Perfluorocarbon Nanoemulsions Research Articles

Perfluorocarbon nanoemulsions with fluorescent, colloidal and magnetic properties

Bimodal imaging agents that combine magnetic resonance imaging (MRI) and nearinfrared (NIR) imaging formulated as nanoemulsions became increasingly popular for imaging inflammation in vivo. Quality of in vivo imaging using nanoemulsions is directly dependent on their integrity and stability. Here we report the design of nanoemulsions for bimodal imaging, where both photostability and colloidal stability are equally addressed. A highly chemically and photo stable quaterrylenediimide dye was introduced into perfluoro-15-crown-5 ether (PCE) nanoemulsions. The nanoemulsions were prepared with PCE and Miglyol 812N mixed at 1:1 v/v ratio as internal phase stabilized by non-ionic surfactants. Data shows exceptional colloidal stability demonstrated as unchanged droplet size (∼130 nm) and polydispersity (<0.15) after 182 days follow up at both 4 and 25 °C. Nanoemulsions also sustained the exposure to mechanical and temperature stress, and prolonged exposure to light without changes in droplet size, 19F signal or fluorescence signal. No toxicity was observed in vitro in model inflammatory cells upon 24 h exposure while confocal microscopy showed that nanoemulsions droplets accumulated in the cytoplasm. Overall, our data demonstrates that design of bimodal imaging agents requires consideration of stability of each imaging component and that of the nanosystem as a whole to achieve excellent imaging performance.

(400) Theranostics nanoemulsions as new tools for studying inflammation in chronic pain preclinical models

Inflammatory cells (e.g. macrophages) infiltrate the affected tissue in response to neuronal injury at varied levels. As the neuronal healing process progresses, inflammatory cells undergo profound activity and phenotypic changes. New methods to assess inflammatory cell phenotype and quantify in vivo aspects of the neuroinflammatory processes in neuronal injury are needed. Theranostic nanomedicine represents a new nanotechnology strategy where therapeutic and imaging agents are combined into a single nanosystem. Our group recently designed and developed novel perfluorocarbon nanoemulsions for treatment and imaging of inflammation (Patel et al, PLOS One 2013; Janjic et al, SPIE 2013, Patel et al, JBO 2013). These nanodroplets are designed to specifically accumulate in inflammatory cells (e.g. macrophages) and deliver anti-inflammatory drugs. Therefore, these novel nanoemulsions allow formonitoring of inflammation response to the drug using combination of F magnetic resonance and near-infrared imaging. In this report, we present in detail theranostic nanoemulsions development adapted for neuropathic chronic pain preclinical studies. Design, development and formulation strategies are presented. Nanoemulsions presented are in size range of 85-150 nm. They are highly stable under biologically relevant conditions, safe and biocompatible. They can deliver varied anti-inflammatory agents (e.g. celecoxib, curcumin, resveratrol, and others). In short term, the presented work will lead to unique biological nanoprobes for studying the effects of inflammation in neuronal injury and the role of inflammatory cells in prolonged and intractable neuropathic pain development. In long term, the proposed nanomedicine can serve as the basis for a novel approaches to both prevent and treat chronic neuropathic pain, where molecular imaging can help to individualize therapeutic treatment. We present design of novel theranostic nanoemulsions and offer a starting point for not fully unexplored area of research and we hope this example prompts others to utilize theranostic nanosystems in chronic pain research.

Imaging neuroinflammation in vivo in a neuropathic pain rat model with near-infrared fluorescence and ¹⁹F magnetic resonance.

Chronic neuropathic pain following surgery represents a serious worldwide health problem leading to life-long treatment and the possibility of significant disability. In this study, neuropathic pain was modeled using the chronic constriction injury (CCI). The CCI rats exhibit mechanical hypersensitivity (typical neuropathic pain symptom) to mechanical stimulation of the affected paw 11 days post surgery, at a time when sham surgery animals do not exhibit hypersensitivity. Following a similar time course, TRPV1 gene expression appears to rise with the hypersensitivity to mechanical stimulation. Recent studies have shown that immune cells play a role in the development of neuropathic pain. To further explore the relationship between neuropathic pain and immune cells, we hypothesize that the infiltration of immune cells into the affected sciatic nerve can be monitored in vivo by molecular imaging. To test this hypothesis, an intravenous injection of a novel perfluorocarbon (PFC) nanoemulsion, which is phagocytosed by inflammatory cells (e.g. monocytes and macrophages), was used in a rat CCI model. The nanoemulsion carries two distinct imaging agents, a near-infrared (NIR) lipophilic fluorescence reporter (DiR) and a 19F MRI (magnetic resonance imaging) tracer, PFC. We demonstrate that in live rats, NIR fluorescence is concentrated in the area of the affected sciatic nerve. Furthermore, the 19F MRI signal was observed on the sciatic nerve. Histological examination of the CCI sciatic nerve reveals significant infiltration of CD68 positive macrophages. These results demonstrate that the infiltration of immune cells into the sciatic nerve can be visualized in live animals using these methods.

Intracellular pH measurements using perfluorocarbon nanoemulsions.

We report the synthesis and formulation of unique perfluorocarbon (PFC) nanoemulsions enabling intracellular pH measurements in living cells via fluorescent microscopy and flow cytometry. These nanoemulsions are formulated to readily enter cells upon coincubation and contain two cyanine-based fluorescent reporters covalently bound to the PFC molecules, specifically Cy3-PFC and CypHer5-PFC conjugates. The spectral and pH-sensing properties of the nanoemulsions were characterized in vitro and showed the unaltered spectral behavior of dyes after formulation. In rat 9L glioma cells loaded with nanoemulsion, the local pH of nanoemulsions was longitudinally quantified using optical microscopy and flow cytometry and displayed a steady decrease in pH to a level of 5.5 over 3 h, indicating rapid uptake of nanoemulsion to acidic compartments. Overall, these reagents enable real-time optical detection of intracellular pH in living cells in response to pharmacological manipulations. Moreover, recent approaches for in vivo cell tracking using magnetic resonance imaging (MRI) employ intracellular PFC nanoemulsion probes to track cells using (19)F MRI. However, the intracellular fate of these imaging probes is poorly understood. The pH-sensing nanoemulsions allow the study of the fate of the PFC tracer inside the labeled cell, which is important for understanding the PFC cell loading dynamics, nanoemulsion stability and cell viability over time.

Cell Labeling for 19F MRI: New and Improved Approach to Perfluorocarbon Nanoemulsion Design.

This report describes novel perfluorocarbon (PFC) nanoemulsions designed to improve ex vivo cell labeling for 19F magnetic resonance imaging (MRI). 19F MRI is a powerful non-invasive technique for monitoring cells of the immune system in vivo, where cells are labeled ex vivo with PFC nanoemulsions in cell culture. The quality of 19F MRI is directly affected by the quality of ex vivo PFC cell labeling. When co-cultured with cells for longer periods of time, nanoemulsions tend to settle due to high specific weight of PFC oils (1.5–2.0 g/mL). This in turn can decrease efficacy of excess nanoemulsion removal and reliability of the cell labeling in vitro. To solve this problem, novel PFC nanoemulsions are reported which demonstrate lack of sedimentation and high stability under cell labeling conditions. They are monodisperse, have small droplet size (~130 nm) and low polydispersity (<0.15), show a single peak in the 19F nuclear magnetic resonance spectrum at −71.4 ppm and possess high fluorine content. The droplet size and polydispersity remained unchanged after 160 days of follow up at three temperatures (4, 25 and 37 °C). Further, stressors such as elevated temperature in the presence of cells, and centrifugation, did not affect the nanoemulsion droplet size and polydispersity. Detailed synthetic methodology and in vitro testing for these new PFC nanoemulsions is presented.

Phase transitions of perfluorocarbon nanoemulsion induced with ultrasound: A mathematical model

While ultrasound has been used in many medical and industrial applications, only recently has research been done on phase transformations induced by ultrasound. This paper presents a numerical model and the predicted results of the phase transformation of a spherical nanosized droplet of perfluorocarbon in water. Such a model has applications in acoustic droplet vaporization, the generation of gas bubbles for medical imaging, therapeutic delivery and other biomedical applications.The formation of a gas phase and the subsequent bubble dynamics were studied as a function of acoustic parameters, such as frequency and amplitude, and of the physical aspects of the perfluorocarbon nanodroplets, such as chemical species, temperature, droplet size and interfacial energy. The model involves simultaneous applications of mass, energy and momentum balances to describe bubble formation and collapse, and was developed and solved numerically. It was found that, all other parameters being constant, the maximum bubble size and collapse velocity increases with increasing ultrasound amplitude, droplet size, vapor pressure and temperature. The bubble size and collapse velocity decreased with increasing surface tension and frequency. These results correlate with experimental observations of acoustic droplet vaporization.

Two-color fluorescent (near-infrared and visible) triphasic perfluorocarbon nanoemulsions

Design and development of a new formulation as a unique assembly of distinct fluorescent reporters with nonoverlapping fluorescence spectra and a F19 magnetic resonance imaging agent into colloidally and optically stable triphasic nanoemulsion are reported. Specifically, a cyanine dye-perfluorocarbon (PFC) conjugate was introduced into the PFC phase of the nanoemulsion and a near-infrared dye was introduced into the hydrocarbon (HC) layer. To the best of our knowledge, this is the first report of a triphasic nanoemulsion system where each oil phase, HC, and PFC are fluorescently labeled and formulated into an optically and colloidally stable nanosystem. Having, each oil phase separately labeled by a fluorescent dye allows for improved correlation between in vivo imaging and histological data. Further, dual fluorescent labeling can improve intracellular tracking of the nanodroplets and help assess the fate of the nanoemulsion in biologically relevant media. The nanoemulsions were produced by high shear processing (microfluidization) and stabilized with biocompatible nonionic surfactants resulting in mono-modal size distribution with average droplet size less than 200 nm. Nanoemulsions demonstrate excellent colloidal stability and only moderate changes in the fluorescence signal for both dyes. Confocal fluorescence microscopy of macrophages exposed to nanoemulsions shows the presence of both fluorescence agents in the cytoplasm.

Indocyanine green-loaded perfluorocarbon nanoemulsions for bimodal (19)F-magnetic resonance/nearinfrared fluorescence imaging and subsequent phototherapy.

We have developed an indocyanine green-loaded perfluorocarbon (ICG/PFCE) nanoemulsion as a multifunctional theranostic nanomedicine which enables not only (19)F magnetic resonance (MR)/near-infrared fluorescence (NIRF) bimodal imaging but also subsequent photodynamic/photothermal dual therapy of cancer. The hydrodynamic size of ICG/PFCE nanoemulsions was 164.2 nm. The stability of indocyanine green (ICG) in aqueous solution was significantly improved when loaded on perfluorocarbon nanoemulsions. In addition, ICG/PFCE nanoemulsions showed good dispersion stability in aqueous media containing 10% fetal bovine serum, for at least 14 days. (19)F-MRI of ICG/PFCE nanoemulsions showed that the signal intensity increased with increasing nanoemulsion concentration with no signal observed from the surrounding background. Using NIRF imaging with perfluorocarbon nanoemulsion alone, without ICG, did not produce NIRF, while clear and bright fluorescent images were obtained with ICG/PFCE nanoemulsions at 10-µM ICG equivalent. The capacity of ICG-loaded nanoemulsions to generate heat following light irradiation by using an 810-nm laser was comparable to that of free ICG, while singlet oxygen generation of ICG-loaded nanoemulsions was significantly better than that of free ICG. In vitro cytotoxicity tests and fluorescence microscopy confirmed biocompatibility of the nanoemulsion. Upon light irradiation, U87MG glioblastoma cells incubated with ICG/PFCE nanoemulsions underwent necrotic cell death. The therapeutic mechanism during light illumination appears to be mainly due to the photodynamic effect at lower ICG concentrations, whilst the photothermal effect became more obvious at increased ICG concentrations, enabling combined photodynamic/photothermal therapy of cancer cells.

Bimodal Perfluorocarbon Nanoemulsions for Nasopharyngeal Carcinoma Targeting

The aim of this study was to perform the detection of folate receptor (FR)-positive tumors with a bimodal imaging contrast agent, a perfluorocarbon (PFC)/rhodamine nanoemulsion, providing both 19F-based magnetic resonance imaging (MRI) and fluorescence imaging capabilities. The PFC/rhodamine nanoemulsion was further infused with phospholipid-anchored folate to improve the ability to target FR-expressing tumors. The preferential accumulation of the FR-targeted bimodal nanoemulsion in FR-positive tumor sites was monitored by both 19F-MRI and optical imaging. The FR-targeted PFC nanoemulsion had no significant effect on cell viability, and the size and fluorescence signal of PFC nanoemulsion were very stable. These nanoprobes were successfully delivered into FR-positive tumor xenograft models and showed significantly enhanced signal intensities of 19F-MRI and fluorescence imaging in the tumor area. The folate-PFC/rhodamine nanoemulsion has a great potential to serve as a useful optical and 19F-MRI agent for the diagnosis and targeting of FR-positive tumor.

Imaging of Intratumoral Inflammation during Oncolytic Virotherapy of Tumors by 19F-Magnetic Resonance Imaging (MRI)

BackgroundOncolytic virotherapy of tumors is an up-coming, promising therapeutic modality of cancer therapy. Unfortunately, non-invasive techniques to evaluate the inflammatory host response to treatment are rare. Here, we evaluate 19F magnetic resonance imaging (MRI) which enables the non-invasive visualization of inflammatory processes in pathological conditions by the use of perfluorocarbon nanoemulsions (PFC) for monitoring of oncolytic virotherapy.Methodology/Principal FindingsThe Vaccinia virus strain GLV-1h68 was used as an oncolytic agent for the treatment of different tumor models. Systemic application of PFC emulsions followed by 1H/19F MRI of mock-infected and GLV-1h68-infected tumor-bearing mice revealed a significant accumulation of the 19F signal in the tumor rim of virus-treated mice. Histological examination of tumors confirmed a similar spatial distribution of the 19F signal hot spots and CD68+-macrophages. Thereby, the CD68+-macrophages encapsulate the GFP-positive viral infection foci. In multiple tumor models, we specifically visualized early inflammatory cell recruitment in Vaccinia virus colonized tumors. Furthermore, we documented that the 19F signal correlated with the extent of viral spreading within tumors.Conclusions/SignificanceThese results suggest 19F MRI as a non-invasive methodology to document the tumor-associated host immune response as well as the extent of intratumoral viral replication. Thus, 19F MRI represents a new platform to non-invasively investigate the role of the host immune response for therapeutic outcome of oncolytic virotherapy and individual patient response.

Nucleating and sustaining acoustic cavitation for biomedical applications

Wes Nyborg was a pioneer in the field of biomedical ultrasound. In particular, Nyborg conducted extensive studies that provided insight into how bubbles oscillating in liquids generated forces that could alter the anatomy and/or physiology of cells. While these studies demonstrated that acoustic cavitation is instrumental in a variety of bioeffects, nucleating and sustaining acoustic cavitation in a controlled manner has proven to be a challenge, particularly in vivo. Studies have shown that these challenges can be addressed with the use of novel materials, particles, and/or acoustic pulsing schemes. In particular, pH-sensitive polymers with varying hydrophobicity have been used to sustain cavitation/cell interactions and vaporizable perfluorocarbon nanoemulsions have been used to reduce the pressure threshold for cavitation nucleation. The impact of these materials and particles on cavitation-mediated bioeffects will be discussed.

Phase‐shift, stimuli‐responsive perfluorocarbon nanodroplets for drug delivery to cancer

This review focuses on phase-shift perfluorocarbon nanoemulsions whose action depends on an ultrasound-triggered phase shift from a liquid to gas state. For drug-loaded perfluorocarbon nanoemulsions, microbubbles are formed under the action of tumor-directed ultrasound and drug is released locally into tumor volume in this process. This review covers in detail mechanisms involved in the droplet-to-bubble transition as well as mechanisms of ultrasound-mediated drug delivery.

Self-assembling perfluorocarbon nanoemulsions for drug delivery and imaging

The purpose of this project is to create a stable, perfluorocarbonbased drug delivery agent that can be imaged by magnetic resonance in vivo. Perfluorocarbon (PFC)-based nanoemulsions have a long research history as blood substitutes, MRI tracers, and ultrasound agents. PFCs can also provide a biologically inert platform for controlled drug delivery and release and act as a highly specific 19F MRI tracer for in vivo monitoring. PFCs alone cannot serve as drug delivery vehicles due to their high lipophobicity and hydrophobicity. To improve their drug carrying potential, PFCs are formulated with surfactants or hydrocarbons into nanoemulsions. In this study, a previously synthesized compound, perfluoropolyether-tyramide (PFPE-tyramide, 4-(2-aminoethyl) phenol conjugated to perfluoropolyether)1 was formulated into nanoemulsions in the presence of various surfactants by selfassembly or high shear techniques such as sonication and microfluidization. Olive oil was introduced into selected formulations as a potential carrier of lipophilic drugs. Preliminary toxicity and stability data are also shown.

Visualization of Abscess Formation in a Murine Thigh Infection Model of Staphylococcus aureus by 19F-Magnetic Resonance Imaging (MRI)

BackgroundDuring the last years, 19F-MRI and perfluorocarbon nanoemulsion (PFC) emerged as a powerful contrast agent based MRI methodology to track cells and to visualize inflammation. We applied this new modality to visualize deep tissue abscesses during acute and chronic phase of inflammation caused by Staphylococcus aureus infection.Methodology and Principal FindingsIn this study, a murine thigh infection model was used to induce abscess formation and PFC or CLIO (cross linked ironoxides) was administered during acute or chronic phase of inflammation. 24 h after inoculation, the contrast agent accumulation was imaged at the site of infection by MRI. Measurements revealed a strong accumulation of PFC at the abscess rim at acute and chronic phase of infection. The pattern was similar to CLIO accumulation at chronic phase and formed a hollow sphere around the edema area. Histology revealed strong influx of neutrophils at the site of infection and to a smaller extend macrophages during acute phase and strong influx of macrophages at chronic phase of inflammation.Conclusion and SignificanceWe introduce 19F-MRI in combination with PFC nanoemulsions as a new platform to visualize abscess formation in a murine thigh infection model of S. aureus. The possibility to track immune cells in vivo by this modality offers new opportunities to investigate host immune response, the efficacy of antibacterial therapies and the influence of virulence factors for pathogenesis.

Ultrasound-mediated tumor imaging and nanotherapy using drug loaded, block copolymer stabilized perfluorocarbon nanoemulsions

Perfluorocarbon nanoemulsions can deliver lipophilic therapeutic agents to solid tumors and simultaneously provide for monitoring nanocarrier biodistribution via ultrasonography and/or 19F MRI. In the first generation of block copolymer stabilized perfluorocarbon nanoemulsions, perfluoropentane (PFP) was used as the droplet forming compound. Although manifesting excellent therapeutic and ultrasound imaging properties, PFP nanoemulsions were unstable at storage, difficult to handle, and underwent hard to control phenomenon of irreversible droplet-to-bubble transition upon injection. To solve the above problems, perfluoro-15-crown-5-ether (PFCE) was used as a core forming compound in the second generation of block copolymer stabilized perfluorocarbon nanoemulsions. PFCE nanodroplets manifest both ultrasound and fluorine (19F) MR contrast properties, which allows using multimodal imaging and 19F MR spectroscopy for monitoring nanodroplet pharmacokinetics and biodistribution. In the present paper, acoustic, imaging, and therapeutic properties of unloaded and paclitaxel (PTX) loaded PFCE nanoemulsions are reported. As manifested by the 19F MR spectroscopy, PFCE nanodroplets are long circulating, with about 50% of the injected dose remaining in circulation 2h after the systemic injection. Sonication with 1-MHz therapeutic ultrasound triggered reversible droplet-to-bubble transition in PFCE nanoemulsions. Microbubbles formed by acoustic vaporization of nanodroplets underwent stable cavitation. The nanodroplet size (200nm to 350nm depending on a type of the shell and conditions of emulsification) as well as long residence in circulation favored their passive accumulation in tumor tissue that was confirmed by ultrasonography. In the breast and pancreatic cancer animal models, ultrasound-mediated therapy with paclitaxel-loaded PFCE nanoemulsions showed excellent therapeutic properties characterized by tumor regression and suppression of metastasis. Anticipated mechanisms of the observed effects are discussed.

New pharmacological preparations based on perfluorocarbon nanoemulsions

The technology and some physicochemical and biomedical characteristics of perfluorocarbon (PFC) nanoemulsions are considered. These nanosystems can act as universal nanocarriers under certain technological conditions for preparing biologically active emulsions based on PFC. These emulsions contain nanoparticles that can absorb, penetrate, and affect any systems of an organism and parts of organs and carry on their surface various substances such as gases, organic compounds, solid particles, markers, pharmaceuticals, and active substances. PFC emulsions can perform as blood-plasma-substituting solutions with gas-transport function.

Perfluorocarbon nanoemulsions as contrast agents for imaging and ultrasound‐mediated targeted chemotherapy.

During the last decade, achievements of nanomedicine have made possible image‐guided drug targeting to disease sites. In oncology, tumor targeting of chemotherapeutic agents can be realized by drug encapsulation in nanocarriers such as liposomes, polymeric micelles, nanoemulsions, etc. A degree of tumor targeting may be increased by developing stimuli responsive nanocarriers that release their drug load in response to external stimuli such as heat, light, or ultrasound. Using ultrasound allows combining imaging and therapy. However, many mechanistic aspects of the ultrasound‐mediated drug delivery remain elusive. Which mode of ultrasound, mechanical or thermal, is optimal for drug delivery? Which is the optimal timing of ultrasound application in relation to drug injection? What would be the best target for molecularly targeted carriers? These problems will be discussed for paclitaxel‐loaded perfluorocarbon nanoemulsions that manifest a unique combination of properties (acoustic droplet vaporization, cavi...

Theragnostics for tumor and plaque angiogenesis with perfluorocarbon nanoemulsions

Molecular imaging agents are extending the potential of noninvasive medical diagnosis from basic gross anatomical descriptions to complicated phenotypic characterizations based upon the recognition of unique cell-surface biochemical signatures. Although originally the purview of nuclear medicine, "molecular imaging" is now studied in conjunction with all clinically relevant imaging modalities. Of the myriad of particles that have emerged as prospective candidates for clinical translation, perfluorocarbon nanoparticles offer great potential for combining targeted imaging with drug delivery, much like the "magic bullet" envisioned by Paul Ehrlich 100 years ago. Perfluorocarbon nanoparticles, once studied in Phase III clinical trials as blood substitutes, have found new life for molecular imaging and drug delivery. The particles have been adapted for use with all clinically relevant modalities and for targeted drug delivery. In particular, their intravascular constraint due to particle size provides a distinct advantage for angiogenesis imaging and antiangiogenesis therapy. As perfluorocarbon nanoparticles have recently entered Phase I clinical study, this review provides a timely focus on the development of this platform technology and its application for angiogenesis-related pathologies.

Mechanisms of nucleotide trafficking during siRNA delivery to endothelial cells using perfluorocarbon nanoemulsions

RNA interference (RNAi) is a useful in vitro research tool, but its application as a safe and effective therapeutic agent may benefit from improved understanding of mechanisms of exogenous siRNA delivery, including cell trafficking and sorting patterns. We report the development of a transfection reagent for siRNA delivery which employs a distinctive non-digestive mode of particle-cell membrane interaction through the formation of a hemifusion complex resulting in lipid raft transport of cargo to the cytosol, bypassing the usual endosomal nanoparticle uptake pathway. We further demonstrate markedly enhanced efficacy over conventional transfection agents for suppressing endothelial cell expression of upregulated vascular adhesion molecules.

Fluorine-containing nanoemulsions for MRI cell tracking.

In this article we review the chemistry and nanoemulsion formulation of perfluorocarbons used for in vivo(19)F MRI cell tracking. In this application, cells of interest are labeled in culture using a perfluorocarbon nanoemulsion. Labeled cells are introduced into a subject and tracked using (19)F MRI or NMR spectroscopy. In the same imaging session, a high-resolution, conventional ((1)H) image can be used to place the (19)F-labeled cells into anatomical context. Perfluorocarbon-based (19)F cell tracking is a useful technology because of the high specificity for labeled cells, ability to quantify cell accumulations, and biocompatibility. This technology can be widely applied to studies of inflammation, cellular regenerative medicine, and immunotherapy.