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Perfluorocarbon particle size influences magnetic resonance signal and immunological properties of dendritic cells.

Waiczies H, Lepore S, Janitzek N, Hagen U, Seifert F, Ittermann B, Purfürst B, Pezzutto A, Paul F, Niendorf T, Waiczies S - PLoS ONE (2011)

Bottom Line: Dendritic cells (DC) are professional antigen presenting cells and with respect to impact of PFCE particles on DC function, we observed that markers of maturation for these cells (CD80, CD86) were also significantly elevated following labeling with larger PFCE particles (560 nm).When labeled with these larger particles that also gave an optimal signal in MRS, DC presented whole antigen more robustly to CD8+ T cells than control cells.Our data suggest that increasing particle size is one important feature for optimizing cell labeling by PFCE particles, but may also present possible pitfalls such as alteration of the immunological status of these cells.

View Article: PubMed Central - PubMed

Affiliation: Max Delbrück Center for Molecular Medicine and Charité Medical Faculty, Berlin, Germany.

ABSTRACT
The development of cellular tracking by fluorine ((19)F) magnetic resonance imaging (MRI) has introduced a number of advantages for following immune cell therapies in vivo. These include improved signal selectivity and a possibility to correlate cells labeled with fluorine-rich particles with conventional anatomic proton ((1)H) imaging. While the optimization of the cellular labeling method is clearly important, the impact of labeling on cellular dynamics should be kept in mind. We show by (19)F MR spectroscopy (MRS) that the efficiency in labeling cells of the murine immune system (dendritic cells) by perfluoro-15-crown-5-ether (PFCE) particles increases with increasing particle size (560>365>245>130 nm). Dendritic cells (DC) are professional antigen presenting cells and with respect to impact of PFCE particles on DC function, we observed that markers of maturation for these cells (CD80, CD86) were also significantly elevated following labeling with larger PFCE particles (560 nm). When labeled with these larger particles that also gave an optimal signal in MRS, DC presented whole antigen more robustly to CD8+ T cells than control cells. Our data suggest that increasing particle size is one important feature for optimizing cell labeling by PFCE particles, but may also present possible pitfalls such as alteration of the immunological status of these cells. Therefore depending on the clinical scenario in which the (19)F-labeled cellular vaccines will be applied (cancer, autoimmune disease, transplantation), it will be interesting to monitor the fate of these cells in vivo in the relevant preclinical mouse models.

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Perfluorocarbon particle size determines MR signal amplitude of DC.(A) DC were labeled with different concentrations and incubation times of 560 nm PFCE particles and their 19F signal was measured in a 3 T MRI scanner using a 90° block excitation pulse with 10 kHz bandwidth. (B) Different numbers of DC were labeled with 1 mM 560 nm PFCE particles over a period of 24 h and their 19F signal was measured in a 9.4 T MRI scanner. The inset depicts cross-sectional scans of the cell pellets within the NMR tubes made using a FLASH 3D Sequence on the 9.4 T scanner. (C) DC were labeled with 1 mM 560 nm PFCE particles, loaded with (right) or without (left) antigen and administered intradermally in hind limb. Shown is a coronal overlay of 19F cellular (red) and 1H anatomical (grayscale) MR images. (D) DC were labeled with different-sized particles and a constant PFCE concentration (1 mM) and their 19F signal amplitude was measured in a 9.4 T MRI scanner. This experiment is representative of 3 independent experiments
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pone-0021981-g001: Perfluorocarbon particle size determines MR signal amplitude of DC.(A) DC were labeled with different concentrations and incubation times of 560 nm PFCE particles and their 19F signal was measured in a 3 T MRI scanner using a 90° block excitation pulse with 10 kHz bandwidth. (B) Different numbers of DC were labeled with 1 mM 560 nm PFCE particles over a period of 24 h and their 19F signal was measured in a 9.4 T MRI scanner. The inset depicts cross-sectional scans of the cell pellets within the NMR tubes made using a FLASH 3D Sequence on the 9.4 T scanner. (C) DC were labeled with 1 mM 560 nm PFCE particles, loaded with (right) or without (left) antigen and administered intradermally in hind limb. Shown is a coronal overlay of 19F cellular (red) and 1H anatomical (grayscale) MR images. (D) DC were labeled with different-sized particles and a constant PFCE concentration (1 mM) and their 19F signal amplitude was measured in a 9.4 T MRI scanner. This experiment is representative of 3 independent experiments

Mentions: Using the direct sonication method we obtained particles with an average size of 245 nm±1.97 nm (±S.E.M.) that increased to 560 nm±6.36 nm following steam sterilization. The increase in particle size was also observed when we prepared PFCE particles using high pressure homogenization. Although particles doubled in size following steam sterilization, the size of particles achieved by both sonication and high pressure homogenization remained constant over several days under cell culture conditions (data not shown). Using the 560 nm particles, we labeled DC with different concentrations of PFCE together with different incubation times. The 19F signal amplitude was directly proportional to labeling time and PFCE concentration (Fig. 1A). The area under the curve (AUC) for DC samples incubated with particles containing 1 mM PFCE over a period of 3 h, 24 h and 48 h was calculated to be 9.28*107, 1.43*108 and 2.65*108 respectively, while the AUC for DC samples incubated for 24 h with particles containing 0.4 mM and 2 mM PFCE was calculated to be 1.18*108 and 2.22*108 respectively. To extrapolate the 19F signal amplitude generated in vitro to the extent of DC localizing to specific regions in vivo, a calibration curve was made with different numbers of DC labeled with the same concentration of 560 nm particles (1 mM PFCE) and over a period of 24 h. The 19F signal amplitude (Fig. 1B) was calculated from 19F-MRS using the loop coil for the 9.4T MRI (Supp. Fig. S1B). The signal achieved was compared with the cellular in vitro cross-sectional images (FLASH 3D Sequence) of the 19F-labeled cell pellets within the NMR tubes (Fig. 1B, inset) using a commercial 1H/19F dual-tunable volume birdcage resonator for the 9.4T MRI (see Methods). The 19F signal amplitude to cell number curve gave a linear fit indicating that the signal of the spectroscopy data correlate linearly with the number of labeled cells (Fig. 1B). With the setup used the minimum amount of labeled cells detectable in MRS was 0.6*106 DC corresponding to about 1018 fluorine atoms. For MRI this translates into a minimum cell number of 106 DC within the volume of interest (Fig. 1B). Using the same concentration of 560 nm particles (1 mM PFCE) we could follow the migration of 10*106 19F-labeled DC that were loaded with whole chicken ovalbumin (OVA) antigen. Following 18 h intradermal application of these cells in the right hind limb we observed a migration of the 19F-labeled cells from footpad into the draining popliteal lymph node, in contrast to cells that were not loaded with antigen and applied to the left hind limb (Fig. 1C). In the latter case the cells remained localized to the footpad.


Perfluorocarbon particle size influences magnetic resonance signal and immunological properties of dendritic cells.

Waiczies H, Lepore S, Janitzek N, Hagen U, Seifert F, Ittermann B, Purfürst B, Pezzutto A, Paul F, Niendorf T, Waiczies S - PLoS ONE (2011)

Perfluorocarbon particle size determines MR signal amplitude of DC.(A) DC were labeled with different concentrations and incubation times of 560 nm PFCE particles and their 19F signal was measured in a 3 T MRI scanner using a 90° block excitation pulse with 10 kHz bandwidth. (B) Different numbers of DC were labeled with 1 mM 560 nm PFCE particles over a period of 24 h and their 19F signal was measured in a 9.4 T MRI scanner. The inset depicts cross-sectional scans of the cell pellets within the NMR tubes made using a FLASH 3D Sequence on the 9.4 T scanner. (C) DC were labeled with 1 mM 560 nm PFCE particles, loaded with (right) or without (left) antigen and administered intradermally in hind limb. Shown is a coronal overlay of 19F cellular (red) and 1H anatomical (grayscale) MR images. (D) DC were labeled with different-sized particles and a constant PFCE concentration (1 mM) and their 19F signal amplitude was measured in a 9.4 T MRI scanner. This experiment is representative of 3 independent experiments
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3139612&req=5

pone-0021981-g001: Perfluorocarbon particle size determines MR signal amplitude of DC.(A) DC were labeled with different concentrations and incubation times of 560 nm PFCE particles and their 19F signal was measured in a 3 T MRI scanner using a 90° block excitation pulse with 10 kHz bandwidth. (B) Different numbers of DC were labeled with 1 mM 560 nm PFCE particles over a period of 24 h and their 19F signal was measured in a 9.4 T MRI scanner. The inset depicts cross-sectional scans of the cell pellets within the NMR tubes made using a FLASH 3D Sequence on the 9.4 T scanner. (C) DC were labeled with 1 mM 560 nm PFCE particles, loaded with (right) or without (left) antigen and administered intradermally in hind limb. Shown is a coronal overlay of 19F cellular (red) and 1H anatomical (grayscale) MR images. (D) DC were labeled with different-sized particles and a constant PFCE concentration (1 mM) and their 19F signal amplitude was measured in a 9.4 T MRI scanner. This experiment is representative of 3 independent experiments
Mentions: Using the direct sonication method we obtained particles with an average size of 245 nm±1.97 nm (±S.E.M.) that increased to 560 nm±6.36 nm following steam sterilization. The increase in particle size was also observed when we prepared PFCE particles using high pressure homogenization. Although particles doubled in size following steam sterilization, the size of particles achieved by both sonication and high pressure homogenization remained constant over several days under cell culture conditions (data not shown). Using the 560 nm particles, we labeled DC with different concentrations of PFCE together with different incubation times. The 19F signal amplitude was directly proportional to labeling time and PFCE concentration (Fig. 1A). The area under the curve (AUC) for DC samples incubated with particles containing 1 mM PFCE over a period of 3 h, 24 h and 48 h was calculated to be 9.28*107, 1.43*108 and 2.65*108 respectively, while the AUC for DC samples incubated for 24 h with particles containing 0.4 mM and 2 mM PFCE was calculated to be 1.18*108 and 2.22*108 respectively. To extrapolate the 19F signal amplitude generated in vitro to the extent of DC localizing to specific regions in vivo, a calibration curve was made with different numbers of DC labeled with the same concentration of 560 nm particles (1 mM PFCE) and over a period of 24 h. The 19F signal amplitude (Fig. 1B) was calculated from 19F-MRS using the loop coil for the 9.4T MRI (Supp. Fig. S1B). The signal achieved was compared with the cellular in vitro cross-sectional images (FLASH 3D Sequence) of the 19F-labeled cell pellets within the NMR tubes (Fig. 1B, inset) using a commercial 1H/19F dual-tunable volume birdcage resonator for the 9.4T MRI (see Methods). The 19F signal amplitude to cell number curve gave a linear fit indicating that the signal of the spectroscopy data correlate linearly with the number of labeled cells (Fig. 1B). With the setup used the minimum amount of labeled cells detectable in MRS was 0.6*106 DC corresponding to about 1018 fluorine atoms. For MRI this translates into a minimum cell number of 106 DC within the volume of interest (Fig. 1B). Using the same concentration of 560 nm particles (1 mM PFCE) we could follow the migration of 10*106 19F-labeled DC that were loaded with whole chicken ovalbumin (OVA) antigen. Following 18 h intradermal application of these cells in the right hind limb we observed a migration of the 19F-labeled cells from footpad into the draining popliteal lymph node, in contrast to cells that were not loaded with antigen and applied to the left hind limb (Fig. 1C). In the latter case the cells remained localized to the footpad.

Bottom Line: Dendritic cells (DC) are professional antigen presenting cells and with respect to impact of PFCE particles on DC function, we observed that markers of maturation for these cells (CD80, CD86) were also significantly elevated following labeling with larger PFCE particles (560 nm).When labeled with these larger particles that also gave an optimal signal in MRS, DC presented whole antigen more robustly to CD8+ T cells than control cells.Our data suggest that increasing particle size is one important feature for optimizing cell labeling by PFCE particles, but may also present possible pitfalls such as alteration of the immunological status of these cells.

View Article: PubMed Central - PubMed

Affiliation: Max Delbrück Center for Molecular Medicine and Charité Medical Faculty, Berlin, Germany.

ABSTRACT
The development of cellular tracking by fluorine ((19)F) magnetic resonance imaging (MRI) has introduced a number of advantages for following immune cell therapies in vivo. These include improved signal selectivity and a possibility to correlate cells labeled with fluorine-rich particles with conventional anatomic proton ((1)H) imaging. While the optimization of the cellular labeling method is clearly important, the impact of labeling on cellular dynamics should be kept in mind. We show by (19)F MR spectroscopy (MRS) that the efficiency in labeling cells of the murine immune system (dendritic cells) by perfluoro-15-crown-5-ether (PFCE) particles increases with increasing particle size (560>365>245>130 nm). Dendritic cells (DC) are professional antigen presenting cells and with respect to impact of PFCE particles on DC function, we observed that markers of maturation for these cells (CD80, CD86) were also significantly elevated following labeling with larger PFCE particles (560 nm). When labeled with these larger particles that also gave an optimal signal in MRS, DC presented whole antigen more robustly to CD8+ T cells than control cells. Our data suggest that increasing particle size is one important feature for optimizing cell labeling by PFCE particles, but may also present possible pitfalls such as alteration of the immunological status of these cells. Therefore depending on the clinical scenario in which the (19)F-labeled cellular vaccines will be applied (cancer, autoimmune disease, transplantation), it will be interesting to monitor the fate of these cells in vivo in the relevant preclinical mouse models.

Show MeSH
Related in: MedlinePlus