether cholesterol was homogenously mixed with the DOTAP in our lipid formulations. Our 73 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


initial studies incorporated fluorescently-labelled lipids (NBD-cholesterol and rhodamine-PE) to assess fluorescence resonance energy transfer (FRET) between these two components as cholesterol contents were increased. We reasoned that any heterogeneity in lipid mixing would be detected as a change in FRET efficiency between these two components. As shown in Figure 1, FRET efficiency remained fairly constant (i.e., 0.35 – 0.45) as the mole percent of cholesterol was increased from 10 to 60 percent, but a sharp reduction was observed in liposomes formulated with cholesterol contents above 60 mole percent. The cholesterol contents where we observed altered FRET efficiency were consistent with those at which enhanced serum stability and transfection were observed. More specifically, the formation of a cholesterol domain within our preparations should cause an increase in the average distance between cholesterol and diacyl lipids due to sequestration of cholesterol molecules into a region that excludes diacyl lipids. Accordingly, FRET quenching profiles were obtained at different cholesterol contents, and the data were used to calculate the distance between labelled lipids in liposomes composed of DOTAP and cholesterol as described by Yguerabide (36). As shown in Figure 2, the distance between the labelled diacyl lipid and cholesterol remained fairly constant up to 60% mol/mol cholesterol, but a significant increase in distance was observed above 60%, consistent with the formation of a cholesterol domain at high cholesterol contents. As noted above for the FRET efficiency data, the marked changes in molecular distance occur at the same cholesterol concentrations where we observed enhanced serum stability and transfection in vitro, and are consistent with the formation of a cholesterol domain.

Figure 1. FRET efficiency measured in DOTAP/cholesterol liposomes (0.1 mM DOTAP in dH2O) formulated with cholesterol at 10, 20, 40, 60, 70 and 80 mole percent. Samples were labeled with NBD-cholesterol and Rhodamine-PE. Symbols and error bars represent the mean ± one standard deviation of three replicates. 74 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


The FRET experiments described above offer the advantage of being able to calculate molecular distances that provide a more intuitive feel to the findings. However, such experiments are labor-intensive, and the potential for a fluorescent label to modify interactions between molecules is always a concern. Accordingly, we performed additional experiments with differential scanning calorimetry to assess the formation of cholesterol domains within our formulations (37). The advantage of this technique is that it does not require modifications/labelling of the different lipid components, and previous studies have utilized this approach to characterize the formation of cholesterol domains in other lipid systems (34, 38, 39). More specifically, the formation of domains containing anhydrous cholesterol is evident by a lipid transition/melt at 38-40 °C in the thermogram (38). Such a transition was clearly observed in our calorimetric experiments, but only at cholesterol concentrations above 66 mole percent (50 % w/w) (37). As demonstrated in our earlier studies, formulation of lipoplexes at these high cholesterol concentrations correlated with a distinct increase in transfection in vitro (17, 37). In an attempt to provide a mechanism for the increased transfection observed in lipoplexes possessing a cholesterol domain, we characterized the binding of bovine serum albumin to lipoplexes formulated at different cholesterol contents. Briefly, these experiments involved incubation of lipoplexes in serum at room temperature for 30 min, followed by centrifugation and protein determination on the pellet. Accordingly, our measurements reflect proteins that bind somewhat tightly to the lipoplexes. Our results demonstrated that the amount of bound protein per cationic lipid was constant above 60% mol/mol, suggesting that reduced protein binding cannot explain the sharp increase in transfection concomitant with domain formation (37). However, the fact that protein binding per cationic lipid did not increase above 60% mol/mol cholesterol indicates that the incorporation of additional surface area in the form of a cholesterol domain does not result in measurable increases in protein binding. In order to determine whether this observation was specific for albumin, similar studies were conducted in fetal bovine serum, and the extent of bound protein was quantified at different cholesterol contents. As shown in Figure 3, the results from studies in serum are very consistent with previous studies conducted with albumin, and protein binding appears to decrease slightly at the highest cholesterol contents. In order to put the amount of bound protein into perspective, we should point out that DOTAP has a molecular weight of 663. Therefore, the levels of protein bound in each experiment indicate that each gram of unneutralized DOTAP binds approximately an equal weight of protein even at high cholesterol contents when adsorption is reduced. In contrast to the high level of protein adsorption to the region of the lipoplex possessing the cationic lipid and bound plasmid, additional protein binding is not observed when a cholesterol domain is incorporated into the lipoplex despite the significant increases in particle size (87, 120, 166 nm diameter at 60, 70, and 80% mol/mol cholesterol, respectively) that would be needed to accommodate the cholesterol domain within the membrane (37).

75 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 2. Calculated distance of closest approach of NBD-cholesterol to Rhodamine-PE by FRET analysis. Upper panel - Quenching profiles for NBD-cholesterol in DOTAP/cholesterol liposomes (0.1 mM DOTAP in dH2O) containing increasing amounts of Rhodamine-PE at cholesterol mole percent of 10% (diamonds), 20% (squares), 40% (closed triangles), 60% (open circles), 70% (open triangles) and 80% (closed circles). Lower panel Calculated distance of closest approach of NBD-cholesterol to Rhodamine-PE in DOTAP/cholesterol liposomes as a function of cholesterol mole percent. The values were calculated from the quenching profiles (upper panel); the data represent the mean ± one standard error of three replicates. The asterisk denotes a statistically significant difference between two groups of samples (p < 0.05). It is well-known that nanoparticles injected intravenously accumulate significant amounts of serum proteins, and our more recent studies have utilized modern proteomic methods to identify the different proteins that bind to various lipoplex formulations (40, 41). Surprisingly, we observe that domain-containing formulations bind different serum proteins than lipoplexes that lack a domain. In our constant search for a solid mechanism to explain the effects of domain 76 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


formation on transfection, we postulated that the specific proteins that are bound to domain-containing lipoplexes (as opposed to the amount of protein bound) might be responsible for the enhanced transfection we consistently observe. However, a careful comparison of proteins bound to different formulations and their corresponding transfection efficiency did not reveal any protein that could potentially enhance or attenuate transfection, at least in cell culture experiments (40, 41).

Figure 3. Effect of cholesterol content on serum protein binding. Lipoplexes (+/= 4) formulated at different cholesterol contents were incubated in 50% FBS for 30 min, and the amount of bound protein was determined as described by Xu and Anchordoquy (2008). The data represent the mean ± one standard deviation of triplicate samples. Despite the inability to identify specific proteins responsible for the enhanced transfection we observe with lipoplexes possessing a cholesterol domain, the observation that cholesterol domains do not adsorb detectable levels of serum proteins is analogous to a “bald spot” on the delivery system that offers some potential advantages. In addition to the potential for the protein-free domain to present an accessible surface that could facilitate binding and/or fusion to a cell membrane, it is possible to conjugate ligands to cholesterol such that ligands preferentially partition into the domain. Considering previous studies documenting the fouling/masking of ligands by bound protein, it follows that location of a ligand in a protein-free cholesterol domain might allow improved presentation and ligand efficacy (42). This is particularly relevant to targeted delivery systems that utilize small molecules and/or short peptides that are more amenable to commercial development (43). Unfortunately, the small ligand size that decreases the chance for immune recognition also increases the probability of being masked by adsorbed proteins. To test this hypothesis, we incorporated folate-conjugated components into lipoplexes formulated at different cholesterol contents, i.e., with and without a domain. In the first set of experiments, folate-cholesterol was incorporated into lipoplexes possessing 36% and 69% wt/wt cholesterol. Because these lipoplexes exhibit a large difference in their transfection efficiency in the absence of a targeting ligand, the effect of incorporating the folate ligand on transfection of KB cells that overexpress the folate receptor was compared to untargeted lipoplexes in each case. It is important to recognize (as stated earlier), that all transfection 77 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


experiments involved a pre-incubation in 50% serum prior to exposure to cells in culture. The data show that incorporation of the folate ligand in lipoplexes possessing a cholesterol domain resulted in a significant increase in transfection as might be expected (44). In contrast, incorporation of the folate ligand into lipoplexes that do not possess a cholesterol domain had no effect on transfection, consistent with previous studies demonstrating the potential for adsorbed protein (“corona”) to inhibit ligand targeting (42, 44). Additional studies were designed to assess the role of the cholesterol domain in promoting effective ligand presentation after exposure to serum. In these experiments, the folate ligand was conjugated to either cholesterol (as above) or a diacyl lipid, phosphatidylethanolamine (PE). In contrast to the experiments described above, these conjugates were incorporated into the identical lipoplex that contained a cholesterol domain, i.e., 69% wt/wt cholesterol (44). Our hypothesis was simply that the folate-cholesterol conjugate would partition into the domain and be available for targeting whereas the folate-PE conjugate would be excluded from the cholesterol domain, and thus susceptible to fouling/masking by adsorbed protein. Consistent with this hypothesis, transfection rates were enhanced with the folate-cholesterol conjugate, but not with the folate-PE conjugate (44). In fact, transfection was dramatically reduced when the folate-PE conjugate was incorporated into the lipoplex; an effect we attribute to the polyethyleneglycol linker that was used to conjugate folate to PE. Indeed, the incorporation of a conjugate of polyethyleneglycol and PE (i.e., no folate) caused similar reductions in transfection, consistent with our assertion that the polyethyleneglycol linker is responsible for this effect. Although the concentration of conjugate in the lipoplexes was only 0.4%, studies have shown that even these low levels of polyethyleneglycol conjugates can significantly reduce transfection rates in vitro and in vivo (21, 29, 43, 44). While the same PEG linker was also used to conjugate folate to cholesterol, no decrease in transfection rate was observed (44). It is tempting to conclude that folate-mediated transfection by domain-containing lipoplexes would have been further enhanced if the folate-cholesterol conjugate had not employed a polyethyleneglycol linker, but our studies have shown that PEG-cholesterol can actually enhance transfection in vitro in some cell lines (45). Regardless, other concerns about utilizing polyethyleneglycol conjugates (e.g., accelerated clearance upon repeat injection, promotion of tumor growth, anaphylactic shock) (22, 24–30, 46) have motivated us to develop methods for effectively conjugating peptide ligands directly to cholesterol. As described above, we have demonstrated enhanced transfection at high cholesterol contents that is coincident with formation of a cholesterol domain. In addition, we have exploited the finding that protein adsorption to the cholesterol domain is minimal, and thus location of a ligand within the domain appears to be critical for achieving maximum transfection in vitro. To test folate targeting in vivo, we utilized human KB cells (HeLa derivative) that over-express the folate receptor. Male, athymic nude mice were inoculated subcutaneously in the flank, and lipoplexes were administered intravenously once the tumors had reached 100 mm3 (47). Our results are very consistent with what we observed in cell culture, i.e., folate-cholesterol enhanced delivery to the tumor (KB cells) by approximately 3-fold whereas folate-PE did not increase delivery beyond that observed with 78 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


non-targeted lipoplexes (47). These results are entirely consistent with our previous conclusions regarding the role of the cholesterol domain in preventing fouling/masking due to serum protein adsorption, and promoting effective ligand presentation that results in more effective delivery. It is typically assumed that enhanced uptake by the target cells should increase transfection rates even though several studies have documented a poor correlation between uptake efficiency and transfection. Curiously, in contrast to the higher transfection observed when the ligand was located within the cholesterol domain, our in vitro studies clearly demonstrated that cell uptake was not increased under these conditions (44). In fact, uptake was significantly higher in cells treated with lipoplexes incorporating the folate-PE conjugate even though transfection was the lowest under these conditions. While the lack of correlation between uptake and transfection is not uncommon, the fact that uptake was highest when the ligand was excluded from the domain appears to contradict our suggestion that location of the ligand within the domain promotes more effective presentation. We have obtained similar results when quantifying uptake via confocal microscopy (unpublished data). The fact that cell uptake is highest under conditions where transfection efficiency remains low suggests that the various uptake pathways (e.g., clathrin-mediated endocytosis, caveolae, macropinocytosis, phagocytosis) have different trafficking efficiencies in terms of their ability to result in expression of the nucleic acid cargo. In this sense, quantification of “cell uptake” via confocal microscopy or flow cytometry is a fairly crude measurement that does not consider efficiencies associated with different uptake pathways. In considering our results showing greater transfection when ligands are located within the domain despite lower cell uptake, we conclude that ligand presentation plays a role in determining the uptake and/or trafficking pathway, and suggest that the domain serves to direct targeted lipoplexes toward more efficient pathways. Regardless of the mechanisms by which domains promote ligand-mediated transfection, we agree with the conclusions by Pozzi et al. that “the existence of such cholesterol nanodomains within lipoplex membranes could lead to a general rethinking of current targeting strategies” (48). In addition to enhancing the effect of ligands to promote transfection, the high cholesterol content used to facilitate domain formation increases serum stability in vitro and in vivo (17, 37, 47, 49). However, such high cholesterol contents (> 50% by weight) greatly rigidifies the bilayer, making it difficult to prepare liposomes by conventional extrusion methods. In addition, the supersaturated cholesterol is metastable, and cholesterol crystal formation can occur in hydrated suspensions during storage. Perhaps the most concerning issue is that infusion reactions and the formation of antibodies against cholesterol have been reported when animals were administered liposomes containing cholesterol contents above 30% (50–52). Taken together, it would be desirable to reduce the cholesterol content of our lipoplexes, but the fact that domain formation requires > 50% weight cholesterol would appear to represent an intractable predicament. Biophysical studies on lipid mixing have demonstrated that the tension within bilayers is responsible for domain formation, and that lipids with saturated acyl chains can promote the formation of cholesterol domains (53). In addition to chain saturation, the length of the hydrocarbon chains affects the 79 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


interaction with cholesterol and the formation of phase-separated cholesterol domains. Accordingly, we formulated lipoplexes with different amounts of phosphatidylcholines possessing progressively longer, saturated acyl chains (i.e., 14:0, 16:0, 18:0, 20:0, 22:0, 24:0) and monitored the formation of cholesterol domains with differential scanning calorimetry (41). The results were consistent with biophysical studies and showed that lipid chain lengths ≥ 20 carbons were effective at causing domain formation in lipoplexes. Surprisingly, the inclusion of these saturated phosphatidylcholines was so effective at promoting cholesterol domains that lipoplexes formulated with cholesterol contents as low as only 10% possessed domains (41)! In addition to calorimetric measurements, transfection was also monitored, and a significant enhancement was noted only when lipoplexes were formulated with lipids possessing sufficiently long acyl chains to cause domain formation. These results confirmed our previous conclusions regarding the role of cholesterol domain formation in enhancing transfection. In investigating the potential for saturated phosphatidylcholines to promote domain formation, experiments were designed to determine if the lipid components affected the protein corona and to what extent adsorbed proteins affected transfection. For this reason, transfection experiments were conducted in serum-free media in addition to our standard protocol involving pre-incubation in 50% serum. The results from the different transfection experiments were quite comparable in terms of observing a significant boost in transfection with formulations possessing a cholesterol domain (lipid chains ≥ 20 carbons) as compared to those lacking a domain (lipid chains ≤ 16 carbons). However, lipoplexes formulated with phosphatidylcholine possessing 18 carbons gave different effects in terms of transfection. More specifically, lipoplexes formulated with phosphatidylcholine possessing chains with 18 carbons showed relatively low transfection in the absence of serum, but were capable of boosting transfection after incubation in serum (41). In other words, transfection with lipoplexes that incorporated lipids with 18-carbon chain lengths was comparable to that observed with domain-containing lipoplexes, but only after serum exposure. Furthermore, calorimetric analysis of this formulation did not detect a domain, consistent with the relatively low transfection observed when experiments were conducted in the absence of serum. In considering this inconsistency, it occurred to us that our calorimetric measurements did not include lipoplexes that had been exposed to serum, and so additional calorimetric studies were conducted on lipoplexes after serum exposure. The results from these calorimetric studies were consistent with that seen in transfection experiments, and we observed that serum exposure caused domain formation in lipoplexes formulated with distearylphosphatidylcholine (saturated 18-carbon chains) (41). No effect on domain formation was observed for shorter chain lengths, suggesting that the membrane tension in formulations possessing 18-carbon lipid chains is sufficiently close to the threshold required for domain formation that serum protein binding can trigger this phenomenon. Although this “switch” may be useful at some point in development, further studies utilized phosphatidylcholine bearing 20-carbon chains (diarachidoyl phosphatidylcholine; DAPC) and 20% mol/mol cholesterol. This formulation is much easier to handle than the high cholesterol formulations described earlier, and is more amenable to preparation by extrusion. Most importantly, this formulation 80 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


does not appear to elicit an antibody response upon repetitive injection in mice (54). By incorporating lipids possessing long, saturated chains to promote domain formation, we have overcome one of the fundamental problems associated with the high cholesterol contents used in our earlier studies. However, it is well-recognized that the cationic agents used for nucleic acid delivery are toxic, and this compromises their potential for therapeutic use (55–59). While many synthetic agents are not readily biodegradable, the use of ester bonds that mimic naturally-occurring membrane lipids is typically correlated with lower toxicities. For this reason, our previous work has utilized DOTAP; a cationic agent that is considered to be minimally toxic, and one that has been used in clinical trials (60). However, there is no question that even DOTAP exhibits toxicity that is routinely observed in cell culture studies. It is important to recognize that cationic agent toxicity is generally assessed in vivo by monitoring blood levels of liver enzymes (typically ALT and/or AST). Similar to that seen with conventional liposome formulations, liver enzymes typically increase 2-5 fold after IV administration in animal models, and then return to normal levels within a couple days. This level of toxicity is considered tolerable, especially considering that treatments are often designed for cancer patients, and typically tested in late-stage cancer patients that are not concerned with a temporary elevation in liver enzymes. Although this attitude is understandable in terms of treating patients with a traditional chemotherapeutic, one must also appreciate that the use of nucleic acids as therapeutics is fundamentally different than traditional small molecule therapeutics. More specifically, gene-based therapies rely on the recipient cell to uptake and express the delivered gene such that the ultimate product (shRNA, protein) is produced and available to elicit its therapeutic effect. It follows that toxicity to the target cell governs expression and the subsequent therapeutic effect. Therefore, the common practice of monitoring toxicity solely by assessing effects on the liver does not take into account the role of target cells in producing/expressing the molecule that ultimately exhibits the therapeutic effect. As mentioned above, it is well-known that cationic agents exhibit significant cell toxicity (55–59). What is generally not appreciated is the role that the mechanism of toxicity plays in facilitating transfection. More specifically, complexes of nucleic acids and cationic agents are typically depicted as well-organized, spherical nanoparticles that are designed to interact with cell membranes and facilitate cytoplasmic delivery via fusion and/or lysis. While most researchers would agree that membrane disruption must occur in order to escape cellular degradation processes (e.g., lysosome), few have considered the effect that such disruption would have on the long-term viability of the cell and its ability to express the therapeutic gene. In this sense, the ability of cationic agents to disturb normal membrane structure and function is linked to both transfection and toxicity. Furthermore, most investigations of gene delivery are initiated in cell culture studies that simply measure reporter gene expression after 24 - 48 h without assessing toxicity, therefore the link between transfection and toxicity is typically not observed. Even in studies that do assess toxicity via viability assays, experiments often involve washing away dead cells such that 81 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


viability measurements are artificially inflated, especially after only 24 - 48 h. If both transfection and viability are monitored in all cells, it can be observed that greater amounts of cationic agent (i.e., higher +/- charge ratios) cause progressive increases in both transfection and toxicity (55). Furthermore, administration of uncomplexed cationic components (i.e., not associated with the delivery vehicle) to cells results in a similar effect on transfection and toxicity, consistent with the idea that permeabilization by cationic agents is the predominant mechanism by which transfection is achieved. It is not generally appreciated that the toxic effects of cationic components are not always evident within the timeframe of a typical in vitro transfection experiment (i.e., 24 - 48 h). Scientists tend to think of cells being either alive or dead, with a rapid/immediate transition between these two states. One could argue that this perception comes from watching movies in which actors collapse immediately after being shot and/or poisoned. However, just like the true process of dying from most diseases (e.g., cancer), cells typically become compromised and slowly die over a protracted period. Accordingly, the ultimate effects of exposure to cationic lipids may not be evident for up to a week. In our recent experiments, we monitored cell viability and reporter gene expression for seven days after transfection with different formulations (55). What we observe is that cells in culture show minimal losses in viability 24 h after transfection as measured by a standard MTT assay, but viability progressively declines in these cells over seven days (as compared to untreated controls) (55). Not surprisingly, we observe a corresponding reduction in reporter gene expression (luciferase) with the progressive loss in viability, presumably due to the inability of dead/dying cells to synthesize protein. Incidentally, we conducted viability measurements with both a live/dead assay and an MTT assay. As shown in Figure 4, the live/dead assay measured lower viability than did the MTT assay, although the trends were consistent. Considering that the live/dead assay essentially quantifies plasma membrane integrity as compared to the mitochondrial activity assessed by MTT, this suggests that mitochondria maintain at least some level of activity despite a compromised plasma membrane. Regardless of the precise timing of events associated with cell death, it is clear from our studies that the effects of exposure to cationic lipids are not fully evident within the 24-48 h timeframe of a typical in vitro experiment (55). It is clear from the experiments described above that cells exposed to cationic components are compromised, and that expression of an exogenous (e.g., therapeutic) gene can be limited by the toxicity of the delivery vehicle. While it is recognized that monovalent, biodegradable cationic agents possess reduced toxicity, our data and those of others have clearly shown that even cations that are considered less-toxic (e.g., DOTAP) do compromise cell viability to a significant extent. With the goal of achieving prolonged gene expression in vivo, we considered other cationic agents that could be incorporated into our domain-containing lipoplexes. Since there are no known naturally-occurring cationic lipids, we investigated sphingosine (a cationic breakdown product of sphingomyelin that is present in every mammalian cell) as a potential replacement for cationic lipids in our formulations. Previous studies had shown that sphingosine was capable of binding DNA and facilitating gene delivery 82 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


in cell culture (61–64), so we substituted sphingosine for DOTAP in our formulations. In contrast to the toxicity seen with DOTAP-based formulations described above, cells treated with lipoplexes prepared with sphingosine did not exhibit a progressive decrease in viability over time (55). In fact, cells treated with sphingosine-based lipoplexes appeared to recover from an initial (24 h) decrease in viability, suggesting that the membrane damage inflicted during the transfection process was reversible/reparable to some extent. Furthermore, in vitro transfection levels after 24 h were comparable to that observed with DOTAP-based formulations. Similar results were obtained when these formulations were administered intravenously to tumor-bearing mice, i.e., gene delivery and expression in tumors after 24 h was similar to that observed with DOTAP-based formulations (54, 65).

Figure 4. Comparison of viability measurements conducted with different assays. MCF-7 cells were treated with lipid/DNA complexes formulated at different charge ratios. After a 4-hour exposure, complexes were removed and cells were incubated in fresh media for 48 h before assessing viability by a live/dead or MTT assay.

Although the levels of reporter gene expression in tumors were comparable between sphingosine- and DOTAP- based lipoplexes, the distribution of expression throughout the rest of the body was altered significantly. In vivo imaging of mice bearing 4T1 tumors revealed that intravenous administration of the sphingosine-based formulations resulted in expression that was largely confined to the tumor 72 h after administration (54). In contrast, gene expression after treatment with DOTAP-based formulations was distributed throughout the major organs and the intestine (Figure 5). 83 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 5. Effect of lipoplex formulation on the distribution of reporter gene expression. Mice bearing 4T1 tumors in their shoulder were administered a plasmid encoding luciferase incorporated into different formulations. Luciferase expression was imaged 72 h after intravenous administration. It is important to point out that all formulations resulted in expression that was broadly distributed throughout the body after 24 h, but that expression outside the tumor was noticeably diminished 72 h after administration (54). The mechanism responsible for this improved localization is currently being investigated in our laboratory, but these results suggest that sphingosine-based formulations allow exogenous gene expression to be primarily limited to the tumor as opposed to DOTAP-based formulations. Considering the reduction in toxicity observed by substituting sphingosine for DOTAP, we sought to further reduce toxicity in vivo by investigating the doses of lipid needed to achieve robust gene expression. Accordingly, we optimized the quantity of lipid (i.e., +/- charge ratio) used for delivery with our formulation composed of sphingosine, cholesterol, and DAPC at a 3:2:5 mole ratio. Our results were consistent with similar studies we have conducted previously with DOTAP-based formulations, and showed that transfection levels in vitro exhibited a distinct optimum at +/- = 4 (65). However, we also found a second, and equally effective, transfection optimum at a charge ratio of 0.5 (i.e., anionic particles with a negative zeta potential of - 24.4 ± 2.9 mV). Both these charge ratios were tested in vivo and found to provide similar, high levels of transfection (65). By reducing the charge ratio from 4 to 0.5, we decrease the lipid amount needed to deliver a dose of DNA by 8-fold! Not surprisingly, we observe a 84 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


concomitant reduction in liver toxicity (ALT levels) by 2-3-fold to levels below that measured for intravenous administration of phosphatidylcholine liposomes (65). Therefore, our current formulation has significantly lower toxicity as compared to the DOTAP:cholesterol lipoplexes used in our earlier work due to both the substitution of sphingosine for DOTAP and the lower amounts of lipid (+/- = 0.5) used for delivery. Although we are able to consistently achieve high levels of reporter gene expression in subcutaneous tumors in our animal models, it is desirable to find approaches that further increase delivery to the tumor with the goal of achieving therapeutic levels of expression. Considering a recent review of nanoparticle technology showing that only 1% of the intravenously-injected dose of nanoparticles actually reaches the tumor, approaches that enhance tumor delivery are desperately needed (66). One method of delivering more cargo to tumors is to utilize repetitive administration, but the potential to utilize this approach to prolong therapeutic expression levels and/or progressively increase delivery is limited by the immune response to nanoparticles (67–70). For example, the immune response to PEGylated nanoparticles is well characterized, and involves an IgM response to the initial injection (23, 24). If a second dose of nanoparticles is administered within two weeks (“refractory period”), an IgG response is elicited, and the particles are rapidly cleared; i.e, “accelerated blood clearance”. It is common for studies to design dosing intervals such that subsequent injections do not occur within the refractory period (e.g., 2-3 weeks) (70). Although our lipoplexes do not utilize PEGylated components, the ability of relatively simple particles (e.g., liposomes) to activate the innate immune system (e.g., complement) is well-established (71). One approach to preventing an immune response is to co-administer a liposomal chemotherapeutic that destroys cells involved in the immune response to the nanoparticle (67–69). Such studies have shown that the immune-associated cytokine response is greatly diminished under these conditions, and this correlates with improved delivery and efficacy after multiple administration. In an attempt to avoid the co-administration of a chemotherapeutic, we investigated the cytokine response to different lipoplex formulations. In these experiments, immunocompetent Balb/c mice were intravenously-administered a lipoplex formulation, and then re-administered the same formulation three days later. Blood was collected two hours after the second injection, and analyzed for 37 different cytokines/chemokines, and levels were compared to that observed in mice injected with saline. Mice administered lipofectamine-based lipoplexes possessed elevated levels of every cytokine/chemokine (54). In contrast, blood from mice injected with our formulation composed of sphingosine, cholesterol, and DAPC incorporating a plasmid with minimal CpG motifs contained only slightly elevated levels of a single chemokine, keratinocyte chemokine (54). This chemokine is not associated with inflammation and/or a danger response that would be elicited in response to an invading “non-self” entity (72). Instead, keratinocyte chemokine recruits leukocytes in response to liver injury, consistent with the mild elevation of liver enzymes we have measured in response to intravenous administration (54, 65, 73). Interestingly, we saw elevation of three additional cytokines (IL-1α, IL-7, IL-13) if the same lipid formulation incorporated a plasmid containing extensive 85 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


CpG sequences, demonstrating the importance of reducing immunostimulation by the nucleic acid component of the lipoplex (54). The effect of PEGylation on the cytokine response was dramatic, with 30/37 cytokines/chemokines being elevated in mice when our formulation incorporated 5% PEG-DSPE! The effects of DOTAP were less dramatic but still significant, with 11/37 cytokines/chemokines being elevated when our formulation was prepared with DOTAP instead of sphingosine (54). The absence of a cytokine response after repeated administration of this formulation suggests that our delivery system does not elicit the typical immune/inflammatory response that is observed with other nanoparticles. Accordingly, this (in addition to the low toxicity described above) should permit repetitive injection of our delivery system without the accelerated blood clearance observed in other studies. Furthermore, no refractory period between injections is required, and thus frequent dosing should allow for progressively higher levels of gene expression in the tumor. To test this hypothesis, immunocompetent mice bearing murine tumors (4T1) were administered four doses of lipoplexes at 3-day intervals. Reporter gene expression (luciferase) was imaged after each injection and quantified via luciferase assay by extracting tissues 24 h after the first and fourth injection. Consistent with our hypothesis, the images indicate a progressive increase in expression within tumors after each injection, and analysis of extracted tumors shows that plasmid delivery was 26-fold higher after the fourth injection as compared to a single injection (54). In contrast to that observed in the tumors, plasmid accumulation in the other tissues was increased by less than 4-fold, consistent with similar delivery efficiency after each of the four doses, with some plasmid degradation/loss occurring within tissues during the course of the experiment. The mechanism by which delivery was increased by more than four-fold in tumors is unclear, but these data suggest that the lack of an immune response allows frequent, repetitive dosing to achieve markedly higher delivery and expression than that possible with other gene delivery systems (54). It is well-established that delivery to tumors can be enhanced by incorporating targeting ligands that allow for retention of particles at the target site (74). Although many studies have utilized ligands that target receptors that are overexpressed on tumors (e.g,, folate, transferrin, herceptin), it should be recognized that only a tiny fraction of particles are able to directly access tumor cells through extravasation (66, 75). Considering that the vast majority of particulate delivery vehicles remain within the vasculature, a more effective strategy might be to utilize ligands that target proteins highly expressed on tumor vasculature (e.g., integrins) that is more accessible from the blood. It has been shown that a specific peptide known as iRGD targets the αν β3/β5 integrin on the vasculature of many tumor types, and that this binding triggers a translocation across the vascular endothelium and a proteolytic cleavage that results in a peptide ligand for Neuropilin-1 that is expressed on many cancer cells (76). Furthermore, both the inhibitory effects of PEGylated components on intracellular delivery and the potential for triggering an immune response provide a strong rationale for developing conjugation methods that do not utilize PEG as a linker (21, 22, 31, 44, 47, 77). Accordingly, our most recent work has developed methods for the specific conjugation of peptides through their terminal amine to 86 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


the hydroxyl group of cholesterol. This approach not only avoids the use of PEG as a linker, but it also allows direct conjugation to cholesterol thereby locating the ligand within the particle’s cholesterol domain for improved delivery (44, 47). Our data in immunocompetent, tumor-bearing mice demonstrate that very low levels of the iRGD-cholesterol conjugate (0.05%) are capable of enhancing tumor delivery by more than 25-fold after a single injection! Increased ligand concentrations (up to 3%) did not enhance tumor delivery, consistent with the suggestion that the number of receptors on the tumor cells and/or vasculature may limit the effectiveness of ligand-mediated uptake (78). Regardless of the potential limitations of ligand-mediated delivery, our experiments demonstrate that DNA delivery and reporter gene expression in tumors can be dramatically enhanced by utilizing iRGD conjugated to cholesterol, consistent with our earlier work showing the ability of ligands located within the cholesterol domain to improve delivery as compared to ligands located outside of the domain (44, 47). In conclusion, early studies with liposomes and lipoplexes have demonstrated that high levels of cholesterol can be used to impart resistance to serum-induced destabilization (17, 32, 33). Considering the dramatic reductions in transfection/intracellular delivery due to the use of PEGylated components as well as the life-threatening immune reactions observed in clinical trials resulting from pre-existing anti-PEG antibodies, we were motivated to avoid PEGylation (21, 22, 29, 31, 44, 46, 77, 79). Our initial studies demonstrated the effects of cholesterol in imparting serum-resistance and maintaining particle size and transfection activity in the presence of physiological levels of serum (17, 37, 49). Subsequent studies documented that the high cholesterol levels that impart these favorable properties correlate with the formation of a cholesterol nanodomain within the lipid component of the delivery vehicle (37, 41, 44). Furthermore, location of ligands within the cholesterol nanodomain results in greater delivery as compared to lipoplexes incorporating the identical ligand that is excluded from the domain (44, 47). More recent studies have identified methods for achieving the enhanced stability and formation of a cholesterol domain at lower cholesterol contents (≥ 10% cholesterol), while also using low amounts of only naturally-occurring lipids that greatly reduce toxicity and the cytokine response typically associated with particulate delivery systems (41, 54, 55, 65). Finally, our most recent work has demonstrated that these formulations can be administered repeatedly without the accelerated blood clearance observed with other systems, and be targeted with iRGD to greatly enhance gene delivery to tumors in animal models (54). The next phase of this research is to deliver therapeutic genes for cancer treatment with the hope of significantly shrinking tumors and/or modifying their phenotype. While our experiments to date have largely focused on delivery to the tumor, other applications of this delivery technology (e.g., immunotherapy, antiviral) are currently being explored.

87 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Acknowledgments The views and opinions presented here represent those of the authors and should not be considered to represent advice, policy or guidance on behalf of the Food and Drug Administration. The authors are grateful for the support of this work by numerous sources throughout the years including the National Institutes of Health and collaborators within the pharmaceutical industry.


References 1.

Abraham, S. A.; McKenzie, C.; Masin, D.; Ng, R.; Harasym, T. O.; Mayer, L. D.; Bally, M. B. In vitro and in vivo characterization of doxorubicin and vincristine coencapsulated within liposomes through use of transition metal ion complexation and pH gradient loading. Clin. Cancer Res. 2004, 10, 728–738. 2. Bangham, A. D. Membrane models with phospholipids. Prog. Biophys. Mol. Biol. 1968, 18, 29–95. 3. Bangham, A. D.; Horne, R. W. Negative staining of phospholipids and their structural modification by surface-active agents as observed in the electron microscope. J. Mol. Biol. 1964, 8, 660–8. 4. Bangham, A. D.; Standish, M. M.; Weissmann, G. The action of steroids and streptolysin S on the permeability of phospholipid structures to cations. J. Mol. Biol. 1965, 13, 253–9. 5. Felgner, P. L. Nonviral strategies for gene therapy. Sci. Am. 1997, 276, 102–106. 6. Felgner, P. L.; Gadek, T. R.; Holm, M.; Roman, R.; Chan, H. W.; Wenz, M.; Northrop, J. P.; Ringold, G. M.; Danielsen, M. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl. Acad. Sci. U. S. A. 1987, 84, 7413–7. 7. Fraley, R.; Straubinger, R. M.; Rule, G.; Springer, E. L.; Papahadjopoulos, D. Liposome-mediated delivery of deoxyribonucleic acid to cells: enhanced efficiency of delivery related to lipid composition and incubation conditions. Biochemistry 1981, 20, 6978–87. 8. Fraley, R.; Subramani, S.; Berg, P.; Papahadjopoulos, D. Introduction of liposome-encapsulated SV40 DNA into cells. J. Biol. Chem. 1980, 255, 10431–5. 9. Straubinger, R. M.; Papahadjopoulos, D. Liposomes as carriers for intracellular delivery of nucleic acids. Methods Enzymol. 1983, 101, 512–27. 10. Fraley, R. T.; Dellaporta, S. L.; Papahadjopoulos, D. Liposome-mediated delivery of tobacco mosaic virus RNA into tobacco protoplasts: a sensitive assay for monitoring liposome-protoplast interactions. Proc. Natl. Acad. Sci. U. S. A. 1982, 79, 1859–63. 11. Felgner, P. L.; Ringold, G. M. Cationic liposome-mediated transfection. Nature 1989, 337, 387–388. 88 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


12. Li, S.; Tseng, W. C.; Stolz, D. B.; Wu, S. P.; Watkins, S. C.; Huang, L. Dynamic changes in the characteristics of cationic lipidic vectors after exposure to mouse serum: implications for intravenous lipofection. Gene Ther. 1999, 6, 585–94. 13. Thierry, A. R.; Rabinovich, P.; Peng, B.; Mahan, L. C.; Bryant, J. L.; Gallo, R. C. Characterization of liposome-mediated gene delivery: expression, stability and pharmacokinetics of plasmid DNA. Gene Ther. 1997, 4, 226–37. 14. Yang, J. P.; Huang, L. Overcoming the inhibitory effect of serum on lipofection by increasing the charge ratio of cationic liposome to DNA. Gene Ther. 1997, 4, 950–60. 15. Yang, J. P.; Huang, L. Time-dependent maturation of cationic liposome-DNA complex for serum resistance. Gene Ther. 1998, 5, 380–7. 16. Zelphati, O.; Uyechi, L. S.; Barron, L. G.; Szoka, F. C., Jr. Effect of serum components on the physico-chemical properties of cationic lipid/oligonucleotide complexes and on their interactions with cells. Biochim. Biophys. Acta 1998, 1390, 119–33. 17. Zhang, Y.; Anchordoquy, T. J. The role of lipid charge density in the serum stability of cationic lipid/DNA complexes. Biochim. Biophys. Acta 2004, 1663, 143–57. 18. Ogris, M.; Brunner, S.; Schuller, S.; Kircheis, R.; Wagner, E. PEGylated DNA/transferrin-PEI complexes: reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery. Gene Ther. 1999, 6, 595–605. 19. Wheeler, J. J.; Palmer, L.; Ossanlou, M.; MacLachlan, I.; Graham, R. W.; Zhang, Y. P.; Hope, M. J.; Scherrer, P.; Cullis, P. R. Stabilized plasmid-lipid particles: construction and characterization. Gene Ther. 1999, 6, 271–81. 20. Papahadjopoulos, D.; Allen, T. M.; Gabizon, A.; Mayhew, E.; Matthay, K.; Huang, S. K.; Lee, K. D.; Woodle, M. C.; Lasic, D. D.; Redemann, C.; et al. Sterically stabilized liposomes: improvements in pharmacokinetics and antitumor therapeutic efficacy. Proc. Natl. Acad. Sci. U. S. A. 1991, 88, 11460–4. 21. Harvie, P.; Wong, F. M. P.; Bally, M. B. Use of poly(ethylene glycol)-lipid conjugates to regulate the surface attributes and transfection activity of lipidDNA particles. J. Pharm. Sci. 2000, 89, 652–663. 22. Ganson, N. J.; Povsic, T. J.; Sullenger, B. A.; Alexander, J. H.; Zelenkofske, S. L.; Sailstad, J. M.; Rusconi, C. P.; Hershfield, M. S. Pre-existing anti-polyethylene glycol antibody linked to first-exposure allergic reactions to pegnivacogin, a PEGylated RNA aptamer. J. Allergy Clin. Immunol. 2016, 137, 1610–1613. 23. Ishida, T.; Ichihara, M.; Wang, X.; Yamamoto, K.; Kimura, J.; Majima, E.; Kiwada, H. Injection of PEGylated liposomes in rats elicits PEG-specific IgM, which is responsible for rapid elimination of a second dose of PEGylated liposomes. J. Controlled Release 2006, 112, 15–25. 24. Ishida, T.; Kiwada, H. Accelerated blood clearance (ABC) phenomenon upon repeated injection of PEGylated liposomes. Int. J. Pharmaceutics 2008, 354, 56–62. 89 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


25. Moghimi, S. M. Cancer nanomedicine and the complement system activation paradigm: anaphylaxis and tumour growth. J. Controlled Release 2014, 190, 556–562. 26. Saadati, R.; Dadashzadeh, S.; Abbasian, Z.; Soleimanjahi, H. Accelerated blood clearance of PEGylated PLGA nanoparticles following repeated injections: effects of polymer dose, PEG coating, and encapsulated anticancer drug. Pharm. Res. 2013, 30, 985–995. 27. Sabnani, M. K.; Rajan, R.; Rowland, B.; Mavinkurve, V.; Wood, L. M.; Gabizon, A. A.; La-Beck, N. M. Liposome promotion of tumor growth is associated with angiogenesis and inhibition of antitumor immune responses. Nanomedicine 2015, 11, 259–62. 28. Tagami, T.; Uehara, Y.; Moriyoshi, N.; Ishida, T.; Kiwada, H. Anti-PEG IgM production by siRNA encapsulated in a PEGylated lipid nanocarrier is dependent on the sequence of the siRNA. J. Controlled Release 2011, 151, 149–154. 29. Verhoef, J. J. F.; Anchordoquy, T. J. Questioning the use of PEGylation for drug delivery. Drug Delivery Trans. Res. 2013, 3, 499–503. 30. Wang, X.; Ishida, T.; Kiwada, H. Anti-PEG IgM elicited by injection of liposomes is involved in the enhanced blood clearance of a subsequent dose of PEGylated liposomes. J. Controlled Release 2007, 119, 236–44. 31. Povsic, T. J.; Lawrence, M. G.; Lincoff, A. M.; Mehran, R.; Rusconi, C. P.; Zelenkofske, S. L.; Huang, Z.; Sailstad, J.; Armstrong, P. W.; Steg, P. G.; Bode, C.; Becker, R. C.; Alexander, J. H.; Adkinson, N. F.; Levinson, A. I.; Investigators, R.-P. Pre-existing anti-PEG antibodies are associated with severe immediate allergic reactions to pegnivacogin, a PEGylated aptamer. J. Allergy Clin. Immunol. 2016, 138, 1712–1715. 32. Allen, T. M.; Cleland, L. G. Serum-induced leakage of liposome contents. Biochim. Biophys. Acta 1980, 597, 418–26. 33. Crook, K.; Stevenson, B. J.; Dubouchet, M.; Porteous, D. J. Inclusion of cholesterol in DOTAP transfection complexes increases the delivery of DNA to cells in vitro in the presence of serum. Gene Ther. 1998, 5, 137–43. 34. Epand, R. M.; Hughes, D. W.; Sayer, B. G.; Borochov, N.; Bach, D.; Wachtel, E. Novel properties of cholesterol-dioleoylphosphatidylcholine mixtures. Biochim. Biophys. Acta, Biomembr. 2003, 1616, 196–208. 35. Huang, J.; Buboltz, J. T.; Feigenson, G. W. Maximum solubility of cholesterol in phosphatidylcholine and phosphatidylethanolamine bilayers. Biochim. Biophys. Acta 1999, 1417, 89–100. 36. Yguerabide, J. Theory for establishing proximity relations in biologicalmembranes by excitation-energy transfer measurements. Biophys. J. 1994, 66, 683–693. 37. Xu, L.; Anchordoquy, T. J. Cholesterol domains in cationic lipid/DNA complexes improve transfection. Biochim. Biophys. Acta 2008, 1778, 2177–81. 38. Epand, R. M.; Bach, D.; Borochov, N.; Wachtel, E. Cholesterol crystalline polymorphism and the solubility of cholesterol in phosphatidylserine. Biophys. J. 2000, 78, 866–873. 90 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


39. Loomis, C. R.; Shipley, G. G.; Small, D. M. Phase-behavior of hydrated cholesterol. J. Lipid Res. 1979, 20, 525–535. 40. Betker, J. L.; Gomez, J.; Anchordoquy, T. J. The effects of lipoplex formulation variables on the protein corona and comparisons with in vitro transfection efficiency. J. Controlled Release 2013, 171, 261–8. 41. Betker, J. L.; Kullberg, M.; Gomez, J.; Anchordoquy, T. J. Cholesterol domains enhance transfection. Ther. Delivery 2013, 4, 453–62. 42. Salvati, A.; Pitek, A. S.; Monopoli, M. P.; Prapainop, K.; Bombelli, F. B.; Hristov, D. R.; Kelly, P. M.; Aberg, C.; Mahon, E.; Dawson, K. A. Transferrin-functionalized nanoparticles lose their targeting capabilities when a biomolecule corona adsorbs on the surface. Nature Nanotechnology 2013, 8, 137–143. 43. Xu, L.; Anchordoquy, T. Drug delivery trends in clinical trials and translational medicine: challenges and opportunities in the delivery of nucleic acid-based therapeutics. J. Pharm. Sci. 2011, 100, 38–52. 44. Xu, L.; Anchordoquy, T. J. Effect of cholesterol nanodomains on the targeting of lipid-based gene delivery in cultured cells. Mol. Pharmaceutics 2010, 7, 1311–1317. 45. Xu, L.; Wempe, M. F.; Anchordoquy, T. J. The effect of cholesterol domains on PEGylated liposomal gene delivery in vitro. Ther. Delivery 2011, 2, 451–60. 46. Verhoef, J. J. F.; Carpenter, J. F.; Anchordoquy, T. J.; Schellekens, H. Potential induction of anti-PEG antibodies and complement activation toward PEGylated therapeutics. Drug Discovery Today 2014, 19, 1945–1952. 47. Xu, L.; Betker, J.; Yin, H.; Anchordoquy, T. J. Ligands located within a cholesterol domain enhance gene delivery to the target tissue. J. Controlled Release 2012, 160, 57–63. 48. Pozzi, D.; Marchini, C.; Cardarelli, F.; Amenitsch, H.; Garulli, C.; Bifone, A.; Caracciolo, G. Transfection efficiency boost of cholesterol-containing lipoplexes. Biochim. Biophys. Acta, Biomembr. 2012, 1818, 2335–2343. 49. Zhang, Y.; Bradshaw-Pierce, E. L.; Delille, A.; Gustafson, D. L.; Anchordoquy, T. J. In vivo comparative study of lipid/DNA complexes with different in vitro serum stability: effects on biodistribution and tumor accumulation. J. Pharm. Sci. 2008, 97, 237–50. 50. Alving, C. R.; Swartz, G. M. Antibodies to cholesterol, cholesterol conjugates, and liposomes - implications for atherosclerosis and autoimmunity. Crit. Rev. Immunol. 1991, 10, 441–453. 51. Swartz, G. M.; Gentry, M. K.; Amende, L. M.; Blanchettemackie, E. J.; Alving, C. R. Antibodies to Cholesterol. Proc. Natl. Acad. Sci. U. S. A. 1988, 85, 1902–1906. 52. Szebeni, J.; Baranyi, L.; Savay, S.; Bodo, M.; Morse, D. S.; Basta, M.; Stahl, G. L.; Bunger, R.; Alving, C. R. Liposome-induced pulmonary hypertension: properties and mechanism of a complement-mediated pseudoallergic reaction. Am. J. Physiol.: Heart Circ. Physiol. 2000, 279, H1319–H1328. 91 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


53. Bach, D.; Wachtel, E. Phospholipid/cholesterol model membranes: formation of cholesterol crystallites. Biochim. Biophys. Acta, Biomembr. 2003, 1610, 187–197. 54. Betker, J. L.; Anchordoquy, T. J. Nonadditive effects of repetitive administration of lipoplexes in immunocompetent mice. J. Pharm. Sci. 2017, 106, 872–881. 55. Betker, J. L.; Anchordoquy, T. J. Relating toxicity to transfection: using sphingosine to maintain prolonged expression in vitro. Mol. Pharmaceutics 2015, 12, 264–73. 56. Freedland, S. J.; Malone, R. W.; Borchers, H. M.; Zadourian, Z.; Malone, J. G.; Bennett, M. J.; Nantz, M. H.; Li, J. H.; Gumerlock, P. H.; Erickson, K. L. Toxicity of cationic lipid-ribozyme complexes in human prostate tumor cells can mimic ribozyme activity. Biochem. Mol. Med. 1996, 59, 144–53. 57. San, H.; Yang, Z. Y.; Pompili, V. J.; Jaffe, M. L.; Plautz, G. E.; Xu, L.; Felgner, J. H.; Wheeler, C. J.; Felgner, P. L.; Gao, X.; et al. Safety and shortterm toxicity of a novel cationic lipid formulation for human gene therapy. Hum. Gene Ther. 1993, 4, 781–8. 58. Scheule, R. K.; St George, J. A.; Bagley, R. G.; Marshall, J.; Kaplan, J. M.; Akita, G. Y.; Wang, K. X.; Lee, E. R.; Harris, D. J.; Jiang, C.; Yew, N. S.; Smith, A. E.; Cheng, S. H. Basis of pulmonary toxicity associated with cationic lipid-mediated gene transfer to the mammalian lung. Hum. Gene Ther. 1997, 8, 689–707. 59. Yew, N. S.; Scheule, R. K. Toxicity of Cationic Lipid-DNA Complexes. In Non-Viral Vectors for Gene Therapy, 2nd ed., Part 1; Advances in Genetics Series; 2005, Volume 53, pp 189−214. 60. Simberg, D.; Weisman, S.; Talmon, Y.; Barenholz, Y. DOTAP (and other cationic lipids): chemistry, biophysics, and transfection. Crit. Rev. Ther. Drug Carrier Syst. 2004, 21, 257–317. 61. Baraldo, K.; Leforestier, N.; Bureau, M.; Mignet, N.; Scherman, D. Sphingosine-based liposome as DNA vector for intramuscular gene delivery. Pharm. Res. 2002, 19, 1144–1149. 62. Koiv, A.; Kinnunen, P. K. Binding of DNA to liposomes containing different derivatives of sphingosine. Chem. Phys. Lipids 1994, 72, 77–86. 63. Koiv, A.; Mustonen, P.; Kinnunen, P. K. Differential scanning calorimetry study on the binding of nucleic acids to dimyristoylphosphatidylcholinesphingosine liposomes. Chem. Phys. Lipids 1994, 70, 1–10. 64. Paukku, T.; Lauraeus, S.; Huhtaniemi, I.; Kinnunen, P. K. Novel cationic liposomes for DNA-transfection with high efficiency and low toxicity. Chem. Phys. Lipids 1997, 87, 23–9. 65. Betker, J. L.; Anchordoquy, T. J. Effect of charge ratio on lipoplex-mediated gene delivery and liver toxicity. Ther. Delivery 2015, 6, 1243–1253. 66. Wilhelm, S.; Tavares, A. J.; Dai, Q.; Ohta, S.; Audet, J.; Dvorak, H. F.; Chan, W. C. W. Analysis of nanoparticle delivery to tumours. Nat. Rev. 2016, 1, 1–12 (May 2016). 67. Abu Lila, A. S.; Doi, Y.; Nakamura, K.; Ishida, T.; Kiwada, H. Sequential administration with oxaliplatin-containing PEG-coated cationic liposomes 92 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

68.

69.


70.

71.

72. 73.

74. 75.

76.

77.

78.

79.

promotes a significant delivery of subsequent dose into murine solid tumor. J. Controlled Release 2010, 142, 167–173. Abu Lila, A. S.; Eldin, N. E.; Ichihara, M.; Ishida, T.; Kiwada, H. Multiple administration of PEG-coated liposomal oxaliplatin enhances its therapeutic efficacy: A possible mechanism and the potential for clinical application. Int. J. Pharm. 2012, 438, 176–183. Alaaeldin, E.; Abu Lila, A. S.; Moriyoshi, N.; Sarhan, H. A.; Ishida, T.; Khaled, K. A.; Kiwada, H. The co-delivery of oxaliplatin abrogates the immunogenic response to PEGylated siRNA-lipoplex. Pharm. Res. 2013, 30, 2344–2354. Lindberg, M. F.; Le Gall, T.; Carmoy, N.; Berchel, M.; Hyde, S. C.; Gill, D. R.; Jaffres, P. A.; Lehn, P.; Montier, T. Efficient in vivo transfection and safety profile of a CpG-free and codon optimized luciferase plasmid using a cationic lipophosphoramidate in a multiple intravenous administration procedure. Biomaterials 2015, 59, 1–11. Semple, S. C.; Harasym, T. O.; Clow, K. A.; Ansell, S. M.; Klimuk, S. K.; Hope, M. J. Immunogenicity and rapid blood clearance of liposomes containing polyethylene glycol-lipid conjugates and nucleic acid. J Pharmacol. Exp. Ther. 2005, 312, 1020–1026. Matzinger, P. Tolerance, danger, and the extended family. Annu. Rev. Immunol. 1994, 12, 991–1045. Son, D. S.; Parl, A. K.; Rice, V. M.; Khabele, D. Keratinocyte chemoattractant (KC)/human growth-regulated oncogene (GRO) chemokines and pro-inflammatory chemokine networks in mouse and human ovarian epithelial cancer cells. Cancer Biol. Ther. 2007, 6, 1302–1312. Kullberg, M.; McCarthy, R.; Anchordoquy, T. J. Systemic tumor-specific gene delivery. J. Controlled Release 2013, 172, 730–6. Thurston, G.; McLean, J. W.; Rizen, M.; Baluk, P.; Haskell, A.; Murphy, T. J.; Hanahan, D.; McDonald, D. M. Cationic liposomes target angiogenic endothelial cells in tumors and chronic inflammation in mice. J. Clin. Invest. 1998, 101, 1401–1413. Sugahara, K. N.; Teesalu, T.; Karmali, P. P.; Kotamraju, V. R.; Agemy, L.; Girard, O. M.; Hanahan, D.; Mattrey, R. F.; Ruoslahti, E. Tissue-penetrating delivery of compounds and nanoparticles into tumors. Cancer Cell 2009, 16, 510–520. Bao, Y. J.; Jin, Y.; Chivukula, P.; Zhang, J.; Liu, Y.; Liu, J.; Clamme, J. P.; Mahato, R. I.; Ng, D.; Ying, W. B.; Wang, Y. T.; Yu, L. Effect of PEGylation on biodistribution and gene silencing of siRNA/lipid nanoparticle complexes. Pharm. Res. 2013, 30, 342–351. Hussain, S.; Rodriguez-Fernandez, M.; Braun, G. B.; Doyle, F. J., 3rd; Ruoslahti, E. Quantity and accessibility for specific targeting of receptors in tumours. Sci. Rep. 2014, 4, 5232. Armstrong, J. K.; Hempel, G.; Koling, S.; Chan, L. S.; Fisher, T.; Meiselman, H. J.; Garratty, G. Antibody against poly(ethylene glycol) adversely affects PEG-asparaginase therapy in acute lymphoblastic leukemia patients. Cancer 2007, 110, 103–111. 93

Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Utilizing Cholesterol Nanodomains for Nucleic Acid Delivery - ACS

Recommend Documents