Construction of Cyclic Cell-Penetrating Peptides for Enhanced Penetration of Biological Barriers

A serious obstacle for cancer treatment is the limited access of therapeutics to deeper tumor cells due to physiological barriers. Thus, the development of novel molecular transporters capable of treating biological barriers is strongly desired to enhance the drug delivery efficiency. The purpose of this procedure is to synthesize the cyclic cell-penetrating peptides for enhanced cell-to-cell penetration.

The advantage of this method is that the peptide cyclization can be easily achieved through substitution reactions with cysteines and resin without requiring any metal catalysts. The incorporation of aromatic cross-links improves the overall hydrophobicity of the peptides compared to the hydrophilic lactam or triazole cross-links, thereby enhancing their permeability. To begin, assemble the manual peptide synthesis apparatus in the fume hood.

Place the three-way stopcocks onto the vacuum manifold and connect them to nitrogen. Make sure to cap the unused inlets. Attach a 10-milliliter polypropylene column onto the three-way stopcocks and drain the reaction mixture or solvents from the polypropylene column using a rubber pipette bulb or a vacuum via a waste trap.

To prepare the resin for peptide synthesis, add 4 to 5 milliliters of DMF to the required amount of resin and transfer it to the polypropylene column with gentle nitrogen bubbling for 30 minutes to swell the resin adequately. Drain the DMF and add 4 to 5 milliliters of 50%morpholine/DMF to the resin. Gently bubble nitrogen for 30 minutes to remove the N-terminal Fmoc group, and then drain the mixture.

Wash the resin thoroughly three times by adding 4 to 5 milliliters of DMF to the column and bubbling with nitrogen for at least 1 minute each time. In the same way, wash the resin with dichloromethane three times and DMF three times. Next, dissolve 648.8 milligrams of Fmoc and PBF-protected arginine and 372.6 milligrams of HATU in 5 milliliters of DMF in a centrifuge tube.

Add 348.4 microliters of DIPEA to activate the coupling reaction, and transfer the reaction mixture to the polypropylene column with resin. Gently agitate the mixture with nitrogen bubbling for 1 to 2 hours, and then repeat the coupling reaction once. Drain the reaction mixture and wash the resin sequentially with DMF, dichloromethane, and again DMF three times each for at least 1 minute each time.

Remove the Fmoc-protecting group and wash the resin following the procedure shown earlier before proceeding to couple the next amino acid. Next, couple the beta-alanine as a spacer for FITC labeling using the same process shown for amino acid coupling, and then add a mixture of FITC, DIPEA, and DMF to the polypropylene column in the dark to perform FITC labeling of peptides on the resin. The reaction would take almost 8 hours.

To perform the cyclization of the linear peptide, add a mixture of trifluoroacetic acid, triisopropylsilane, and dichloromethane to the polypropylene column for 2 minutes to selectively remove the trityl-protecting group of cysteine. Drain the mixture and repeat the procedure until the yellowish solution becomes colorless to completely remove the trityl-protecting group. Perform sequential washes of the resin with DMF and dichloromethane at least three times and dissolve 4, 4'bis-bromomethyl-biphenyl in DMF with DIPEA.

Add the solution to the column and react for 4 hours. Wash the resin with 4 to 5 milliliters of methanol twice for five minutes each, and dry it with a continuous flow of nitrogen. For peptides containing cysteines, treat the resin with an effective cleavage cocktail of trifluoroacetic acid, triisopropylsilane, and water or trifluoroacetic acid, triisopropylsilane, 1, 2-ethanedithiol, and water.

Treat the peptide-bound resin for 2 to 3 hours to cleave the peptide, and then remove the trifluoroacetic acid carefully with a stream of nitrogen. To obtain the crude peptides, add 4 to 5 milliliters of diethyl ether to the cleaved peptide preparation to precipitate the crude peptides, and centrifuge at 10, 000 x g for 4 minutes. Carefully discard the supernatant and air-dry the peptide for 3 minutes in an effective fume hood.

Dissolve the small-scale crude peptide in 800 microliters of acetonitrile, and then analyze it using reverse-phase high-performance liquid chromatography and liquid chromatography-mass spectrometry. Inoculate 100, 000 HeLa cells in 2 milliliters of DMEM in a 12-well chamber with a tissue culture plate insert, and incubate for 24 hours in a 37 degrees Celsius humidified incubator containing 5%carbon dioxide. Remove the medium and incubate the cells in the chambers with 1 milliliter of 10 micromolar FITC-R8 or FITC-sR8-4 in FBS-free DMEM for 1 hour.

After that, remove the medium containing the peptides and wash the cells three times with 1 milliliter of PBS. Add 1 milliliter of fresh FBS-free DMEM to the chambers, and then co-incubate the HeLa cells in the chamber with the tissue culture plate insert, with the HeLa cells on the round coverslips at the bottom for 2 hours. Fix the HeLa cells on the round coverslips with 2.5%glutaraldehyde for 15 minutes, and then stain the cells with DAPI for 15 minutes.

Finally, observe the HeLa cells on the coverslips under a fluorescence microscope. A schematic diagram of the synthesis of FITC-labeled linear R8 peptide and FITC-labeled stapled R8 peptide is presented here. The HPLC and MS spectra of the linear R8 peptide and the stapled R8 peptide are shown in this figure.

The retention time of the stapled peptide was substantially longer than that of the linear analog, indicating an enhanced overall hydrophobicity of the peptide after cyclization with the hydrophobic cross-link. This graphical image represents the stability of the linear peptide and the stapled peptide in the presence of 25%FBS. The cyclic R8 remains 77.3%intact after incubation with FBS for 4 hours, while its linear counterpart was mostly degraded, suggesting enhanced proteolytic stability of the cyclic R8 peptide.

Live-cell fluorescence microscopy images of HeLa cells and 4T1 cells after 1 hour incubation with 3 micromolar FITC-labeled linear R8 peptide and FITC-labeled stapled R8 peptide are shown here. It can be seen that the cells treated with cyclic R8 with aromatic cross-link exhibited higher intracellular fluorescence than those treated with their linear counterpart. Similar results were obtained with flow cytometry analysis.

To further investigate whether cyclic R8 confers enhanced cell-to-cell penetration, transwell models were used to simulate the barrier permeability of the peptides from one cell layer to another. The cyclic R8 clearly exhibited higher trans-barrier penetration than the linear R8 peptide, as indicated by a significant increase in intracellular fluorescence. Complete deprotection of the trityl group of cysteine and resin is important for the following cyclization step.

Besides, the cyclization efficiency also depends on the specific sequences and the lengths of the peptides due to steric effects. In such a case, using a resin with lower loading capacity or cyclization the peptides under dilute concentrations in solution phase would be helpful. These cyclic peptides showed an enhanced cell-to-cell permeability compared to their linear counterparts.

We believe they hold great promise for conquering biological barriers and will be used for a molecular for further applications in the field of drug delivery.