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Interaction between cell-penetrating peptides and acid-sensitive anionic oligopeptides as a model for the design of targeted drug carriers.

Sun C, Shen WC, Tu J, Zaro JL - Mol. Pharm. (2014)

Bottom Line: YGR6G6 with clustered arginine residues exhibited greater pH sensitivity in cellular uptake than YG(RG)6 with separated arginine residues.This β-sheet conformation presumably stabilized the association of (HE)10 with YG(RG)6, leading to weakened pH sensitivity of (HE)10-YG(RG)6.The data presented in this study provide a basis for the future design of pH-sensitive HE-CPP carrier for targeted drug delivery.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Natural Medicines, Department of Pharmaceutics, School of Pharmacy, China Pharmaceutical University , 24 Tong Jia Xiang, Nanjing 210009, China.

ABSTRACT
Overcoming the nonspecific cellular uptake of cell-penetrating peptides (CPPs) is a major hurdle in their clinical application. Using pH as the activation switch, histidine-glutamic acid (HE) dipeptide repeats were fused to CPPs to trigger the membrane-penetrating activity at mildly acidic pH environments (i.e., pH 6.5 or below) while masking the internalization at neutral pH (i.e., pH 7.0 or above). In this study, a series of recombinant GST-fusion proteins containing an HE oligopeptide sequence (i.e., (HE)n with n = 8, 10, or 12) and a cationic CPP (i.e., YG(RG)6, YGR6G6, or Tat) were engineered for a pH-sensitive study comparing their cellular uptake and surface binding in cultured HeLa cells. Circular dichroism (CD) spectroscopy was performed to correlate differences between CPPs in secondary structure with the pH sensitivity. YGR6G6 with clustered arginine residues exhibited greater pH sensitivity in cellular uptake than YG(RG)6 with separated arginine residues. Increasing the stretch of HE repeats decreased cellular uptake and surface binding for both YG(RG)6 and YGR6G6. The ratio of cellular internalization at pH 7.5 vs 6.0 was not changed by the presence of serum. CD spectral data revealed that both (HE)10-Tat and (HE)10-YGR6G6 exhibited an unordered secondary structure, whereas (HE)10-YG(RG)6 adopted an antiparallel β-sheet conformation. This β-sheet conformation presumably stabilized the association of (HE)10 with YG(RG)6, leading to weakened pH sensitivity of (HE)10-YG(RG)6. On the other hand, the random-coiled structures, that is, (HE)10-YGR6G6 and (HE)10-Tat, both showed higher pH sensitivity as determined in cell experiments. The data presented in this study provide a basis for the future design of pH-sensitive HE-CPP carrier for targeted drug delivery.

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Comparison of cell uptake (A) and surface binding (B) between GST-(HE)10-YGR6G6 (open circle) and GST-(HE)12-Tat (closed diamond) in HeLa cells at diverse pH. Upon confluence,HeLa cell monolayers were incubated with 5 μg/mL 125I-GST-(HE)10-YGR6G6 and 125I-GST-(HE)12-Tat for 1h at 37 °C at pH 6.0, 6.5,7.0, and 7.5. Cell uptake and surface binding were determined as describedin the Experimental Section. All data werenormalized by total cell protein, and presented as averages ±standard deviation from three independent measurements per condition.
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fig4: Comparison of cell uptake (A) and surface binding (B) between GST-(HE)10-YGR6G6 (open circle) and GST-(HE)12-Tat (closed diamond) in HeLa cells at diverse pH. Upon confluence,HeLa cell monolayers were incubated with 5 μg/mL 125I-GST-(HE)10-YGR6G6 and 125I-GST-(HE)12-Tat for 1h at 37 °C at pH 6.0, 6.5,7.0, and 7.5. Cell uptake and surface binding were determined as describedin the Experimental Section. All data werenormalized by total cell protein, and presented as averages ±standard deviation from three independent measurements per condition.

Mentions: Cell-penetratingTat[47–57] peptide is one of the most extensively studied CPPs.The cell internalization and binding of GST-(HE)12-Tatwas compared to the blocked oligoarginine construct at a ∼1:1.5ratio of cationic amino acids in the CPP sequence (R, K) to numberof (HE) repeats (i.e., GST-(HE)12-Tat vs GST-(HE)10-YGR6G6). In the cell experiments (Figure 4), GST-(HE)12-Tat exhibited a strongerpH sensitivity in both cell uptake (4.4-fold increase at pH 6.0 versuspH 7.5) and surface binding (2.0-fold increase at pH 6.0 versus pH7.5). Therefore, the pH sensitivity was comparable to the argininein a clustered configuration, which is consistent with the Tat peptidesequence.


Interaction between cell-penetrating peptides and acid-sensitive anionic oligopeptides as a model for the design of targeted drug carriers.

Sun C, Shen WC, Tu J, Zaro JL - Mol. Pharm. (2014)

Comparison of cell uptake (A) and surface binding (B) between GST-(HE)10-YGR6G6 (open circle) and GST-(HE)12-Tat (closed diamond) in HeLa cells at diverse pH. Upon confluence,HeLa cell monolayers were incubated with 5 μg/mL 125I-GST-(HE)10-YGR6G6 and 125I-GST-(HE)12-Tat for 1h at 37 °C at pH 6.0, 6.5,7.0, and 7.5. Cell uptake and surface binding were determined as describedin the Experimental Section. All data werenormalized by total cell protein, and presented as averages ±standard deviation from three independent measurements per condition.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4012841&req=5

fig4: Comparison of cell uptake (A) and surface binding (B) between GST-(HE)10-YGR6G6 (open circle) and GST-(HE)12-Tat (closed diamond) in HeLa cells at diverse pH. Upon confluence,HeLa cell monolayers were incubated with 5 μg/mL 125I-GST-(HE)10-YGR6G6 and 125I-GST-(HE)12-Tat for 1h at 37 °C at pH 6.0, 6.5,7.0, and 7.5. Cell uptake and surface binding were determined as describedin the Experimental Section. All data werenormalized by total cell protein, and presented as averages ±standard deviation from three independent measurements per condition.
Mentions: Cell-penetratingTat[47–57] peptide is one of the most extensively studied CPPs.The cell internalization and binding of GST-(HE)12-Tatwas compared to the blocked oligoarginine construct at a ∼1:1.5ratio of cationic amino acids in the CPP sequence (R, K) to numberof (HE) repeats (i.e., GST-(HE)12-Tat vs GST-(HE)10-YGR6G6). In the cell experiments (Figure 4), GST-(HE)12-Tat exhibited a strongerpH sensitivity in both cell uptake (4.4-fold increase at pH 6.0 versuspH 7.5) and surface binding (2.0-fold increase at pH 6.0 versus pH7.5). Therefore, the pH sensitivity was comparable to the argininein a clustered configuration, which is consistent with the Tat peptidesequence.

Bottom Line: YGR6G6 with clustered arginine residues exhibited greater pH sensitivity in cellular uptake than YG(RG)6 with separated arginine residues.This β-sheet conformation presumably stabilized the association of (HE)10 with YG(RG)6, leading to weakened pH sensitivity of (HE)10-YG(RG)6.The data presented in this study provide a basis for the future design of pH-sensitive HE-CPP carrier for targeted drug delivery.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Natural Medicines, Department of Pharmaceutics, School of Pharmacy, China Pharmaceutical University , 24 Tong Jia Xiang, Nanjing 210009, China.

ABSTRACT
Overcoming the nonspecific cellular uptake of cell-penetrating peptides (CPPs) is a major hurdle in their clinical application. Using pH as the activation switch, histidine-glutamic acid (HE) dipeptide repeats were fused to CPPs to trigger the membrane-penetrating activity at mildly acidic pH environments (i.e., pH 6.5 or below) while masking the internalization at neutral pH (i.e., pH 7.0 or above). In this study, a series of recombinant GST-fusion proteins containing an HE oligopeptide sequence (i.e., (HE)n with n = 8, 10, or 12) and a cationic CPP (i.e., YG(RG)6, YGR6G6, or Tat) were engineered for a pH-sensitive study comparing their cellular uptake and surface binding in cultured HeLa cells. Circular dichroism (CD) spectroscopy was performed to correlate differences between CPPs in secondary structure with the pH sensitivity. YGR6G6 with clustered arginine residues exhibited greater pH sensitivity in cellular uptake than YG(RG)6 with separated arginine residues. Increasing the stretch of HE repeats decreased cellular uptake and surface binding for both YG(RG)6 and YGR6G6. The ratio of cellular internalization at pH 7.5 vs 6.0 was not changed by the presence of serum. CD spectral data revealed that both (HE)10-Tat and (HE)10-YGR6G6 exhibited an unordered secondary structure, whereas (HE)10-YG(RG)6 adopted an antiparallel β-sheet conformation. This β-sheet conformation presumably stabilized the association of (HE)10 with YG(RG)6, leading to weakened pH sensitivity of (HE)10-YG(RG)6. On the other hand, the random-coiled structures, that is, (HE)10-YGR6G6 and (HE)10-Tat, both showed higher pH sensitivity as determined in cell experiments. The data presented in this study provide a basis for the future design of pH-sensitive HE-CPP carrier for targeted drug delivery.

Show MeSH
Related in: MedlinePlus