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Tissue and cellular localization of nanoparticles using 35S labeling and light microscopic autoradiography
BASIC SCIENCE Nanomedicine: Nanotechnology, Biology, and Medicine 9 (2013) 465 – 468
Short Communication
nanomedjournal.com
Tissue and cellular localization of nanoparticles using 35 S labeling and light microscopic autoradiography Cornelia Holzhausen, DVM a, 1 , Dominic Gröger, MSc b , Lars Mundhenk, PhD, DVM a , Pia Welker, PhD c , Rainer Haag, PhD b, 2 , Achim D. Gruber, PhD, DVM a,⁎ b
a Department of Veterinary Pathology, Freie Universität Berlin, Berlin, Germany Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany c mivenion GmbH, Berlin, Germany Received 26 November 2012; accepted 12 February 2013
Abstract Microscopical visualization of nanoparticles in tissues is essential for assessing their distribution in whole organisms and their interaction with the cellular microenvironment, including possible toxic effects. However, labeling of nanoparticles with fluorescent dyes may affect their physicochemical properties. Moreover, the detection of organic nanoparticles in their tissue context often poses a particular challenge due to their closer similarities with biomolecules. As part of a biodistribution and toxicity study on organic anti-inflammatory nanoscaled dendritic polyglycerol sulfate amine (dPGS amine) we have established light microscopic autoradiography (LMA) for the tracking of 35S labeled dPGS in standard histopathological tissue samples following intravenous injection in mice. The dPG 35S amine was specifically localized in hepatic Kupffer cells with no histopathologic evidence of toxic, degenerate or inflammatory side effects. The combination of radiolabeling of organic nanoparticles with LMA offers a novel approach for their localization in microscopical slides, also allowing for a simultaneous standard toxicopathology analysis. From the Clinical Editor: In this study, a novel light microscopic autoradiography utilizing 35S isotope demonstrates a combined approach to visualize nanoparticle locations in microscopic slides with no obvious toxicity to the studied cells and with minimal external hazard. © 2013 Elsevier Inc. All rights reserved. Key words: Nanoparticles; Light microscopy; Radioisotope; Mouse model; Toxicopathology
The polymer dendritic polyglycerol sulfate (dPGS) embraces potential opportunities in therapy and imaging of inflammatory diseases. Its high binding affinity to L- and P-selectins inhibits leucocyte extravasation and dPGS was found to interact with
Abbreviations: dPGS, dendritic polyglycerol sulfate; LMA, light microscopic autoradiography; NP, Nanoparticles; TEM, transmission electron microscopy; HE, hematoxylin and eosin; AB, Alcian blue. No conflicts of interest. This work was supported by the Helmholtz Virtual Institute on “Multifunctional Polymers in Medicine” and the FU Focus Area ‘Nanoscale’, the collaborative research center SFB 765 of the German Science Foundation (DFG) and the DFG Priority Program 1313 Biological Responses to Nanoscale Particles. ⁎ Corresponding author: Department of Veterinary Pathology, Freie Universität Berlin, Robert-von-Ostertag-Str. 15, 14163 Berlin, Germany. E-mail address: [email protected] (A.D. Gruber). 1 This article is part of the PhD thesis of CH. 2 RH is a consultant for mivenion GmbH.
complement factors C3 and C5a. In a mouse dermatitis model, the anti-inflammatory effect of dPGS was as effective as that of prednisolone. 1 The tissue and cellular localization of nanoparticles (NP) following their application to the whole organism is of critical interest for understanding their in vivo interactions with desired and unwanted target structures, and, finally for predicting, their efficacy and tolerability in novel therapeutic approaches. 2 Standard light microscopic histopathology has proven useful for decades in the evaluation of xenobiotic effects in humans and animal models. In this context, however, the detection of NP at the single particle level is problematic due to limited light microscopic resolution which, per definition of NP size, does not allow for NP recognition. 3 The visualization of organic NP such as dPGS poses another particular challenge because they provide little contrast in tissue context in transmission electron microscopy (TEM). 3 Hence organic NP have previously been tagged with fluorochromes followed by microscopic tissue examination under ultraviolet light. 4–6 However, addition of a
1549-9634/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nano.2013.02.003 Please cite this article as: Holzhausen C, et al, Tissue and cellular localization of nanoparticles using Nanomedicine: NBM 2013;9:465-468, http://dx.doi.org/10.1016/j.nano.2013.02.003
35
S labeling and light microscopic autoradiography.
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C. Holzhausen et al / Nanomedicine: Nanotechnology, Biology, and Medicine 9 (2013) 465–468
fluorochrome, e.g. indocarbocyanine3 with a molecular weight of 767 Da, to nanoscaled macromolecules such as dPGS with a molecular weight of 1300 Da 7 is likely to alter their physical and chemical properties, possibly resulting in a different interaction with biomolecules, cells and tissues. 8 Here, we have established a novel approach for the histological, light microscopical visualization of the organic NP dPGS in mouse tissue sections as part of a biodistribution and toxicity study. We hypothesized that replacing sulfur atoms in the shell of dPGS with the radioisotope 35S would allow for its visualization by light microscopic autoradiography (LMA) without addition of a fluorescent tag and thus with no risk of changing its interaction with biomolecules.
Materials and methods Unlabeled dPGS amine or 35S labeled dPGS (dPG 35S amine) of 6 ± 1.5 nm size was diluted in 0.9% NaCl solution to a final concentration of 3 mg/100 μl. 1 Synthesis and radiolabeling of dPG 35 S particle are described elsewhere (Groeger et al., submitted). NMRI mice received intravenous injections of dPG 35S amine (group 1), non-radioactively labeled dPGS amine (group 2) or solvent (group 3). The study-protocol was approved by the State Office of Health and Social Affairs, Berlin (G-0028/10) and conducted according to national guidelines and the human care of the animals followed the guidelines. Each mouse received 0.03 mg dPGS amine or dPG 35S amine per g body weight (n = 3 mice per group, respectively), the latter with a radioactivity of 8880 Bq per g body weight. All animals were observed for clinical signs of illness. Mice were sacrificed at different time-points after administration. Animals were autopsied and tissue samples of the organs were immersion-fixed in 4% formalin for routine histopathology. The majority of radioactive signals was obtained from the livers five days after administration, as assessed by liquid scintillation counting of whole organ homogenates (mean 35.38± 12.01 Bq/mg, n = 3 mice, each sample measured three times, data not shown). Liver samples from mice sacrificed five days after injection were thus embedded in paraffin, sectioned at 2 μm, mounted on microscopic glass slides, deparaffinized and stained with hematoxylin and eosin,(HE) according to standard procedures. A gelatin based autoradiographic emulsion (Ilford nuclear emulsion K5D, prediluted) was incubated in a 40 °C water-bath for 45 min. Tissue sections were covered with the emulsion immediately after HE staining by dipping the glass medium with the mounted tissue section into the emulsion and allowing the surplus to drain off. Sections were exposed at total darkness for three weeks at 25 °C and a humidity of 75% following the supplier’s recommendations (Harman Technology Ltd.). The emulsion-covered slides were developed using IlfordPhenisol-developer for 4 min at 20 °C, rinsed with distilled water and fixed with Ilford-rapid-fixator. The whole procedure was conducted under safelight conditions and concluded in washing the slides with tap-water for 15 min. The slides were finally covered with hydrophilic Kaiser’s glycerol and examined under a light microscope (Olympus BX42) equipped with a camera (Colorview II, SIS) and a digital image analysis software
(AnalySIS, Vers.). Consecutive serial sections from the same tissue blocks were stained with Alcian blue (AB). 3
Results No clinical, pathological or histological abnormalities were observed in any mouse examined. Histologically, signals indicating radioactive decay in tissues from group 1 appeared as black dots of approximately 1 μm size that were almost exclusively located over or adjacent to hepatic Kupffer cells that belong to the mononuclear phagocytic system, MPS, (Figure 1, A, left panel). This cell type was clearly identified by its unique comma-shaped nuclei and concomitant association with hepatic sinusoids. No association of the dots was observed with other cells or structures, including hepatocytes, bile ducts, vascular endothelial cells or the lumen of blood vessels. On average, between 5 and 50 dots were associated with a single Kupffer cell. The dots were located within the photoemulsion layer, slightly above the optical plane of the tissue sections, requiring different foci planes while microscoping and photographing the sections. Merging of the digital images generated a single optical image (Figure 1, A, third row). Dots were scarce and randomly distributed in sections from groups 2 and 3 (Figure 1, A, mid and right panels), interpreted as the result of background radioactivity, without any association with a specific cell type or tissue structure. Tissue sections stained with AB, clearly developed blue signals over the cytosol of Kupffer cells from livers of groups 1 and 2 but not group 3. This confirmed that the radiolabeled and the unlabeled dPGS amine had similarly accumulated selectively in the cytosol of Kupffer cells but no other cell types or tissue structures (Figure 1, B).
Discussion The addition of fluorescent tags for visualization of organic nanoparticles in cells or tissues inevitably leads to changes in size, molecular weight and structure that may result in changed biochemical interactions of the tagged molecule. 8 In contrast, radiolabeling offers the advantage that no significant changes in size, molecular weight or chemical reactivity are to be expected that may influence the properties of the labeled molecule in vivo. The radioisotope 35S, for example, may be used to replace the naturally occurring, non-radioactive 32S isotope in compounds containing sulfur such as dPGS. Labeling sulfur atoms in the anionic sulphated shell of the dPGS molecule with 35S resulted in a radioactivity of 300,000 Bq/mg dPG 35S amine. 35S emits beta radiation at a maximum energy of 0.167 MeV 9 that can be used for detection by standard autoradiography on X-ray film, histologic photoemulsion and digital autoradiographic techniques. Moreover, the physical half-life of 35S of 87.4 days facilitates experimental procedures over several weeks and still allows for prolonged and thus highly sensitive exposures in autoradiographic detection systems. Radioisotopes other than 35 S may certainly be used for radiolabeling of different elements, however, the nature and energy of the emitted radiation as well as
C. Holzhausen et al / Nanomedicine: Nanotechnology, Biology, and Medicine 9 (2013) 465–468
467
Figure 1. Visualization of 35S radiolabeled nanoscaled dendritic polyglycerol amone (dPG 35S amine) by light microscopical autoradiography (LMA) in the livers of mice 5 days after intravenous injection. (A) Radioactive signals were identified in tissue sections from group 1 (left column) as punctate black staining in the photoemulsion layer (top row) associated with hepatic Kupffer cells that were identified in the tissue sections (second row) by the comma shaped nuclei and their association with hepatic sinusoids (red circles in the merged plane). Sparse signals in groups 2 (unlabeled dPGS amine, mid column) and group 3 (solvent, right column) that were not associated with any specific cell type or tissue structure were interpreted as the result of background radiation. HE-stain, bars = 20 μm. (B) Blue colour signals specifically associated with the cytosol of Kupffer cells (red circles) were observed in Alcian blue stained liver sections in groups 1 and 2 (left and mid column) but not in mice injected with solvent (right column), supporting the specificity of radioactive signals for the detection of dPG 35S amine in panel (A), bars = 20 μm.
its half-life will largely affect its suitability for biomedical studies and autoradiographic detection systems. Accumulation of intravenously injected radiolabeled and unlabeled dPGS amine in hepatic Kupffer cells was confirmed by staining the tissue sections with AB, a cationic dye for polysaccharides that also stains sulfate esters including dPGS amine 10, albeit at a sensitivity that cannot be expected to visualize dPGS amine or other nanoscaled molecules in low numbers. 10 Hepatic Kupffer cells belong to the MPS and act as resident
macrophages that clear biogenic and xenobiotic macromolecules from the circulation. A toxicohistopathological analysis of the slides that were also used for the autoradiographic detection of dPG 35S amine failed to yield any evidence of degenerative, inflammatory or other pathological cellular or tissue changes, suggesting that their accumulation had no adverse effect on Kupffer cells or other cell types in the livers five days after injection. Moreover, no cellular changes could be attributed to the radioactive decay of the dPG 35S amine which was not surprising
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C. Holzhausen et al / Nanomedicine: Nanotechnology, Biology, and Medicine 9 (2013) 465–468
since the administered radioactivity of approximately 200 kBq per mouse was not expected to cause any radiation damage. 11 In summary, we have established a novel approach for the localization of untagged, nanoscaled organic molecules in microscopical sections from formalin-fixed, paraffin-embedded tissues commonly used for histopathological analysis. Radioactive labelling may also facilitate precise particle tracking and quantification using more quantitative detection methods in lysed tissues, such as liquid scintillation counting.
Acknowledgment We thank Jörg Schnorr for the excellent support by implementing the animal study protocol.
References 1. Dernedde J, Rausch A, Weinhart M, Enders S, Tauber R, Licha K, et al. Dendritic polyglycerol sulfates as multivalent inhibitors of inflammation. Proc Natl Acad Sci 2010;107:19679-84. 2. Oberdörster Günter OE, Oberdörster J. Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles. Environ Health Perspect 2005;113:823-39.
3. Terje D, Werner HD, Mah LNM, Jan-Thorsten S. Techniques in microscopy for biomedical applications. In: J-T S, editor. Manuals in biomedical research: World Scientific Publishing Co. Pte. Ltd; 2006. 4. Almutairi A, Akers WJ, Berezin MY, Achilefu S, Fréchet JMJ. Monitoring the biodegradation of dendritic near-infrared nanoprobes by in vivo fluorescence imaging. Mol Pharm 2008;5:1103-10. 5. Saxena V, Sadoqi M, Shao J. Polymeric nanoparticulate delivery system for indocyanine green: biodistribution in healthy mice. Int J Pharm 2006;308:200-4. 6. Janib SM, Moses AS, MacKay JA. Imaging and drug delivery using theranostic nanoparticles. Adv Drug Deliv Rev 2010;62:1052-63. 7. Weinhart M, Gröger D, Enders S, Riese SB, Dernedde J, Kainthan RK, et al. The role of dimension in multivalent binding events: structure– activity relationship of dendritic polyglycerol sulfate binding to l-selectin in correlation with size and surface charge density. Macromol Biosci 2011;11:1088-98. 8. Marquis BJ, Love SA, Braun KL, Haynes CL. Analytical methods to assess nanoparticle toxicity. Analyst 2009;134:425-39. 9. Kocher DC. Radioactive decay data tables: a handbook of decay data for application to radiation dosimetry and radiological assessments. [Oak Ridge, Tenn.]; Springfield, Va.: Technical Information Center, U.S. Dept. of Energy; Available from National Technical Information Service, U.S. Dept. of Commerce; 1981. 10. Scott JE. Histochemistry of alcian blue. Histochem Cell Biol 1972;30: 215-34. 11. Agrawal S, Temsamani J, Tang JY. Pharmacokinetics, biodistribution, and stability of oligodeoxynucleotide phosphorothioates in mice. PNAS 1991;88:7595-9.
Short Communication
nanomedjournal.com
Tissue and cellular localization of nanoparticles using 35 S labeling and light microscopic autoradiography Cornelia Holzhausen, DVM a, 1 , Dominic Gröger, MSc b , Lars Mundhenk, PhD, DVM a , Pia Welker, PhD c , Rainer Haag, PhD b, 2 , Achim D. Gruber, PhD, DVM a,⁎ b
a Department of Veterinary Pathology, Freie Universität Berlin, Berlin, Germany Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany c mivenion GmbH, Berlin, Germany Received 26 November 2012; accepted 12 February 2013
Abstract Microscopical visualization of nanoparticles in tissues is essential for assessing their distribution in whole organisms and their interaction with the cellular microenvironment, including possible toxic effects. However, labeling of nanoparticles with fluorescent dyes may affect their physicochemical properties. Moreover, the detection of organic nanoparticles in their tissue context often poses a particular challenge due to their closer similarities with biomolecules. As part of a biodistribution and toxicity study on organic anti-inflammatory nanoscaled dendritic polyglycerol sulfate amine (dPGS amine) we have established light microscopic autoradiography (LMA) for the tracking of 35S labeled dPGS in standard histopathological tissue samples following intravenous injection in mice. The dPG 35S amine was specifically localized in hepatic Kupffer cells with no histopathologic evidence of toxic, degenerate or inflammatory side effects. The combination of radiolabeling of organic nanoparticles with LMA offers a novel approach for their localization in microscopical slides, also allowing for a simultaneous standard toxicopathology analysis. From the Clinical Editor: In this study, a novel light microscopic autoradiography utilizing 35S isotope demonstrates a combined approach to visualize nanoparticle locations in microscopic slides with no obvious toxicity to the studied cells and with minimal external hazard. © 2013 Elsevier Inc. All rights reserved. Key words: Nanoparticles; Light microscopy; Radioisotope; Mouse model; Toxicopathology
The polymer dendritic polyglycerol sulfate (dPGS) embraces potential opportunities in therapy and imaging of inflammatory diseases. Its high binding affinity to L- and P-selectins inhibits leucocyte extravasation and dPGS was found to interact with
Abbreviations: dPGS, dendritic polyglycerol sulfate; LMA, light microscopic autoradiography; NP, Nanoparticles; TEM, transmission electron microscopy; HE, hematoxylin and eosin; AB, Alcian blue. No conflicts of interest. This work was supported by the Helmholtz Virtual Institute on “Multifunctional Polymers in Medicine” and the FU Focus Area ‘Nanoscale’, the collaborative research center SFB 765 of the German Science Foundation (DFG) and the DFG Priority Program 1313 Biological Responses to Nanoscale Particles. ⁎ Corresponding author: Department of Veterinary Pathology, Freie Universität Berlin, Robert-von-Ostertag-Str. 15, 14163 Berlin, Germany. E-mail address: [email protected] (A.D. Gruber). 1 This article is part of the PhD thesis of CH. 2 RH is a consultant for mivenion GmbH.
complement factors C3 and C5a. In a mouse dermatitis model, the anti-inflammatory effect of dPGS was as effective as that of prednisolone. 1 The tissue and cellular localization of nanoparticles (NP) following their application to the whole organism is of critical interest for understanding their in vivo interactions with desired and unwanted target structures, and, finally for predicting, their efficacy and tolerability in novel therapeutic approaches. 2 Standard light microscopic histopathology has proven useful for decades in the evaluation of xenobiotic effects in humans and animal models. In this context, however, the detection of NP at the single particle level is problematic due to limited light microscopic resolution which, per definition of NP size, does not allow for NP recognition. 3 The visualization of organic NP such as dPGS poses another particular challenge because they provide little contrast in tissue context in transmission electron microscopy (TEM). 3 Hence organic NP have previously been tagged with fluorochromes followed by microscopic tissue examination under ultraviolet light. 4–6 However, addition of a
1549-9634/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nano.2013.02.003 Please cite this article as: Holzhausen C, et al, Tissue and cellular localization of nanoparticles using Nanomedicine: NBM 2013;9:465-468, http://dx.doi.org/10.1016/j.nano.2013.02.003
35
S labeling and light microscopic autoradiography.
466
C. Holzhausen et al / Nanomedicine: Nanotechnology, Biology, and Medicine 9 (2013) 465–468
fluorochrome, e.g. indocarbocyanine3 with a molecular weight of 767 Da, to nanoscaled macromolecules such as dPGS with a molecular weight of 1300 Da 7 is likely to alter their physical and chemical properties, possibly resulting in a different interaction with biomolecules, cells and tissues. 8 Here, we have established a novel approach for the histological, light microscopical visualization of the organic NP dPGS in mouse tissue sections as part of a biodistribution and toxicity study. We hypothesized that replacing sulfur atoms in the shell of dPGS with the radioisotope 35S would allow for its visualization by light microscopic autoradiography (LMA) without addition of a fluorescent tag and thus with no risk of changing its interaction with biomolecules.
Materials and methods Unlabeled dPGS amine or 35S labeled dPGS (dPG 35S amine) of 6 ± 1.5 nm size was diluted in 0.9% NaCl solution to a final concentration of 3 mg/100 μl. 1 Synthesis and radiolabeling of dPG 35 S particle are described elsewhere (Groeger et al., submitted). NMRI mice received intravenous injections of dPG 35S amine (group 1), non-radioactively labeled dPGS amine (group 2) or solvent (group 3). The study-protocol was approved by the State Office of Health and Social Affairs, Berlin (G-0028/10) and conducted according to national guidelines and the human care of the animals followed the guidelines. Each mouse received 0.03 mg dPGS amine or dPG 35S amine per g body weight (n = 3 mice per group, respectively), the latter with a radioactivity of 8880 Bq per g body weight. All animals were observed for clinical signs of illness. Mice were sacrificed at different time-points after administration. Animals were autopsied and tissue samples of the organs were immersion-fixed in 4% formalin for routine histopathology. The majority of radioactive signals was obtained from the livers five days after administration, as assessed by liquid scintillation counting of whole organ homogenates (mean 35.38± 12.01 Bq/mg, n = 3 mice, each sample measured three times, data not shown). Liver samples from mice sacrificed five days after injection were thus embedded in paraffin, sectioned at 2 μm, mounted on microscopic glass slides, deparaffinized and stained with hematoxylin and eosin,(HE) according to standard procedures. A gelatin based autoradiographic emulsion (Ilford nuclear emulsion K5D, prediluted) was incubated in a 40 °C water-bath for 45 min. Tissue sections were covered with the emulsion immediately after HE staining by dipping the glass medium with the mounted tissue section into the emulsion and allowing the surplus to drain off. Sections were exposed at total darkness for three weeks at 25 °C and a humidity of 75% following the supplier’s recommendations (Harman Technology Ltd.). The emulsion-covered slides were developed using IlfordPhenisol-developer for 4 min at 20 °C, rinsed with distilled water and fixed with Ilford-rapid-fixator. The whole procedure was conducted under safelight conditions and concluded in washing the slides with tap-water for 15 min. The slides were finally covered with hydrophilic Kaiser’s glycerol and examined under a light microscope (Olympus BX42) equipped with a camera (Colorview II, SIS) and a digital image analysis software
(AnalySIS, Vers.). Consecutive serial sections from the same tissue blocks were stained with Alcian blue (AB). 3
Results No clinical, pathological or histological abnormalities were observed in any mouse examined. Histologically, signals indicating radioactive decay in tissues from group 1 appeared as black dots of approximately 1 μm size that were almost exclusively located over or adjacent to hepatic Kupffer cells that belong to the mononuclear phagocytic system, MPS, (Figure 1, A, left panel). This cell type was clearly identified by its unique comma-shaped nuclei and concomitant association with hepatic sinusoids. No association of the dots was observed with other cells or structures, including hepatocytes, bile ducts, vascular endothelial cells or the lumen of blood vessels. On average, between 5 and 50 dots were associated with a single Kupffer cell. The dots were located within the photoemulsion layer, slightly above the optical plane of the tissue sections, requiring different foci planes while microscoping and photographing the sections. Merging of the digital images generated a single optical image (Figure 1, A, third row). Dots were scarce and randomly distributed in sections from groups 2 and 3 (Figure 1, A, mid and right panels), interpreted as the result of background radioactivity, without any association with a specific cell type or tissue structure. Tissue sections stained with AB, clearly developed blue signals over the cytosol of Kupffer cells from livers of groups 1 and 2 but not group 3. This confirmed that the radiolabeled and the unlabeled dPGS amine had similarly accumulated selectively in the cytosol of Kupffer cells but no other cell types or tissue structures (Figure 1, B).
Discussion The addition of fluorescent tags for visualization of organic nanoparticles in cells or tissues inevitably leads to changes in size, molecular weight and structure that may result in changed biochemical interactions of the tagged molecule. 8 In contrast, radiolabeling offers the advantage that no significant changes in size, molecular weight or chemical reactivity are to be expected that may influence the properties of the labeled molecule in vivo. The radioisotope 35S, for example, may be used to replace the naturally occurring, non-radioactive 32S isotope in compounds containing sulfur such as dPGS. Labeling sulfur atoms in the anionic sulphated shell of the dPGS molecule with 35S resulted in a radioactivity of 300,000 Bq/mg dPG 35S amine. 35S emits beta radiation at a maximum energy of 0.167 MeV 9 that can be used for detection by standard autoradiography on X-ray film, histologic photoemulsion and digital autoradiographic techniques. Moreover, the physical half-life of 35S of 87.4 days facilitates experimental procedures over several weeks and still allows for prolonged and thus highly sensitive exposures in autoradiographic detection systems. Radioisotopes other than 35 S may certainly be used for radiolabeling of different elements, however, the nature and energy of the emitted radiation as well as
C. Holzhausen et al / Nanomedicine: Nanotechnology, Biology, and Medicine 9 (2013) 465–468
467
Figure 1. Visualization of 35S radiolabeled nanoscaled dendritic polyglycerol amone (dPG 35S amine) by light microscopical autoradiography (LMA) in the livers of mice 5 days after intravenous injection. (A) Radioactive signals were identified in tissue sections from group 1 (left column) as punctate black staining in the photoemulsion layer (top row) associated with hepatic Kupffer cells that were identified in the tissue sections (second row) by the comma shaped nuclei and their association with hepatic sinusoids (red circles in the merged plane). Sparse signals in groups 2 (unlabeled dPGS amine, mid column) and group 3 (solvent, right column) that were not associated with any specific cell type or tissue structure were interpreted as the result of background radiation. HE-stain, bars = 20 μm. (B) Blue colour signals specifically associated with the cytosol of Kupffer cells (red circles) were observed in Alcian blue stained liver sections in groups 1 and 2 (left and mid column) but not in mice injected with solvent (right column), supporting the specificity of radioactive signals for the detection of dPG 35S amine in panel (A), bars = 20 μm.
its half-life will largely affect its suitability for biomedical studies and autoradiographic detection systems. Accumulation of intravenously injected radiolabeled and unlabeled dPGS amine in hepatic Kupffer cells was confirmed by staining the tissue sections with AB, a cationic dye for polysaccharides that also stains sulfate esters including dPGS amine 10, albeit at a sensitivity that cannot be expected to visualize dPGS amine or other nanoscaled molecules in low numbers. 10 Hepatic Kupffer cells belong to the MPS and act as resident
macrophages that clear biogenic and xenobiotic macromolecules from the circulation. A toxicohistopathological analysis of the slides that were also used for the autoradiographic detection of dPG 35S amine failed to yield any evidence of degenerative, inflammatory or other pathological cellular or tissue changes, suggesting that their accumulation had no adverse effect on Kupffer cells or other cell types in the livers five days after injection. Moreover, no cellular changes could be attributed to the radioactive decay of the dPG 35S amine which was not surprising
468
C. Holzhausen et al / Nanomedicine: Nanotechnology, Biology, and Medicine 9 (2013) 465–468
since the administered radioactivity of approximately 200 kBq per mouse was not expected to cause any radiation damage. 11 In summary, we have established a novel approach for the localization of untagged, nanoscaled organic molecules in microscopical sections from formalin-fixed, paraffin-embedded tissues commonly used for histopathological analysis. Radioactive labelling may also facilitate precise particle tracking and quantification using more quantitative detection methods in lysed tissues, such as liquid scintillation counting.
Acknowledgment We thank Jörg Schnorr for the excellent support by implementing the animal study protocol.
References 1. Dernedde J, Rausch A, Weinhart M, Enders S, Tauber R, Licha K, et al. Dendritic polyglycerol sulfates as multivalent inhibitors of inflammation. Proc Natl Acad Sci 2010;107:19679-84. 2. Oberdörster Günter OE, Oberdörster J. Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles. Environ Health Perspect 2005;113:823-39.
3. Terje D, Werner HD, Mah LNM, Jan-Thorsten S. Techniques in microscopy for biomedical applications. In: J-T S, editor. Manuals in biomedical research: World Scientific Publishing Co. Pte. Ltd; 2006. 4. Almutairi A, Akers WJ, Berezin MY, Achilefu S, Fréchet JMJ. Monitoring the biodegradation of dendritic near-infrared nanoprobes by in vivo fluorescence imaging. Mol Pharm 2008;5:1103-10. 5. Saxena V, Sadoqi M, Shao J. Polymeric nanoparticulate delivery system for indocyanine green: biodistribution in healthy mice. Int J Pharm 2006;308:200-4. 6. Janib SM, Moses AS, MacKay JA. Imaging and drug delivery using theranostic nanoparticles. Adv Drug Deliv Rev 2010;62:1052-63. 7. Weinhart M, Gröger D, Enders S, Riese SB, Dernedde J, Kainthan RK, et al. The role of dimension in multivalent binding events: structure– activity relationship of dendritic polyglycerol sulfate binding to l-selectin in correlation with size and surface charge density. Macromol Biosci 2011;11:1088-98. 8. Marquis BJ, Love SA, Braun KL, Haynes CL. Analytical methods to assess nanoparticle toxicity. Analyst 2009;134:425-39. 9. Kocher DC. Radioactive decay data tables: a handbook of decay data for application to radiation dosimetry and radiological assessments. [Oak Ridge, Tenn.]; Springfield, Va.: Technical Information Center, U.S. Dept. of Energy; Available from National Technical Information Service, U.S. Dept. of Commerce; 1981. 10. Scott JE. Histochemistry of alcian blue. Histochem Cell Biol 1972;30: 215-34. 11. Agrawal S, Temsamani J, Tang JY. Pharmacokinetics, biodistribution, and stability of oligodeoxynucleotide phosphorothioates in mice. PNAS 1991;88:7595-9.