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Efficient Cargo Delivery into Adult Brain Tissue Using Short Cell-Penetrating Peptides.

Kizil C, Iltzsche A, Thomas AK, Bhattarai P, Zhang Y, Brand M - PLoS ONE (2015)

Bottom Line: Therefore, functional analyses of gene function in RGCs and neurons are essential.We also found that polyR peptide can help carry plasmid DNA several cell diameters into the brain tissue after a series of coupling reactions using DBCO-PEG4-maleimide-based Michael's addition and azide-mediated copper-free click reaction.Combined with the advantages of CVMI, such as rapidness, reproducibility, and ability to be used in adult animals, CPPs improve the applicability of the CVMI technique to deeper parts of the central nervous system tissues.

View Article: PubMed Central - PubMed

Affiliation: German Centre for Neurodegenerative Diseases (DZNE) Dresden within the Helmholtz Association, Arnoldstr. 18, 01307, Dresden, Germany; DFG-Center for Regenerative Therapies Dresden (CRTD) - Cluster of Excellence, Technische Universität Dresden, Fetscherstr. 105, 01307, Dresden, Germany.

ABSTRACT
Zebrafish brains can regenerate lost neurons upon neurogenic activity of the radial glial progenitor cells (RGCs) that reside at the ventricular region. Understanding the molecular events underlying this ability is of great interest for translational studies of regenerative medicine. Therefore, functional analyses of gene function in RGCs and neurons are essential. Using cerebroventricular microinjection (CVMI), RGCs can be targeted efficiently but the penetration capacity of the injected molecules reduces dramatically in deeper parts of the brain tissue, such as the parenchymal regions that contain the neurons. In this report, we tested the penetration efficiency of five known cell-penetrating peptides (CPPs) and identified two- polyR and Trans - that efficiently penetrate the brain tissue without overt toxicity in a dose-dependent manner as determined by TUNEL staining and L-Plastin immunohistochemistry. We also found that polyR peptide can help carry plasmid DNA several cell diameters into the brain tissue after a series of coupling reactions using DBCO-PEG4-maleimide-based Michael's addition and azide-mediated copper-free click reaction. Combined with the advantages of CVMI, such as rapidness, reproducibility, and ability to be used in adult animals, CPPs improve the applicability of the CVMI technique to deeper parts of the central nervous system tissues.

No MeSH data available.


Related in: MedlinePlus

Overview of Peptide Translocation in the Telencephalon upon 100 μM Injection, and Dose-dependence.(A) Reaction scheme for the coupling of 5(6)-carboxyfluorescein to peptides. Fluorescein immunohistochemistry (IHC) on rostral telencephalon of peptide (B) control PolyD, (C) PolyR, (D) Antp, (E) Tat 49–57, (F) Trans and (G) CendRP injected brains. Graphs depict the average area of peptide translocation in one telencephalic hemisphere, upon 100 μM dose injection (H) and upon 200 μM dose injection of CPPs (I). Scale bars: 200 μm, n = 3, data are mean ± s.e.m.
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pone.0124073.g001: Overview of Peptide Translocation in the Telencephalon upon 100 μM Injection, and Dose-dependence.(A) Reaction scheme for the coupling of 5(6)-carboxyfluorescein to peptides. Fluorescein immunohistochemistry (IHC) on rostral telencephalon of peptide (B) control PolyD, (C) PolyR, (D) Antp, (E) Tat 49–57, (F) Trans and (G) CendRP injected brains. Graphs depict the average area of peptide translocation in one telencephalic hemisphere, upon 100 μM dose injection (H) and upon 200 μM dose injection of CPPs (I). Scale bars: 200 μm, n = 3, data are mean ± s.e.m.

Mentions: In order to identify peptides that successfully enter deep into the brain tissue, we synthesized five cell penetrating peptides using standard Fmoc solid-phase peptide synthesis chemistry [23–25] (Table 1). Poly-Aspartic Acid (PolyD) was designed as an anionic control peptide that would not penetrate the tissue in contrast to the cationic amino acid residues, which are known to be crucial for the peptide trafficking [26]. Negatively charged compounds are often very difficult to penetrate cell membrane. For example, it is notoriously difficult to deliver phosphorylated peptide or oligonucleotides into cells. The poly-D possesses many negative charges, which prevents its interaction with phospholipid membrane. This concept has been used to prevent drug from entering into cells [27] or to carry out chemical reaction outside cells [28]. Therefore, we used polyD as a control peptide. Poly-Arginine (PolyR) [11], Antennapedia (Antp) [11,29], basic nuclear localization sequence containing region of the transcriptional activator region of the HIV Tat protein 49–57 (Tat(49–57)) [14,17,30,31], Transportan (Trans) [11,32] and C-end rule (CendR) peptide (CendRP) [33,34] were selected based on previous in vivo and in vitro translocation success. To allow the detection of peptides in the tissue, 5(6)-carboxyfluorescein was coupled to the N-terminus of these peptides. (Fig 1A).


Efficient Cargo Delivery into Adult Brain Tissue Using Short Cell-Penetrating Peptides.

Kizil C, Iltzsche A, Thomas AK, Bhattarai P, Zhang Y, Brand M - PLoS ONE (2015)

Overview of Peptide Translocation in the Telencephalon upon 100 μM Injection, and Dose-dependence.(A) Reaction scheme for the coupling of 5(6)-carboxyfluorescein to peptides. Fluorescein immunohistochemistry (IHC) on rostral telencephalon of peptide (B) control PolyD, (C) PolyR, (D) Antp, (E) Tat 49–57, (F) Trans and (G) CendRP injected brains. Graphs depict the average area of peptide translocation in one telencephalic hemisphere, upon 100 μM dose injection (H) and upon 200 μM dose injection of CPPs (I). Scale bars: 200 μm, n = 3, data are mean ± s.e.m.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4403811&req=5

pone.0124073.g001: Overview of Peptide Translocation in the Telencephalon upon 100 μM Injection, and Dose-dependence.(A) Reaction scheme for the coupling of 5(6)-carboxyfluorescein to peptides. Fluorescein immunohistochemistry (IHC) on rostral telencephalon of peptide (B) control PolyD, (C) PolyR, (D) Antp, (E) Tat 49–57, (F) Trans and (G) CendRP injected brains. Graphs depict the average area of peptide translocation in one telencephalic hemisphere, upon 100 μM dose injection (H) and upon 200 μM dose injection of CPPs (I). Scale bars: 200 μm, n = 3, data are mean ± s.e.m.
Mentions: In order to identify peptides that successfully enter deep into the brain tissue, we synthesized five cell penetrating peptides using standard Fmoc solid-phase peptide synthesis chemistry [23–25] (Table 1). Poly-Aspartic Acid (PolyD) was designed as an anionic control peptide that would not penetrate the tissue in contrast to the cationic amino acid residues, which are known to be crucial for the peptide trafficking [26]. Negatively charged compounds are often very difficult to penetrate cell membrane. For example, it is notoriously difficult to deliver phosphorylated peptide or oligonucleotides into cells. The poly-D possesses many negative charges, which prevents its interaction with phospholipid membrane. This concept has been used to prevent drug from entering into cells [27] or to carry out chemical reaction outside cells [28]. Therefore, we used polyD as a control peptide. Poly-Arginine (PolyR) [11], Antennapedia (Antp) [11,29], basic nuclear localization sequence containing region of the transcriptional activator region of the HIV Tat protein 49–57 (Tat(49–57)) [14,17,30,31], Transportan (Trans) [11,32] and C-end rule (CendR) peptide (CendRP) [33,34] were selected based on previous in vivo and in vitro translocation success. To allow the detection of peptides in the tissue, 5(6)-carboxyfluorescein was coupled to the N-terminus of these peptides. (Fig 1A).

Bottom Line: Therefore, functional analyses of gene function in RGCs and neurons are essential.We also found that polyR peptide can help carry plasmid DNA several cell diameters into the brain tissue after a series of coupling reactions using DBCO-PEG4-maleimide-based Michael's addition and azide-mediated copper-free click reaction.Combined with the advantages of CVMI, such as rapidness, reproducibility, and ability to be used in adult animals, CPPs improve the applicability of the CVMI technique to deeper parts of the central nervous system tissues.

View Article: PubMed Central - PubMed

Affiliation: German Centre for Neurodegenerative Diseases (DZNE) Dresden within the Helmholtz Association, Arnoldstr. 18, 01307, Dresden, Germany; DFG-Center for Regenerative Therapies Dresden (CRTD) - Cluster of Excellence, Technische Universität Dresden, Fetscherstr. 105, 01307, Dresden, Germany.

ABSTRACT
Zebrafish brains can regenerate lost neurons upon neurogenic activity of the radial glial progenitor cells (RGCs) that reside at the ventricular region. Understanding the molecular events underlying this ability is of great interest for translational studies of regenerative medicine. Therefore, functional analyses of gene function in RGCs and neurons are essential. Using cerebroventricular microinjection (CVMI), RGCs can be targeted efficiently but the penetration capacity of the injected molecules reduces dramatically in deeper parts of the brain tissue, such as the parenchymal regions that contain the neurons. In this report, we tested the penetration efficiency of five known cell-penetrating peptides (CPPs) and identified two- polyR and Trans - that efficiently penetrate the brain tissue without overt toxicity in a dose-dependent manner as determined by TUNEL staining and L-Plastin immunohistochemistry. We also found that polyR peptide can help carry plasmid DNA several cell diameters into the brain tissue after a series of coupling reactions using DBCO-PEG4-maleimide-based Michael's addition and azide-mediated copper-free click reaction. Combined with the advantages of CVMI, such as rapidness, reproducibility, and ability to be used in adult animals, CPPs improve the applicability of the CVMI technique to deeper parts of the central nervous system tissues.

No MeSH data available.


Related in: MedlinePlus