CPPs (for Cell Penetrating Peptide) are 15-aminoacid-long molecules that WRAP'n ROLL around nucleic acids to form nanoparticles. Those nanoparticles can easily penetrate the cells and deliver their precious genetic construct or SiRNA.
A few years ago... How I met CPP (and its publication)
When I was an intern within the public/private joint unit Sys2DIAG I met with Gudrun, a peptide chemist with plenty of promising projects. Among them were the CPP's : very simple peptides ( less than 15 amino acids mostly leucins, triptophans and arginins) yet with an impressive potential as vector molecules.The team behind CPPs has made a recent paper on this technology which you can read here :
Peptide-Based Nanoparticles to Rapidly and Efficiently “Wrap ’n Roll” siRNA into Cells Bioconjugate Chem. 2019, 30, 592−603
For a quick overview, allow me now to introduce you to those tiny peptidic vectors!
First, why are CPPs important?
When I study a research tool I am always wondering: what challenge is awaking these scientists every morning? What unmet need drives them to innovate?
In the case of CPPs, the challenge is: breaking into the cell unnoticed!
Entering the cell and delivering a cargo is a central aspect in plenty of biotechnologies, say for example: RNA intefererence, Plasmid Transfection, Crisp-Cas9 gene editing, the COVID-19 vaccine (!). A few techniques exist to achieve that (see on the left).
Ideally, you want your entry method to be cheap, efficient while also being minimally disruptive for the cells. Easier said than done...
Calcium phospate and electroporation are simple methods, yet can only be applied in vitro. Lipofectamine and viral methods are compatible with in-vivo models but are trickier to produce and handle.
CPPs are interesting because they are easy to synthesize, quite stable in solution and they can transfect many cell types with little toxicity.
Being peptides, CPPs are also an open platform for functionnalization to increase stability or specificity.
How do they work?
CPPs work in a 3 step process: 1. Assembly into nanoparticles 2. Internalization and 3. Delivery.
1. Nanoparticle Assembly
Thanks to their structure and amino-acids, cell penetrating peptides bind the nucleic acids in non-covalent fashion. They then aggregate into small (100nm) nanoparticles which can be observed using Scanning Electron Microscopy.
When mixed in the presence of cells, the nanoparticules enter the cell's membrane and go inside.
This process is spontaneous due to the charges of the CPP.
3. Gene KnockDown with SiRNA's
Once inside the cell, the cargo SiRNA is released and quickly shuts down gene expression as seen in the graphs above. 20nM SiRNA-anti-
CDK4 in Wrap peptide were incubated for 5 min up to 60 min and CDK4 expression was recorded showing a neat decrease in gene expression even after as few as 5 minutes.
WRAPing up my postI hope that you enjoyed the ride in the world of CPPs and that it helped raise your awareness on transfection techniques that you may not know. Personnaly I enjoyed seeing how those molecules are still in use today and provide a simple and efficient RNA interference modality
I recently asked Gudrun if she’d like to spread the CPP technology to other laboratories. She met with the rest of the team and... I'll tell you next time!