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Cell-penetrating Peptide Synthesis
The process of introducing drugs into cells has always proved a major challenge for scientists. However, cell-penetrating peptides (CPPs) have the ability to enter a cell's plasma membrane independent of a membrane receptor. They are usually small peptides at 10–30 residues in length. The sequences of amino acids are often positively charged.
Tat, the transcription activator of the human immunodeficiency virus type 1 (HIV-1) viral genome was shown to enter cells in a non-toxic and highly efficient manner. Tat became known as the first cell-penetrating peptide.
CPPs have demonstrated themselves to be capable of delivering biologically active cargo to the cell interior. Attached to a CPP, therapeutic cargo could be delivered to an intracellular target, thus overcoming the entry restrictions set by the plasma membrane.
The cell-penetrating peptides have gained a widespread popularity as very promising nonviral transmembrane delivery vectors. Although CPPs have been successfully used for carrying different cargoes that might vary in size and nature (plasmid DNA, peptides, proteins, nanoparticles, quantum dots, etc.), the most rapid progress has been made in the delivery of oligonucleotides (ONs).
- Modifications
- Models
- CPP Examples
- Case Studies
CPPs Peptide Synthesis Modifications:
CPP-based cellular delivery vectors have enabled introduction of a noncovalent strategy for association of ONs with carrier by simple mixing. However the peptide sequence needs to be carefully tuned to the tupe of cargo as well as to the application. The Stearylated poly-arginine peptide was found to be effective with a larger plasmid. However it was ineffective in cellular delivery of a splice-correcting oligonucleotide.
Simple mixing method:
Example: For MPG mediated gene delivery, peptide carrier/DNA complexes were formed in DMEM or phosphate-buffered saline (500 ml of DMEM containing 100 ng of DNA complexed with MPG at a charge ratio of 5:1) and incubated for 30 min at 37°C. Cells grown to 60% confluence were then overlaid with these preformed complexes.
Click this review paper from Nature to read more about the strategies for peptide-mediated delivery.
CPPs pack oligonucleotides into nanocomplexes, which do not dissociate in the presence of serum proteins and transfect cells more efficiently than commercially available cationic lipids.
Many novel CPPs have been shown to transfect and trigger specific biological response in a wide range of cell types, including challenging primary and
suspension cells without being cytotoxic. In vivo experiments have revealed the capacity of CPP-mediated ONs to effectively inhibit tumor growth in tumor-bearing mice without triggering immune response, emphasizing even more the high therapeutic potential of CPP–ON complexes.
Table: Application and effects of CPP-mediated siRNA delivery by using noncovalent strategy
CPP |
Cells/Model |
Effect |
Reference |
MPG
ac-GALFLGFLGAAGSTMGAWSQPKKKRKV-cys
The cysteamide stabilises the carrier/DNA particle, as similarly reported for gene delivery mediated by cysteine-containing peptides. |
HeLa and Cos-7
HS68, NIH-3T3 |
Cellular uptake of single- and double-stranded ONs
50 nmol/l siRNA→85% and 78% luciferase
downregulation |
Simeoni et al. |
|
HS68 |
100 nmol/l siRNA→60% GAPDH downregulation |
Simeoni et al. |
MPGΔNLS
ac-GALFLGFLGAAGSTMGAWSQPKSKRKV-cys |
HeLa and Cos-7 |
50 nmol/l siRNA→95% and 90% luciferase
downregulation |
Simeoni et al. |
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Stearyl-R8
st-RRRRRRRR-NH2
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FITC-Stearyl-R8 Control peptide
Polyarginine peptides, which are very efficient CPPs have been used for siRNA delivery into cells.
Stearylated octaarginine (Stearyl-R8), are able to encapsulate and shuttle nucleic acids through cell membranes without the need to covalently attach the nucleic acid to the arginine-containing carrier peptide. |
EGFP-expressing
hippocampal neurons |
16 nmol/l siRNA→>50% reduction in EGFP activity |
Tönges et al. |
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EB1
LIKLWSHLIHIWFQNRRLKWKKK-amide |
HeLa, HepG2 |
Luciferase downregulation |
Lundberg et al. |
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Tat-DRBD
GRKKRRQRRRPQ-DRBD |
H1299, HUVEC, Jurkat T,
hESC |
>90% reduction in GAPDH mRNA level |
Eguchi et al. |
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PF6
St-AGYLLGK[kk2sa4qn4]INLKALAALAKKIL-NH2 |
Hepatoma, MEF,
HUVEC, mESC |
Significant downregulation of HPRT1 |
El-Andaloussi
et al. |
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There are three proposed routes of CPP entry:
- Model 1: The inverted micelle model.
- Model 2: The direct penetration (pore formation) mechanism.
- Model 3: An endocytic mechanism of uptake. Source: Cell-penetrating peptides and their therapeutic applications, Victoria Sebbage, BioscienceHorizons, Volume 2, Number 1, March 2009.
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Arg-rich or Lys-rich CPPs have been widely used for gene targeting. CPPs, like stearly-RRRRRRRR-amide can be spaced by aminohexanoyl (Ahx or X) and/or β-alanyl (β) moieties to enhance hydrophobicity and proteolytic resistance in serum, such as (R-Ahx-R)4-Ahx-βAla and (R-Ahx-R-R-βAla-R)2-Ahx-βAla called RXR4 and B-peptide respectively. The aminohexanoyl spacer in the R-Ahx-R motif was found to be optimal in the splicing-redirection assay.
Another strategy is to add a second chemical moiety such as a fatty acid to the end of an Arg-rich peptide to enhance delivery of the peptide nucleic acid conjugate.
For example, the addition of a C14 palmitoyl lipid chain to the free terminus of the PNA in a R9-PNA conjugate also increased splice-redirecting activity. Click this review paper from Nature to read more about the strategies for peptide-mediated delivery.
Since the discovery of Tat, the number of known peptides with cell-penetrating capabilities has grown. The following table shows a selection of currently known CPPs, their origins and sequences.
Name |
Origin |
Sequence |
Tat family |
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Tat (48-60) |
HIV-1 protein |
GRKKRRQRRRPPQQ |
Oligoarginine |
Tat derivative |
Rn |
Penetralia family |
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p-Antp |
Antermapedia homeodomain |
RQIKIWFQNRRMKWKK |
plsl |
Igl-1 homeodomain |
RVIRVWFQNKRCKDKK |
Chimeric CPPs |
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Transportan |
Galanin-mastoparan |
GWTLNSAGYLLGKINLKALAALAKKIL |
MPG peptides |
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P-beta |
gp41-SV40 |
GALFLGFLGAAGSTMGAWSQPKKKRKV |
P-alpha |
gp41-SV40 |
GALFLAFLAAALSLMGLWSQPKKKRRV |
Pep-1 |
Trp-rich motif-SV40 |
KETWWETWWTEWSQPKKKRRV |
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| STR-Cpps |
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STR-R8 |
Stearly-RRRRRRRR-amide
(Stearyl = CH3(CH2)16CO-)
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FITC-Stearyl-R8 Control peptide
The Stearylated poly-arginine peptide was found to be effective with a larger plasmid. However it was ineffective in cellular delivery of a splice-correcting oligonucleotide. |
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STR-(RxR)4 |
Stearly-(RxR)4-amide
(X=6-aminohexanoic acid) |
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STR-TP10 |
Stearly-AGYLLGKINLKALAALAKKIL-amide |
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PF14 |
Stearly-AGYLLGKLLOOLLAAAALOOLL-amide (O=ornithine) |
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NickFect1 |
Stearly-AGY(PO3)LLGKTNLKALAALAKKIL-amide |
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HIV TAT cell-penetrating peptides
Cell-penetrating peptides such as the HIV TAT peptides are able to enter cell by direct translocation and endocytosis. HIV TAT or even simple poly-arginines can be effectively designed for drug delivery. However how cell-penetrating peptides, HIV TAT peptide for example, accomplish these cellular molecular transfer has so far been a mystery.
How does simple HIV TAT peptide facilitate mechanisms like direct translocation and multiple endocytotic processes? Researchers from Gerard Wong’s lab found how HIV TAT peptides can have multiple interactions with the cell membrane, the actin cytoskeleton and specific cell-surface receptors to produce multiple pathways of translocation under different conditions. Click here for the publication from Gerard Wong’s lab: http://bit.ly/zQrH6t.
Interestingly, TAT peptide can multiplex different interactions with the same sequence, thus interacting with the membrane, the actin cytoskeleton, and specific receptors to produce multiple pathways of translocation under different conditions.
CPPs entry mechanism is sensitive to the peptide sequence. The addition of a single hydrophobic residue to purely hydrophilic CPPs can drastically modify the translocation mechanism. For example, polyarginine (polyR), the simplest prototypical CPP, can induce the cell membrane pore formation. Hydrophobic amino acids create positive curvature by inserting into the membrane. Arginine simultaneously creates positive and negative curvatures, whereas lysine creates negative curvature along one direction only. This implies a compensatory relation between arginines and lysines/hydrophobes.
Why is the hydrophobic content of the TAT peptide relatively low if hydrophobicity can help generate negative Gaussian curvature? CPPs use less hydrophobic residues to generate saddle-splay curvature. This difference in sequences can potentially only induce transient pore-like translocation structures in the membrane and thus lead to shorter pore lifetimes for CPPs. Because of the amino acid composition for CPPs, the TAT peptide can mediate endocytosis with or without receptors. |
References:
Simeoni, F, Morris, MC, Heitz, F and Divita, G (2003). Insight into the mechanism of the peptide-based gene delivery system MPG: implications for delivery of siRNA into mammalian cells. Nucleic Acids Res 31: 2717–2724.
Tönges, L, Lingor, P, Egle, R, Dietz, GP, Fahr, A and Bähr, M (2006). Stearylated
octaarginine and artificial virus-like particles for transfection of siRNA into primary rat
neurons. RNA 12: 1431–1438.
Morris, MC, Vidal, P, Chaloin, L, Heitz, F and Divita, G (1997). A new peptide vector
for efficient delivery of oligonucleotides into mammalian cells. Nucleic Acids Res 25:
2730–2736.
Andaloussi, SE, Lehto, T, Mäger, I, Rosenthal-Aizman, K, Oprea, II, Simonson, OE et al.
(2011). Design of a peptide-based vector, PepFect6, for efficient delivery of siRNA in
cell culture and systemically in vivo. Nucleic Acids Res 39: 3972–3987.
Eguchi, A, Meade, BR, Chang, YC, Fredrickson, CT, Willert, K, Puri, N et al. (2009).
Efficient siRNA delivery into primary cells by a peptide transduction domain-dsRNA
binding domain fusion protein. Nat Biotechnol 27: 567–571.
Lundberg, P, El-Andaloussi, S, Sütlü, T, Johansson, H and Langel, Ű (2007). Delivery
of short interfering RNA using endosomolytic cell-penetrating peptides. FASEB J 21:
2664–2671.
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