A stable, highly concentrated fluorous nanoemulsion formulation for in vivo cancer imaging via 19F-MRI.

Magnetic resonance imaging (MRI) is a routine diagnostic modality in oncology that produces excellent imaging resolution and tumor contrast without the use of ionizing radiation. However, improved contrast agents are still needed to further increase detection sensitivity and avoid toxicity/allergic reactions associated with paramagnetic metal contrast agents, which may be seen in a small percentage of the human population. Fluorine-19 (19F)-MRI is at the forefront of the developing MRI methodologies due to near-zero background signal, high natural abundance of 100%, and unambiguous signal specificity. In this study, we have developed a colloidal nanoemulsion (NE) formulation that can encapsulate high volumes of the fluorous MRI tracer, perfluoro-[15-crown-5]-ether (PFCE) (35% v/v). These nanoparticles exhibit long-term (at least 100 days) stability and high PFCE loading capacity in formulation with our semifluorinated triblock copolymer, M2F8H18. With sizes of approximately 200 nm, these NEs enable in vivo delivery and passive targeting to tumors. Our diagnostic formulation, M2F8H18/PFCE NE, yielded in vivo 19F-MR images with a high signal-to-noise ratio up to 100 in a tumor-bearing mouse model at clinically relevant scan times. M2F8H18/PFCE NE circulated stably in the vasculature, accumulated in high concentration of an estimated 4-9 × 1017 19F spins/voxel at the tumor site, and cleared from most organs over the span of 2 weeks. Uptake by the mononuclear phagocyte system to the liver and spleen was also observed, most likely due to particle size. These promising results suggest that M2F8H18/PFCE NE is a favorable 19F-MR diagnostic tracer for further development in oncological studies and potential clinical translation.

MCT Nanoemulsions for the Efficient Delivery of siRNA.

In this study, an oil-in-water (o/w) nanoemulsion is used to deliver siRNA targeting Twist1, a protein that contributes to tumor metastasis in a variety of cancers. The FDA-approved oil, medium chain triglycerides (MCT), is used as the hydrophobic phase for the nanoemulsion. The siRNA is paired with dioleoyl-3-trimethylammonium-propane (DOTAP) to form a hydrophobic salt that is soluble at high concentrations in MCT. The resulting MCT/siRNA-DOTAP solution is formulated into a nanoemulsion with an average particle size of 140 nm. The nanoemulsion displays long term stability over the course of 195 days. In an in vivo murine tumor model, the nanoemulsion facilitates a 46% decrease in Twist1 mRNA after 48 h.

Gold nanoparticles in virus detection: Recent advances and potential considerations for SARS-CoV-2 testing development.

Viruses are infectious agents that pose significant threats to plants, animals, and humans. The current coronavirus disease 2019 pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread globally and resulted in over 2 million deaths and immeasurable financial losses. Rapid and sensitive virus diagnostics become crucially important in controlling the spread of a pandemic before effective treatment and vaccines are available. Gold nanoparticle (AuNP)-based testing holds great potential for this urgent unmet biomedical need. In this review, we describe the most recent advances in AuNP-based viral detection applications. In addition, we discuss considerations for the design of AuNP-based SARS-CoV-2 testings. Finally, we highlight and propose important parameters to consider for the future development of effective AuNP-based testings that would be critical for not only this COVID-19 pandemic, but also potential future outbreaks. This article is categorized under: Diagnostic Tools > Biosensing Diagnostic Tools > In Vitro Nanoparticle-Based Sensing.

Preparation, Characterization, and Formulation Optimization of Ionic-Liquid-in-Water Nanoemulsions toward Systemic Delivery of Amphotericin B.

Amphotericin B (AmB) is an antifungal agent that poses a challenge for intravenous drug delivery due to its hydrophobicity and severe side effects that are attributed to the self-aggregation of AmB in aqueous solution. To overcome this problem, we have rationally designed an ionic-liquid-in-water nanoemulsion drug delivery system that harnesses the unique properties of ionic liquids. The complex drug AmB serves as a model pharmaceutical agent to demonstrate the robustness of ionic-liquid-in-water nanoemulsions. High concentrations of AmB were solubilized in a new hydrophobic dicholinium-based ionic liquid. The absorption spectrum of AmB in an ionic liquid mixture and prepared nanoemulsion indicates AmB solubilization in the monomeric form. The hydrophobic ionic liquid exhibits high in vivo biocompatibility with zebrafish. The hemolytic activity of the AmB nanoemulsion was negligible, yet it maintained antifungal activity against Candida albicans. The preliminary results presented in this Communication indicate that ionic-liquid-in-water nanoemulsions may allow for the delivery of a variety of pharmaceuticals intravenously, broadening the scope of ionic liquids in the pharmaceutical sciences.

Droplet Core Intermolecular Interactions and Block Copolymer Composition Heavily Influence Oil-In-Water Nanoemulsion Stability.

Colloidal oil-in-water nanoemulsions are gaining increasing interest as a nanoparticle delivery system because of their large oil droplet core that can carry a large payload. In order to formulate these particles with long-term stability, an appropriate oil media and block copolymer pair must be selected. The interaction between the nanoemulsion core and the polymer shell is critical to forming stable nanoparticles. Herein, we probed how interactions between various polymers with hydrocarbon and perfluorocarbon oil media influenced nanoemulsion formation, stability, and size. Through a series of nanoemulsions with unique polymer/oil media combinations, we examined the effects of oil core hydrophobicity, fluorophilicity, surface charge, and volume as well as the effects of polymer tail composition. Surprisingly, we found that nanoemulsions formulated with pure perfluorocarbon oil cores versus perfluoro poly(ether) oil cores exhibited very different characteristics. We also found that both hydrocarbon and fluorocarbon polymer tails interacted favorably with perfluoro poly(ethers) as well as hydrocarbon oil cores forming stable nanoemulsions. We believe these results are focused on the unique properties of perfluorocarbons especially their rigidity, low polarizability, and near-zero surface charge. Interestingly, we saw that perfluoro poly(ethers) deviated from these expected properties resulting in an increased versatility when formulating nanoemulsions with perfluoro poly(ether) oil cores compared to pure perfluorocarbon oil cores. Nanoemulsion size, stability, growth rate, and life time were explored to probe these factors. Experimental and computational data are presented as a rationale.

Multicompartment Theranostic Nanoemulsions Stabilized by a Triphilic Semifluorinated Block Copolymer.

The presence of a perfluorocarbon block in a multiblock polymer has been shown to be an additional driving force toward nanoparticle assembly. In the preparation of nanoemulsions, this perfluorocarbon block also provides enhanced particle stability. Herein, the synthesis of a new triphilic, semifluorinated copolymer, M2F8H18, is introduced. This ABC type block copolymer can be used to formulate extremely stable nanoemulsions, assembled around a lipophilic droplet, with lifetimes of one year or more. The central oil droplet can stably solubilize high concentrations of hydrophobic drugs, making this system an ideal drug delivery vehicle. The incorporation of the perfluorocarbon block modulates drug release from the lipophilic core via the surrounding fluorous shell. Fluorous imaging agents incorporated into the fluorous shell prolong drug release even further as well as provide potent 19F-MRI contrast ability. In vitro studies show that these nanoemulsions efficiently inhibit cancer cell growth, thus providing a theranostic drug delivery system.

Lipid-free Fluoropolymer-based Propofol Emulsions and Lipid Reversal of Propofol Anesthesia in Rats.

BACKGROUND: Propofol, as a lipid-based emulsion, is effective at inducing anesthesia. It does, however, suffer from several drawbacks, including microbial growth, hyperlipidemia, and pain on injection. In this study, the authors examined the ability of four lipid-free propofol nanoemulsions to induce anesthesia in rats and tested whether a subsequent lipid bolus would accelerate emergence from anesthesia. METHODS: The authors administered five formulations of propofol intravenously to six rats, delivering five different doses five times each, in a repeated-measures randomized crossover design and measured time to loss and recovery of righting reflex. The formulations included (1) Diprivan (AstraZeneca, United Kingdom); (2) L3, incorporating a semifluorinated surfactant plus egg lecithin; (3) B8, incorporating a semifluorinated surfactant only; (4) F8, incorporating a semifluorinated surfactant plus perfluorooctyl bromide; and (5) L80, incorporating egg lecithin only. In a second phase of the study, the authors administered a lipid bolus immediately after a dose of B8 or Diprivan. RESULTS: All formulations except L80 impaired the righting reflex without apparent toxic effects. The authors estimated the threshold dose for induction by determining the x-intercept of the linear regression between time to recovery versus log dose. Threshold doses ranged from 5.8 (95% CI, 5.5 to 6.2) to 8.6 (95% CI, 7.2 to 10.2) mg/kg. A 15 ml/kg lipid bolus resulted in an accelerated clearance. CONCLUSIONS: Three of the four novel lipid-free fluoropolymer-based formulations showed efficacy in producing anesthesia, which was comparable to that of Diprivan, and a lipid bolus hastened recovery. These novel propofol formulations have the potential to avoid complications seen with the existing lipid-based formulation.

Synthesis, emulsification and self-assembly properties of sugar-containing semifluorinated amphiphiles.

Surfactants with two and three monosaccharide-based heads and a perfluoroalkyl tail have been synthesized. Perfluoroalkyl C3-symmetric triol and C2-symmetric diol were conveniently prepared via Cu-catalyzed azide-alkyne cycloaddition between a fluorous alkyne and tertiary and secondary azides, respectively. Glycosylation of the perfluoroalkyl diol and triol led to orthoester-type structures, which were evaluated for their capacity to stabilize aqueous emulsions of highly fluorinated anesthetics. The self-assembly properties of the tri-sugar amphiphile were examined by transmission electron microscopy.

Synthesis and characterization of perfluoro-tert-butyl semifluorinated amphiphilic polymers and their potential application in hydrophobic drug delivery.

Semifluorinated polymer surfactants, composed of a monomethyl poly(ethylene glycol) (mPEG) hydrophilic head group and either 1, 2, or 3 perfluoro-tert-butyl (PFtB) groups as the fluorophilic tail, were synthesized, and their aqueous self-assemblies were investigated as a potential design for theranostic nanoparticles. Polymers with three PFtB groups (PFtBTRI) solely formed stable, spherical micelles, approximately 12 nm in size. These PFtBTRI surfactants demonstrate similar characteristics with those of polymers with linear perfluorocarbon tails, despite large differences in tail structure. For example, PFtB polymer solutions stably emulsified 20 v/v% sevoflurane with perfluorooctyl bromide (PFOB) as a stabilizer. However, these PFtB polymers have the additional potential to serve as F-MRI contrast agents. PFtBTRI micelles gave one narrow 19F-NMR signal in D2O, with T1 and T2 parameters of approximately 500 and 100 ms, respectively. 19F-MR images of PFtB polymer solutions at 1 mM gave intense signal at 4.7 T without sensitizers or selective excitation sequences. These preliminary data demonstrate the potential of PFtB polymers as a basic design, which can be further modified to serve as dual drug-delivery and imaging vehicles.

Base-Induced Instability of Fluorotelomer Alcohols.

The stability of fluorotelomer alcohols under basic conditions was studied. HF elimination across the CF2-CH2 junction is shown to be facilitated by an intramolecular hydrogen bond, while solvation is the key determinant in the stability of alcohols of various perfluoroalkyl lengths. Finally, fluorotelomer alcohols can be rendered kinetically stable if either the alcohol or the base has low solubility in the reaction medium.

Exceptionally stable fluorous emulsions for the intravenous delivery of volatile general anesthetics.

BACKGROUND: IV delivery of volatile fluorinated anesthetics has a number of potential advantages when compared with the current inhalation method of administration. We reported previously that the IV delivery of sevoflurane can be achieved through an emulsion composed of a linear fluorinated diblock copolymer, a stabilizer, and the anesthetic. However, this original emulsion was subject to particle size growth that would limit its potential clinical utility. We hypothesized that the use of bulkier fluorous groups and smaller polyethylene glycol moieties in the polymer design would result in improved emulsion stability while maintaining anesthetic functionality. METHODS: The authors prepared emulsions incorporating sevoflurane, perfluorooctyl bromide as a stabilizing agent, and combinations of linear fluorinated diblock copolymer and a novel dibranched fluorinated diblock copolymer. Emulsion stability was assessed using dynamic light scattering. The ability of the emulsions to induce anesthesia was tested in vivo by administering them intravenously to 15 male Sprague-Dawley rats and measuring loss of the forepaw righting reflex. RESULTS: 20% (volume/volume) sevoflurane emulsions incorporating mixtures of dibranched and linear diblock copolymers had improved stability, with those containing an excess of the dibranched polymers displaying stability of particle size for more than 1 yr. The ED50s for loss of forepaw-righting reflex were all similar, and ranged between 0.55- 0.60 ml/kg body weight. CONCLUSIONS: Hemifluorinated dibranched polymers can be used to generate exceptionally stable sevoflurane nanoemulsions, as required of formulations intended for clinical use. IV delivery of the emulsion in rats resulted in induction of anesthesia with rapid onset and smooth and rapid recovery.

Encapsulation and release of Amphotericin B from an ABC triblock fluorous copolymer.

PURPOSE: PEG-phospholipid-based micelles have been successfully used for the solubilization of several hydrophobic drugs but generally lack sustained stability in blood. Our novel PEG-Fluorocarbon-DSPE polymers were designed to increase stability and improve time-release properties of drug-loaded micelles. METHODS: Novel ABC fluorous copolymers were synthesized, characterized, and used for encapsulation release of amphotericin B. FRET studies were used to study micelle stability. RESULTS: The micelles formed by the new polymers showed lower critical micelle concentrations and higher viscosity cores than those formed by the polymers lacking the fluorous block. FRET studies indicated that fluorocarbon-containing micelles had increased stability in presence of human serum. Physicochemical properties and in vitro release profile of micelles loaded with Amphotericin B (AmB) were studied. CONCLUSIONS: The effect of PEG length and fluorocarbon incorporation were investigated. The shorter hydrophilic PEG2K induced greater stability than PEG5K by decreasing the proportion of hydrophilic block of the polymer. The fluorocarbon placed between hydrophilic and hydrophobic block formed a fluorous shell contributing to the enhanced thermodynamic stability of micelles and to the drug sustained release. Polymer mPEG2K-F(10)-DSPE, bearing both a fluorocarbon block and a shorter mPEG, showed the greatest stability and the longest half-life for AmB release.

Synthesis, characterization, and applications of hemifluorinated dibranched amphiphiles.

Here we describe the synthesis and the physicochemical and preliminary pharmaceutical assessment of a novel class of hemifluorinated dibranched derivatives: M(1)diH(x)F(y). These compounds have the remarkable ability to completely stop the Ostwald ripening commonly associated with nanoemulsions. The developed synthesis is modular and allows easy incremental structural variations in the fluorophilic (fluorous chains), lipophilic (alkyl spacer head), and hydrophilic (polar head) domains. Furthermore, the synthesis can be easily scaled up and highly pure compounds can be readily obtained through silica gel and fluoro-silica gel column chromatography, without any need to use HPLC or other time-consuming techniques. Surface properties such as micelle formation, critical aggregation concentration (CAC), and emulsion stability studies demonstrated the different behavior of the dibranched hemifluorinated surfactant M(1)diH(x)F(y) with respect to that of single-chain semifluorinated analogues M(z)F(y). Remarkably, the new polymer M(1)diH(3)F(8) drastically slowed the ripening of nanoemulsions of the commonly used fluorinated anesthetic sevoflurane over a period of more than 1 year. During this time, the nanodroplet size did not increase to more than 400 nm. This result is very promising for inducing and maintaining general anesthesia through intravenous delivery of volatile anesthetics, eliminating the need for the use of large and costly vaporizers in the operating room.

Histamine release associated with intravenous delivery of a fluorocarbon-based sevoflurane emulsion in canines.

The purpose of this study was to evaluate the effectiveness of a novel fluorocarbon-based sevoflurane emulsion in dogs previously shown to produce short-term rodent anesthesia. On the basis of an unexpected allergic-type clinical reaction, we also tested the hypothesis that this type of formulation causes histamine release and complement activation. Physiological parameters, plasma histamine levels (radioimmunoassay), and complement activation (enzyme immunoassay) were quantified in response to emulsion components, including F13M5 (the emulsion's fluorocarbon-based polymer) and methoxy poly(ethylene glycol) 5000 (the polymer's hydrophilic block). Although the emulsion produced general anesthesia in dogs, they also experienced hypotension and clinical signs suggestive of an allergic-like response (i.e., vasodilation, urticaria, and pruritus upon recovery). Emulsions lacking sevoflurane failed to induce anesthesia but did elicit the allergic response. Plasma histamine levels were significantly increased following injection of micellar solutions of F13M5. Direct complement activation by the emulsion or its components was weak or absent. An allergic response leading to histamine release, likely initiated by the F13M5 component via an immunoglobulin pathway, is associated with an intravenous fluorocarbon-based emulsion of sevoflurane. Subsequently, its usefulness in medicine in its present formulation is limited.

Fluoropolymer-based emulsions for the intravenous delivery of sevoflurane.

BACKGROUND: The intravenous delivery of halogenated volatile anesthetics has been previously achieved using phospholipid-stabilized emulsions, e.g., Intralipid. However, fluorinated volatile anesthetics, such as sevoflurane, are partially fluorophilic and do not mix well with classic nonfluorinated lipids. This effect limits the maximum amount of sevoflurane that can be stably emulsified in Intralipid to 3.5% vol/vol. This is a significant limitation to the potential clinical use of Intralipid-based emulsions. METHODS: The authors prepared a 20% vol/vol sevoflurane emulsion using a novel fluorinated surfactant and tested its effectiveness and therapeutic index by administering it to male Sprague-Dawley rats via intravenous injection into the jugular vein. The median effective dose to induce anesthesia (ED50), the median lethal dose (LD50), and the therapeutic index (LD50/ED50) were determined. Anesthesia was measured by loss of the forepaw righting reflex. RESULTS: The ED50 and LD50 values were found to be 0.41 and 1.05 ml emulsion/kg body weight, respectively. These lead to a therapeutic index of 2.6, which compares favorably with previously determined values of emulsified isoflurane, as well as values for propofol and thiopental. CONCLUSIONS: A novel semifluorinated surfactant was able to considerably increase the maximum amount of stably emulsified sevoflurane compared with Intralipid. These formulations can be used to rapidly induce anesthesia with bolus dosing from which recovery is smooth and rapid.

Minimum sequence requirements for the binding of paromomycin to the rRNA decoding site A.

We have recently introduced a computational methodology that combines molecular dynamics (MD) simulations, free-energy calculations, and in vitro binding assays to predict the minimum RNA structural requirements for selective, high-affinity RNA binding to small-molecule ligands. Here, we show that this methodology can be applied to the conformationally flexible aminoglycoside antibiotic paromomycin. A RNA consisting of an 11-mer:10-mer duplex that contains one 16S ribosome RNA decoding A-site bound to paromomycin was simulated for 4 ns. The methodology predicts that the 11-mer:10-mer duplex binds to paromomycin with high affinity, whereas smaller RNA duplexes lose complex stability and the ability to bind paromomycin. The predicted high-affinity binding to paromomycin of the 11-mer:10-mer duplex was confirmed experimentally (EC(50) = 0.28 microM), as well as the inability of smaller complexes to bind. Our simulations show good agreement with experiment for dynamic and structural properties of the isolated A-site, including hydrogen-bonding networks and RNA structural rearrangements upon ligand binding. The results suggest that MD simulations can supplement in vitro methods as a tool for predicting minimum RNA-binding motifs for both small, rigid ligands, and large, flexible ligands when structural information is available.

Stabilizing and destabilizing effects of phenylalanine --> F5-phenylalanine mutations on the folding of a small protein.

We report a systematic evaluation of phenylalanine-to-pentafluorophenylalanine (Phe --> F5-Phe) mutants for the 35-residue chicken villin headpiece subdomain (c-VHP), the hydrophobic core of which features a cluster of three Phe side chains (residues 6, 10, and 17). Phe --> F5-Phe mutations are interesting because aryl-perfluoroaryl interactions of optimal geometry are intrinsically more favorable than aryl-aryl interactions and because perfluoroaryl units are more hydrophobic than are analogous aryl units. One mutant, Phe-10 --> F5-Phe, provides enhanced tertiary structural stability relative to the native sequence. The other six mutants analyzed caused a decrease in stability.

Binding properties of aromatic carbon-bound fluorine.

A systematic computational analysis of the ability of aromatic carbon-bound fluorine to participate in hydrogen bonding and electrostatic interactions has been completed. The interaction energies between the most common fluoroaromatics used in medicinal chemistry and both water, the prototypical hydrogen bond donor, and several cations have been calculated at different levels of theory (HF, MP2, DFT). Our results show that aromatic fluorine can participate in significant hydrogen bonds and can also interact with charged molecules.

Minimum sequence requirements for selective RNA-ligand binding: a molecular mechanics algorithm using molecular dynamics and free-energy techniques.

In vitro evolution techniques allow RNA molecules with unique functions to be developed. However, these techniques do not necessarily identify the simplest RNA structures for performing their functions. Determining the simplest RNA that binds to a particular ligand is currently limited to experimental protocols. Here, we introduce a molecular-mechanics based algorithm employing molecular dynamics simulations and free-energy methods to predict the minimum sequence requirements for selective ligand binding to RNA. The algorithm involves iteratively deleting nucleotides from an experimentally determined structure of an RNA-ligand complex, performing energy minimizations and molecular dynamics on each truncated structure, and assessing which truncations do not prohibit RNA binding to the ligand. The algorithm allows prediction of the effects of sequence modifications on RNA structural stability and ligand-binding energy. We have implemented the algorithm in the AMBER suite of programs, but it could be implemented in any molecular mechanics force field parameterized for nucleic acids. Test cases are presented to show the utility and accuracy of the methodology.

Identification of a 14mer RNA that recognizes and binds flavin mononucleotide with high affinity.

Aptamers are nucleic acids developed by in vitro evolution techniques that bind to specific ligands with high affinity and selectivity. Despite such high affinity and selectivity, however, in vitro evolution does not necessarily reveal the minimum structure of the nucleic acid required for selective ligand binding. Here, we show that a 35mer RNA aptamer for the cofactor flavin mononucleotide (FMN) identified by in vitro evolution can be computationally evolved to a mere 14mer structure containing the original binding pocket and eight scaffolding nucleotides while maintaining its ability to bind in vitro selectively to FMN. Using experimental and computational methodologies, we found that the 14mer binds with higher affinity to FMN (K(D) approximately 4 microM) than to flavin adenine dinucleotide (K(D) approximately 12 microM) or to riboflavin (K(D) approximately 13 microM),despite the negative charge of FMN. Different hydrogen-bond strengths resulting from differing ring-system electron densities associated with the aliphatic-chain charges appear to contribute to the selectivity observed for the binding of the 14mer to FMN and riboflavin. Our results suggest that high affinity and selectivity in ligand binding is not restricted to large RNAs, but can also be a property of extraordinarily short RNAs.