Cell penetrating peptides: a comparative transport analysis for 474 sequence motifs

Abstract Delivering reagents into cells is a key demand in molecular medicine. The vehicle of choice is often cell penetrating peptides (CPPs), which can ferry conjugated cargo across membranes. Although numerous peptides have been shown to promote such uptake events, there has been no comprehensive comparison of individual performance under standardized conditions. We have devised a method to rapidly analyze the ability of a multitude of CPP conjugates to carry a model cargo into HeLa cells. Sequence information for 474 CPPs was collected from literature sources, and the respective peptides were synthesized and modified with carboxyfluorescein (FAM) as model cargo. All candidates were evaluated in an identical uptake test, and transport was quantified using cellular fluorescence intensities. Substantial differences in the ability to carry the fluorophore into the cells were observed, with transport performance differing by a factor of 70 between the best CPP investigated and cargo alone. Strong correlations were observed between uptake efficiency and both sequence length and the presence of positive net charge. A compilation of the 20 top performers with regard to cargo delivery performance and cell compatibility is provided.

[1]  B. Wiesner,et al.  Structural requirements for cellular uptake of α‐helical amphipathic peptides , 1999 .

[2]  Sergio Grinstein,et al.  Sensors and regulators of intracellular pH , 2010, Nature Reviews Molecular Cell Biology.

[3]  P. Iversen,et al.  Pharmacokinetics, biodistribution, stability and toxicity of a cell-penetrating peptide-morpholino oligomer conjugate. , 2007, Bioconjugate chemistry.

[4]  Igor L. Medintz,et al.  Delivering quantum dot-peptide bioconjugates to the cellular cytosol: escaping from the endolysosomal system. , 2010, Integrative biology : quantitative biosciences from nano to macro.

[5]  B. Wiesner,et al.  Structural requirements for cellular uptake of alpha-helical amphipathic peptides. , 1999, Journal of Peptide Science.

[6]  G. Elliott,et al.  Intercellular Trafficking and Protein Delivery by a Herpesvirus Structural Protein , 1997, Cell.

[7]  K. Pattabiraman,et al.  The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[8]  K. Peremans,et al.  Chemical-Functional Diversity in Cell-Penetrating Peptides , 2013, PloS one.

[9]  G. Divita,et al.  Interactions of amphipathic CPPs with model membranes. , 2011, Methods in molecular biology.

[10]  U. Haberkorn,et al.  The pharmacokinetics of cell-penetrating peptides. , 2010, Molecular pharmaceutics.

[11]  William H. Sawyer,et al.  Spectral properties of the prototropic forms of fluorescein in aqueous solution , 1996, Journal of Fluorescence.

[12]  T. Bártfai,et al.  VE-cadherin-derived cell-penetrating peptide, pVEC, with carrier functions. , 2001, Experimental cell research.

[13]  Jean-Luc Coll,et al.  Targeting cyclin B1 through peptide-based delivery of siRNA prevents tumour growth , 2009, Nucleic acids research.

[14]  Maurice Green,et al.  Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein , 1988, Cell.

[15]  Isabel D. Alves,et al.  Translocation and Endocytosis for Cell-penetrating Peptide Internalization , 2009, The Journal of Biological Chemistry.

[16]  Hans H Gorris,et al.  Synthesis of a fluorescent ganglioside GM1 derivative and screening of a synthetic peptide library for GM1 binding sequence motifs. , 2007, Bioconjugate chemistry.

[17]  M. C. Cardoso,et al.  Cell Entry of Arginine-rich Peptides Is Independent of Endocytosis*S⃞ , 2009, Journal of Biological Chemistry.

[18]  A. Graessmann,et al.  Targeting of nonkaryophilic cell-permeable peptides into the nuclei of intact cells by covalently attached nuclear localization signals. , 2002, Biochemistry.

[19]  S. Futaki,et al.  Arginine-rich Peptides , 2001, The Journal of Biological Chemistry.

[20]  P. Boisguérin,et al.  Comparison of cellular uptake using 22 CPPs in 4 different cell lines. , 2008, Bioconjugate chemistry.

[21]  Rujing Zhang,et al.  The effect of side-chain functionality and hydrophobicity on the gene delivery capabilities of cationic helical polypeptides. , 2014, Biomaterials.

[22]  M. Pooga,et al.  Prediction of Cell-Penetrating Peptides , 2005, International Journal of Peptide Research and Therapeutics.

[23]  S. Futaki,et al.  Delivery of Macromolecules Using Arginine-Rich Cell-Penetrating Peptides: Ways to Overcome Endosomal Entrapment , 2009, The AAPS Journal.

[24]  E. Racker,et al.  Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. , 1979, Biochemistry.

[25]  K. R. Woods,et al.  Prediction of protein antigenic determinants from amino acid sequences. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[26]  T. Mosmann.  Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. , 1983, Journal of immunological methods.

[27]  Gajendra P. S. Raghava,et al.  CPPsite: a curated database of cell penetrating peptides , 2012, Database J. Biol. Databases Curation.

[28]  S. Sagan,et al.  Tracking a new cell-penetrating (W/R) nonapeptide, through an enzyme-stable mass spectrometry reporter tag. , 2007, Analytical chemistry.

[29]  Ülo Langel,et al.  Design of a Tumor-Homing Cell-Penetrating Peptide , 2008 .

[30]  F. Milletti,et al.  Cell-penetrating peptides: classes, origin, and current landscape. , 2012, Drug discovery today.

[31]  C. Flynn,et al.  A novel cell permeant peptide inhibitor of MAPKAP kinase II inhibits intimal hyperplasia in a human saphenous vein organ culture model. , 2010, Journal of vascular surgery.

[32]  S. Sagan,et al.  Cell‐penetrating peptides: 20 years later, where do we stand? , 2013, FEBS letters.

[33]  Astrid Gräslund,et al.  Mechanisms of Cellular Uptake of Cell-Penetrating Peptides , 2011, Journal of biophysics.