How to Design Cell Penetrating Peptides?

Please view this video on how to design cell penetrating peptides. The transcript is listed below.


Slide 1:

Thank you for joining me. My topic today is the cell-penetrating peptides. My main focus will be the peptide design, peptide synthesis, and its applications.

Slide 2:

First, let me briefly introduce LifeTein. LifeTein was founded in 2008. We have been in the peptide industry for more than ten years. We specialize in peptide synthesis, chemical synthesis, antibody production, and protein services.

Slide 3:

Our main focus is peptide synthesis service. However, over the years, we have expanded to other protein-related areas like protein, antibody services, and products.

Slide 4:

So let us quickly get into the topic: cell-penetrating peptides. What is a cell-penetrating peptide or cpp? From the definition, CPP is a short peptide. They can be about 4-40 aa. The short peptide can enter the cell membrane. They can deliver bioactive cargoes.

Slide 5:

CPPs can also be used to deliver bioactive cargos like siRNAs, DNA, polypeptides, liposomes, nanoparticles, and others, in cells for therapeutic or experimental purposes.

Slide 6:

There are a few popular models for CPP’s entry. 1. The inverted micelle model. The CPPs are positively charged. They interact with the negatively charged phospholipids in the membrane. 2. Direct entry or direct translocation. For example, sequences with multiple Arginines can cause a short-time membrane cytolysis and enter the cells directly. 3. By the traditional method of endocytosis. I will not talk about the details.

Slide 7:

Here are a few examples of the CPP. The most famous examples are HIV tat sequence. The TAT peptide is arginine-rich and can directly penetrate the plasma membrane and stabilize DNA.

Another example is the arginine-rich peptide R8 or R9. We can add stearic acid to the N-terminus.  Stearic acid is a saturated fatty acid with an 18-carbon chain. If you would like to do live cell imaging, we can add fluorescent dye such as Fitc, Alexa fluor, or Cy dye at the N-terminus or C-terminus. I will get to the details.

Slide 8:

CPPs can enter the regular cell membranes. Some other peptides are tissue-targeting peptides. For example, this brain-homing peptide can cross the blood-brain barrier. Other peptides can cross the skin as transdermal peptides, target heart tissues as cardiac targeting peptides, and nuclear localization signal peptides.

Slide 9:

On this slide, I will talk more about the peptide design. There are many ways to make the peptide permeable. In the case of DNA or RNA, you can simply mix the CPPs with oligos. Many transfection reagents are using this mechanism. Simply put, DNA is negatively charged and peptide is positively charged. If you mix them together, they will form small micelles for cell penetration.

However, most of our work is to put the CPPs and your target together by covalent links. For this example, we put eight arginines at the N-terminus of your peptide. A linker called Ahx is added as a spacer. Some users prefer no spacers. It seems that both worked for the purpose. The eight arginines can be put at the C-terminus as well. According to the feedback from our users, most of the N-terminal CPP worked well. A few worked well for the C-terminal conjugation. I guess it depends on the projects.

This example is the Npys linker modification. The cysteine is added to your peptide. We conjugate two sequences together to form a disulfide bond. This is especially useful for the cancer study. Cancer cells have a lower pH of 6.7-7.1. Normal cells have a higher pH of 7.4. Under the acidic environment, the disulfide bond can be cleaved. If your target peptide is a cancer drug candidate, the CPP can introduce the drug cargo to the cancer cell and release the target within the cell. These disulfide-based prodrugs are important for cancer therapy.

If the cysteine is not available for your case, we can add a compound called lysine azide. This method needs click chemistry.

Slide 10:

There are two kinds of click chemistry. The one with copper as the catalyst and the one without. The preferred method is copper-free click chemistry. It is called DBCO and azide reactions. The final conjugate will have a large linker. Many scientists have concerns about the bulky size of the linker. However, some drugs contain bulky linkers without issues or side effects.

Back to Slide 9:

Let us go back to the sequence. The design does not have to be this way. The lysine azide can be any place in the sequence. If you have a head-to-tail cyclic peptide, you can add the lysine azide in the middle. The final product will be like a lollipop, with the CPP as the tail. If the N-terminus is very important to you, you can add the azide at the C-terminus.

Slide 11:

Let us move on to other scenarios. If you would like to track the peptides in live cells, fluorescent dyes can be added. We can do Fitc, Fam, Cy3, cy5, Cy7 and Alexa Fluor. This design will give direct evidence that your target is inside the cells.  

There is a different kind of peptide called peptide nucleic acid or PNA. It is DNA or RNA analog. We can synthesize half as peptide and another half as the PNA.  

This structure is the one I just mentioned earlier. The cyclic tumor targeting RGD peptide can be linked with an R8 cell-penetrating peptide to form a lollipop-shaped structure.

Slide 12:

So far, we have mentioned different ways to conjugate the cargo with cell-penetrating peptides. If your targets are nanoparticles or gold particles. Our requirement is to have active groups like a thiol group or a free amine on it. We have to have the active groups react to the cell-penetrating peptides. It is the same requirement for small compounds.

Slide 13:

The last concept I would like to introduce is the antibody-drug conjugate. This concept is widely accepted in the antibody drug industry. There are three important components: an antibody, a cleavable linker, and the drug.  Once the antibody binds to the target, the drug is released after the hydrolysis by protease.

Slide 14:

The same concept can be used for the peptides. For this concept, we need to screen the best drug candidate for cell entry. The CPP can be tumor-homing peptides, brain-homing peptides, or cardiac targeting peptides I mentioned earlier.

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First, we need to modify the compound. It is better to have a free amine in the compound. Then we can modify the amine group to an azide group. Afterward, we can use the click chemistry for the following conjugation.

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LifeTein produced a series of CPPs. They are ready to conjugate your compounds for screening. So far, we have designed and produced more than fifty CPPs.

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Step 3 is conjugate peptides with drug candidates.

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Once the CPP is conjugated with the drug compound using click chemistry, we can send the final product back to you for further screening. The purpose is to find the best drug delivery system.

Slide 19

To summarize today’s topic, I talked about cell-penetrating peptides with different cargos. As long as you have an active chemical group on the nanoparticles, compounds, or liposomes, we can conjugate the target to any peptide.

Slide 20

That is all for today. Please let me know if you have any questions. Please feel free to contact us by email or phone calls.