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Monitoring the Stability of Perfluorocarbon Nanoemulsions by Cryo-TEM Image Analysis and Dynamic Light Scattering.

Grapentin C, Barnert S, Schubert R - PLoS ONE (2015)

Bottom Line: The results indicate that DLS alone cannot reveal the changes in particle size, but can even mislead to a positive estimation of stability.The combination with Cryo-TEM images gives more insight in the particulate evolution, both techniques supporting one another.The study is one further step in the development of analytical tools for the evaluation of a clinically applicable perfluorooctylbromide nanoemulsion.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Technology and Biopharmacy, Albert Ludwig University Freiburg i. Br., Freiburg im Breisgau, Germany.

ABSTRACT
Perfluorocarbon nanoemulsions (PFC-NE) are disperse systems consisting of nanoscale liquid perfluorocarbon droplets stabilized by an emulsifier, usually phospholipids. Perfluorocarbons are chemically inert and non-toxic substances that are exhaled after in vivo administration. The manufacture of PFC-NE can be done in large scales by means of high pressure homogenization or microfluidization. Originally investigated as oxygen carriers for cases of severe blood loss, their application nowadays is more focused on using them as marker agents in 19F Magnetic Resonance Imaging (19F MRI). 19F is scarce in organisms and thus PFC-NE are a promising tool for highly specific and non-invasive imaging of inflammation via 19F MRI. Neutrophils, monocytes and macrophages phagocytize PFC-NE and subsequently migrate to inflamed tissues. This technique has proven feasibility in numerous disease models in mice, rabbits and mini pigs. The translation to clinical trials in human needs the development of a stable nanoemulsion whose droplet size is well characterized over a long storage time. Usually dynamic light scattering (DLS) is applied as the standard method for determining particle sizes in the nanometer range. Our study uses a second method, analysis of transmission electron microscopy images of cryo-fixed samples (Cryo-TEM), to evaluate stability of PFC-NE in comparison to DLS. Four nanoemulsions of different composition are observed for one year. The results indicate that DLS alone cannot reveal the changes in particle size, but can even mislead to a positive estimation of stability. The combination with Cryo-TEM images gives more insight in the particulate evolution, both techniques supporting one another. The study is one further step in the development of analytical tools for the evaluation of a clinically applicable perfluorooctylbromide nanoemulsion.

No MeSH data available.


Related in: MedlinePlus

Sample Cryo-TEM images of perfluorocarbon-nanoemulsions.(A) Image of a nanoemulsion containing 23% w/w perfluorooctylbromide, after heat sterilization. Nanoemulsion droplets appear as dark vesicles, liposomes are visible to a hugh number as smaller, light vesicles. Scale bar is 500 nm. (B) shows the same nanoemulsion after 50 weeks storage at 4°C. Though the scale bar is 1 μm in this image, increased size of droplets can easily be observed. The number of liposomes seems to be stepped up. In (C) a nanoemulsion containing 20% perfluorooctylbromide and 3% perfluorodecylbromide after heat sterilization is depicted. Though a huge number of liposomes can be detected, more and smaller nanoemulsion droplets are visible than compared to image (A). All samples were diluted in the same ratio with sample buffer.
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pone.0130674.g003: Sample Cryo-TEM images of perfluorocarbon-nanoemulsions.(A) Image of a nanoemulsion containing 23% w/w perfluorooctylbromide, after heat sterilization. Nanoemulsion droplets appear as dark vesicles, liposomes are visible to a hugh number as smaller, light vesicles. Scale bar is 500 nm. (B) shows the same nanoemulsion after 50 weeks storage at 4°C. Though the scale bar is 1 μm in this image, increased size of droplets can easily be observed. The number of liposomes seems to be stepped up. In (C) a nanoemulsion containing 20% perfluorooctylbromide and 3% perfluorodecylbromide after heat sterilization is depicted. Though a huge number of liposomes can be detected, more and smaller nanoemulsion droplets are visible than compared to image (A). All samples were diluted in the same ratio with sample buffer.

Mentions: For the nanoemulsion 23, dynamic light scattering indicates a decreasing average size (dz) while PI worsens. The effect of Ostwald ripening in this preparation leads to shrinkage or disappearance of smaller sized droplets, while bigger sized droplets grow and small liposomes are formed by excess phospholipid, leading to a bimodal size distribution. Cryo-TEM data support this view, as the mean number-weighted size (dn) and median size diminish during storage time while the bigger sized droplets become constantly bigger, as can be seen in percentile values. Fig 3 shows sample Cryo-TEM images of NEs containing 23% PFC. It can be observed that nanoemulsion droplets become bigger and the number of liposomes increases during one year for nanoemulsion 23 (Fig 3(A) & 3(B)). Compared to nanoemulsion 20/3 in Fig 3(C), which shows smaller sized and more PFC containing vesicles after heat sterilization (compare with (A)). This NE 20/3, containing three percent of perfluorodecylbromide, is much more stable. DLS data reveal a slightly increasing hydrodynamic size at a slightly fluctuating, yet acceptable, PI below 0.2. Cryo-TEM data show a similarity to nanoemulsion 23 without added perfluorodecylbromide, as the mean and median size decrease and bigger sized droplets become more. Remarkably, this phenomenon is observed to a significantly smaller degree, Ostwald ripening is retarded.


Monitoring the Stability of Perfluorocarbon Nanoemulsions by Cryo-TEM Image Analysis and Dynamic Light Scattering.

Grapentin C, Barnert S, Schubert R - PLoS ONE (2015)

Sample Cryo-TEM images of perfluorocarbon-nanoemulsions.(A) Image of a nanoemulsion containing 23% w/w perfluorooctylbromide, after heat sterilization. Nanoemulsion droplets appear as dark vesicles, liposomes are visible to a hugh number as smaller, light vesicles. Scale bar is 500 nm. (B) shows the same nanoemulsion after 50 weeks storage at 4°C. Though the scale bar is 1 μm in this image, increased size of droplets can easily be observed. The number of liposomes seems to be stepped up. In (C) a nanoemulsion containing 20% perfluorooctylbromide and 3% perfluorodecylbromide after heat sterilization is depicted. Though a huge number of liposomes can be detected, more and smaller nanoemulsion droplets are visible than compared to image (A). All samples were diluted in the same ratio with sample buffer.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4476784&req=5

pone.0130674.g003: Sample Cryo-TEM images of perfluorocarbon-nanoemulsions.(A) Image of a nanoemulsion containing 23% w/w perfluorooctylbromide, after heat sterilization. Nanoemulsion droplets appear as dark vesicles, liposomes are visible to a hugh number as smaller, light vesicles. Scale bar is 500 nm. (B) shows the same nanoemulsion after 50 weeks storage at 4°C. Though the scale bar is 1 μm in this image, increased size of droplets can easily be observed. The number of liposomes seems to be stepped up. In (C) a nanoemulsion containing 20% perfluorooctylbromide and 3% perfluorodecylbromide after heat sterilization is depicted. Though a huge number of liposomes can be detected, more and smaller nanoemulsion droplets are visible than compared to image (A). All samples were diluted in the same ratio with sample buffer.
Mentions: For the nanoemulsion 23, dynamic light scattering indicates a decreasing average size (dz) while PI worsens. The effect of Ostwald ripening in this preparation leads to shrinkage or disappearance of smaller sized droplets, while bigger sized droplets grow and small liposomes are formed by excess phospholipid, leading to a bimodal size distribution. Cryo-TEM data support this view, as the mean number-weighted size (dn) and median size diminish during storage time while the bigger sized droplets become constantly bigger, as can be seen in percentile values. Fig 3 shows sample Cryo-TEM images of NEs containing 23% PFC. It can be observed that nanoemulsion droplets become bigger and the number of liposomes increases during one year for nanoemulsion 23 (Fig 3(A) & 3(B)). Compared to nanoemulsion 20/3 in Fig 3(C), which shows smaller sized and more PFC containing vesicles after heat sterilization (compare with (A)). This NE 20/3, containing three percent of perfluorodecylbromide, is much more stable. DLS data reveal a slightly increasing hydrodynamic size at a slightly fluctuating, yet acceptable, PI below 0.2. Cryo-TEM data show a similarity to nanoemulsion 23 without added perfluorodecylbromide, as the mean and median size decrease and bigger sized droplets become more. Remarkably, this phenomenon is observed to a significantly smaller degree, Ostwald ripening is retarded.

Bottom Line: The results indicate that DLS alone cannot reveal the changes in particle size, but can even mislead to a positive estimation of stability.The combination with Cryo-TEM images gives more insight in the particulate evolution, both techniques supporting one another.The study is one further step in the development of analytical tools for the evaluation of a clinically applicable perfluorooctylbromide nanoemulsion.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Technology and Biopharmacy, Albert Ludwig University Freiburg i. Br., Freiburg im Breisgau, Germany.

ABSTRACT
Perfluorocarbon nanoemulsions (PFC-NE) are disperse systems consisting of nanoscale liquid perfluorocarbon droplets stabilized by an emulsifier, usually phospholipids. Perfluorocarbons are chemically inert and non-toxic substances that are exhaled after in vivo administration. The manufacture of PFC-NE can be done in large scales by means of high pressure homogenization or microfluidization. Originally investigated as oxygen carriers for cases of severe blood loss, their application nowadays is more focused on using them as marker agents in 19F Magnetic Resonance Imaging (19F MRI). 19F is scarce in organisms and thus PFC-NE are a promising tool for highly specific and non-invasive imaging of inflammation via 19F MRI. Neutrophils, monocytes and macrophages phagocytize PFC-NE and subsequently migrate to inflamed tissues. This technique has proven feasibility in numerous disease models in mice, rabbits and mini pigs. The translation to clinical trials in human needs the development of a stable nanoemulsion whose droplet size is well characterized over a long storage time. Usually dynamic light scattering (DLS) is applied as the standard method for determining particle sizes in the nanometer range. Our study uses a second method, analysis of transmission electron microscopy images of cryo-fixed samples (Cryo-TEM), to evaluate stability of PFC-NE in comparison to DLS. Four nanoemulsions of different composition are observed for one year. The results indicate that DLS alone cannot reveal the changes in particle size, but can even mislead to a positive estimation of stability. The combination with Cryo-TEM images gives more insight in the particulate evolution, both techniques supporting one another. The study is one further step in the development of analytical tools for the evaluation of a clinically applicable perfluorooctylbromide nanoemulsion.

No MeSH data available.


Related in: MedlinePlus