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Detection of Peptide-based nanoparticles in blood plasma by ELISA.

Bode GH, Pickl KE, Sanchez-Purrà M, Albaiges B, Borrós S, Pötgens AJ, Schmitz C, Sinner FM, Losen M, Steinbusch HW, Frank HG, Martinez-Martinez P, European NanoBioPharmaceutics Research Initiati - PLoS ONE (2015)

Bottom Line: We were able to accurately measure peptides bound to pentafluorophenyl methacrylate nanoparticles in blood plasma of rats, and similar results were obtained by LC/MS.We detected FITC-labeled peptides on pentafluorophenyl methacrylate nanoparticles after injection in vivo.This method can be extended to detect nanoparticles with different chemical compositions.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, School for Mental Health and Neuroscience, Faculty of Health, Medicine and Life Sciences, Maastricht University, Maastricht, the Netherlands.

ABSTRACT

Aims: The aim of the current study was to develop a method to detect peptide-linked nanoparticles in blood plasma.

Materials & methods: A convenient enzyme linked immunosorbent assay (ELISA) was developed for the detection of peptides functionalized with biotin and fluorescein groups. As a proof of principle, polymerized pentafluorophenyl methacrylate nanoparticles linked to biotin-carboxyfluorescein labeled peptides were intravenously injected in Wistar rats. Serial blood plasma samples were analyzed by ELISA and by liquid chromatography mass spectrometry (LC/MS) technology.

Results: The ELISA based method for the detection of FITC labeled peptides had a detection limit of 1 ng/mL. We were able to accurately measure peptides bound to pentafluorophenyl methacrylate nanoparticles in blood plasma of rats, and similar results were obtained by LC/MS.

Conclusions: We detected FITC-labeled peptides on pentafluorophenyl methacrylate nanoparticles after injection in vivo. This method can be extended to detect nanoparticles with different chemical compositions.

No MeSH data available.


Schematic representation of the nanoparticle and ELISA design.Peptide 5A contains biotin and carboxyfluorescein, lower case letters denote D-amino acids. Nanoparticles were synthesized by a free-radical polymerization method in a microemulsion system. N-isopropylacrylamide, N,N-dimethylacrylamide and acrylic acid were used as monomers with methylenebisacrylamide as cross-linker. Pentafluorophenyl methacrylate (PFM) was added and the nanoparticles were adorned with peptide 5A (B). An ELISA was designed to detect nanoparticles in biological fluids using the biotin and carboxyfluorescein groups present on peptide 5A (C). Streptavidin coated plates were used to capture the peptides by binding the biotin group and bound peptides were detected with a HRP conjugated mAb anti-fluorescein.
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pone.0126136.g001: Schematic representation of the nanoparticle and ELISA design.Peptide 5A contains biotin and carboxyfluorescein, lower case letters denote D-amino acids. Nanoparticles were synthesized by a free-radical polymerization method in a microemulsion system. N-isopropylacrylamide, N,N-dimethylacrylamide and acrylic acid were used as monomers with methylenebisacrylamide as cross-linker. Pentafluorophenyl methacrylate (PFM) was added and the nanoparticles were adorned with peptide 5A (B). An ELISA was designed to detect nanoparticles in biological fluids using the biotin and carboxyfluorescein groups present on peptide 5A (C). Streptavidin coated plates were used to capture the peptides by binding the biotin group and bound peptides were detected with a HRP conjugated mAb anti-fluorescein.

Mentions: The use of therapeutic peptides in neurodegenerative diseases is an active area of investigation. For example, the NAP peptide (NAPVSIPQ), derived from the activity-dependent neuroprotective protein (ADNP) [1], has shown efficacy in in vitro as well as in vivo models of neurodegenerative diseases [2, 3]. However, therapeutic peptides are known to have several limitations such as poor bioavailability, instability and short half-life [4]. A possible way to overcome these limitations is the use of nanoparticles as delivery method. Nanoparticles increase the bioavailability and efficacy of incorporated peptides by facilitating their transfer across biological membranes and protecting bound peptides against enzymatic degradation [5, 6]. Therapeutic nanoparticles are currently being developed for a wide variety of diseases such as cancer [7], cardiovascular disease [8–10] and neurodegenerative diseases [11, 12]. Although significant progress has been made towards organ-specific delivery of nanoparticles, a drawback is that they often do not reach their intended target tissue in the desired quantities due to filtering by the kidney, liver and spleen [7]. This can be improved by decorating the nanoparticles with functionalized targeting peptides that bind to receptors on the target tissue (Fig 1) [13]. Another challenge is the evaluation of the pharmacokinetics and biodistribution of the nanoparticles in vivo. In this respect LC-MS techniques can be used to measure peptides attached to nanoparticles. However, this approach requires specialized infrastructure. Therefore, it is useful to have reliable methods based on commonly used laboratory techniques, to be able to measure nanoparticles in biological fluids and tissues. In this manuscript we attached labeled reporter peptides to nanoparticles [6]. Subsequently, we used an ELISA-based method to detect the reporter peptide bound to the nanoparticles (Fig 1C), allowing quantification of these nanoparticles in blood plasma after injection in vivo. This ELISA enabled us to measure peptide bound to nanoparticles in blood plasma from 1 ng/mL. In parallel, LC/MS analysis of the same samples was performed to measure the plasma concentration over time of acrylamide based nanoparticles loaded with reporter peptides in rats.


Detection of Peptide-based nanoparticles in blood plasma by ELISA.

Bode GH, Pickl KE, Sanchez-Purrà M, Albaiges B, Borrós S, Pötgens AJ, Schmitz C, Sinner FM, Losen M, Steinbusch HW, Frank HG, Martinez-Martinez P, European NanoBioPharmaceutics Research Initiati - PLoS ONE (2015)

Schematic representation of the nanoparticle and ELISA design.Peptide 5A contains biotin and carboxyfluorescein, lower case letters denote D-amino acids. Nanoparticles were synthesized by a free-radical polymerization method in a microemulsion system. N-isopropylacrylamide, N,N-dimethylacrylamide and acrylic acid were used as monomers with methylenebisacrylamide as cross-linker. Pentafluorophenyl methacrylate (PFM) was added and the nanoparticles were adorned with peptide 5A (B). An ELISA was designed to detect nanoparticles in biological fluids using the biotin and carboxyfluorescein groups present on peptide 5A (C). Streptavidin coated plates were used to capture the peptides by binding the biotin group and bound peptides were detected with a HRP conjugated mAb anti-fluorescein.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4440766&req=5

pone.0126136.g001: Schematic representation of the nanoparticle and ELISA design.Peptide 5A contains biotin and carboxyfluorescein, lower case letters denote D-amino acids. Nanoparticles were synthesized by a free-radical polymerization method in a microemulsion system. N-isopropylacrylamide, N,N-dimethylacrylamide and acrylic acid were used as monomers with methylenebisacrylamide as cross-linker. Pentafluorophenyl methacrylate (PFM) was added and the nanoparticles were adorned with peptide 5A (B). An ELISA was designed to detect nanoparticles in biological fluids using the biotin and carboxyfluorescein groups present on peptide 5A (C). Streptavidin coated plates were used to capture the peptides by binding the biotin group and bound peptides were detected with a HRP conjugated mAb anti-fluorescein.
Mentions: The use of therapeutic peptides in neurodegenerative diseases is an active area of investigation. For example, the NAP peptide (NAPVSIPQ), derived from the activity-dependent neuroprotective protein (ADNP) [1], has shown efficacy in in vitro as well as in vivo models of neurodegenerative diseases [2, 3]. However, therapeutic peptides are known to have several limitations such as poor bioavailability, instability and short half-life [4]. A possible way to overcome these limitations is the use of nanoparticles as delivery method. Nanoparticles increase the bioavailability and efficacy of incorporated peptides by facilitating their transfer across biological membranes and protecting bound peptides against enzymatic degradation [5, 6]. Therapeutic nanoparticles are currently being developed for a wide variety of diseases such as cancer [7], cardiovascular disease [8–10] and neurodegenerative diseases [11, 12]. Although significant progress has been made towards organ-specific delivery of nanoparticles, a drawback is that they often do not reach their intended target tissue in the desired quantities due to filtering by the kidney, liver and spleen [7]. This can be improved by decorating the nanoparticles with functionalized targeting peptides that bind to receptors on the target tissue (Fig 1) [13]. Another challenge is the evaluation of the pharmacokinetics and biodistribution of the nanoparticles in vivo. In this respect LC-MS techniques can be used to measure peptides attached to nanoparticles. However, this approach requires specialized infrastructure. Therefore, it is useful to have reliable methods based on commonly used laboratory techniques, to be able to measure nanoparticles in biological fluids and tissues. In this manuscript we attached labeled reporter peptides to nanoparticles [6]. Subsequently, we used an ELISA-based method to detect the reporter peptide bound to the nanoparticles (Fig 1C), allowing quantification of these nanoparticles in blood plasma after injection in vivo. This ELISA enabled us to measure peptide bound to nanoparticles in blood plasma from 1 ng/mL. In parallel, LC/MS analysis of the same samples was performed to measure the plasma concentration over time of acrylamide based nanoparticles loaded with reporter peptides in rats.

Bottom Line: We were able to accurately measure peptides bound to pentafluorophenyl methacrylate nanoparticles in blood plasma of rats, and similar results were obtained by LC/MS.We detected FITC-labeled peptides on pentafluorophenyl methacrylate nanoparticles after injection in vivo.This method can be extended to detect nanoparticles with different chemical compositions.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, School for Mental Health and Neuroscience, Faculty of Health, Medicine and Life Sciences, Maastricht University, Maastricht, the Netherlands.

ABSTRACT

Aims: The aim of the current study was to develop a method to detect peptide-linked nanoparticles in blood plasma.

Materials & methods: A convenient enzyme linked immunosorbent assay (ELISA) was developed for the detection of peptides functionalized with biotin and fluorescein groups. As a proof of principle, polymerized pentafluorophenyl methacrylate nanoparticles linked to biotin-carboxyfluorescein labeled peptides were intravenously injected in Wistar rats. Serial blood plasma samples were analyzed by ELISA and by liquid chromatography mass spectrometry (LC/MS) technology.

Results: The ELISA based method for the detection of FITC labeled peptides had a detection limit of 1 ng/mL. We were able to accurately measure peptides bound to pentafluorophenyl methacrylate nanoparticles in blood plasma of rats, and similar results were obtained by LC/MS.

Conclusions: We detected FITC-labeled peptides on pentafluorophenyl methacrylate nanoparticles after injection in vivo. This method can be extended to detect nanoparticles with different chemical compositions.

No MeSH data available.