This protocol describes the synthesis of cyclic cell-penetrating peptides with aromatic cross-links and the evaluation of their permeability across biological barriers.
Cancer has been a grand challenge in global health. However, the complex tumor microenvironment generally limits the access of therapeutics to deeper tumor cells, leading to tumor recurrence. To conquer the limited penetration of biological barriers, cell-penetrating peptides (CPPs) have been discovered with excellent membrane translocation ability and have emerged as useful molecular transporters for delivering various cargoes into cells. However, conventional linear CPPs generally show compromised proteolytic stability, which limits their permeability across biological barriers. Thus, the development of novel molecular transporters that can penetrate biological barriers and exhibit enhanced proteolytic stability is highly desired to promote drug delivery efficiency in biomedical applications. We have previously synthesized a panel of short cyclic CPPs with aromatic crosslinks, which exhibited superior permeability in cancer cells and tissues compared to their linear counterparts. Here, a concise protocol is described for the synthesis of the fluorescently labeled cyclic polyarginine R8 peptide and its linear counterpart, as well as key steps for investigating their cell permeability.
The past few decades have witnessed rapid advances in the development of cell-penetrating peptides (CPPs) for drug delivery. CPPs have been widely used as molecular transporters for the treatment of a range of life-threatening diseases, including neurological disorders1,2, heart diseases3, diabetes4, dermatosis5, and cancer6,7. Cancer remains a global health burden accompanied by a high rate of morbidity and mortality despite widespread research efforts8. A serious obstacle to treating cancer is the limited access of therapeutics to deeper tumor cells due to physiological barriers such as compact extracellular matrix (ECM), abnormal tumor vasculature, multiple membrane barriers, and high interstitial fluid pressure (IFP)9. Thus, developing novel CPPs with superior ability to deliver cargoes across biological barriers is considered an essential strategy for cancer treatment10,11.
CPPs can be categorized into cationic, amphipathic, and hydrophobic CPPs in terms of their physicochemical properties12. Among these, the positively charged HIV-TAT peptide and the synthetic polyarginine are of considerable importance in biomedical research and have been extensively studied to facilitate intracellular drug delivery13. Tunnemann et al. reported that a minimum length of eight arginines is essential for efficient cell penetration of the synthetic polyarginine peptides, based on a cell permeability study conducted using R3 to R12 peptides14. However, these CPPs generally have short plasma half-lives due to their rapid hydrolysis in vivo. In addition, little is known regarding the optimization of the chemical structure of CPPs to increase their trans-barrier ability as it is challenging to penetrate multiple cell membranes15. Thus, the development of novel molecular transporters capable of penetrating biological barriers is strongly desired to enhance drug delivery efficiency. In 2020, Komin et al.16 discovered a CPP called CL peptide, which contains a helix motif (RLLRLLR) and a polyarginine tail (R7) for crossing the epithelial monolayer. A set of CL peptide variants were also synthesized by altering the helical pattern. This exploration could be a significant guide for the development of novel CPPs for the delivery of cargoes across biological barriers. Moreover, Dietrich et al. optimized the cell permeability of the StAX peptide, inhibiting the Wnt/β-catenin signaling pathway by increasing the overall hydrophobicity of the peptides17.
Conformational restriction of unstructured linear peptides by cyclization is an effective way to enhance their proteolytic stability and permeablity18,19,20. The structural reinforcement increases the protease resistance of cyclic peptides, making them more stable in vivo compared to their linear counterparts. In addition, the cyclization of peptides can potentially mask the polar peptide backbone by promoting intramolecular hydrogen bonding, thus increasing the membrane permeability of the peptides21. In the past two decades, chemoselective cyclization methods have become effective strategies for the construction of cyclic peptides with different architectures, such as all-hydrocarbon, lactam, triazole, m-xylene, perfluoroaryl, and other cross-links22,23. The biological barrier imposed by the sophisticated tumor microenvironment could reduce the penetration of drugs in solid tumors24. We have previously found that the cyclic CPPs displayed superior resistance to enzymatic digestion over their linear counterparts20. Furthermore, the overall hydrophobicity of the peptides is critical for their enhanced cell permeability22. Based on the studies discussed above, the combination of a positively charged pattern, elevated overall hydrophobicity, and enhanced proteolysis stability can be hypothesized to increase the permeability of CPPs across biological barriers. In a recent study, we identified two cyclic CPPs with aromatic crosslinks at positions i and i+7 that exhibit improved permeability in tumor cells and tissues compared to their linear counterparts15. Here, a concise synthetic protocol for the synthesis of fluorescently labeled cyclic CPPs and the key steps to investigate their permeability are presented.
1. Equipment preparation
NOTE: Carry out all the procedures in an operating fume hood with suitable personal protective equipment.
2. Synthesis of FITC-labeled linear R8 peptide (FITC-R8) and FITC-labeled stapled R8 peptide (FITC-sR8-4)
NOTE: The peptides were synthesized according to a standard Fmoc-based solid-phase peptide synthesis (SPPS) protocol25. The 4-(2',4'-Dimethoxypheyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucyl-MBHA resin (rink amide MBHA resin, see Table of Materials) was used throughout the study.
CAUTION: N, N-dimethylformamide (DMF), N, N-diisopropylethylamine (DIPEA), morpholine, and dichloromethane (DCM) are all colorless and are damaging if inhaled or absorbed through the skin. Ether is extremely flammable. 1,2-Ethanedithiol (EDT) is a particularly odorous substance. Trifluoroacetic acid (TFA) is highly corrosive, and its acidity is 105 times that of acetic acid. Consequently, all reagents and chemicals are supposed to be dealt with using protective equipment in a fume hood.
3. Quantification of the FITC-labeled peptides
4. Stability of peptides in fetal bovine serum (FBS)
5. Cellular uptake of the peptides
6. Exploration of the cell-to-cell penetration of the peptides using transwell models
In this protocol, a synthetic procedure to constrain the linear polyarginine R8 into its cyclic form was presented. The SPPS was conducted manually using a simple apparatus (Figure 1). The detailed synthetic process of SPPS is shown in Figure 2. Briefly, the resin was sufficiently swelled, followed by deprotection of the Nα-Fmoc protecting group. Then, the Nα-Fmoc-protected amino acid was anchored on the resin until the completion of the peptide assembly (steps 1-4 in Figure 2). Then, the crude peptides were cleaved from the resin by the cleavage cocktail (step 5 in Figure 2). FITC was used to label the peptides to synthesize fluorescently labeled cyclic CPPs and track their permeability across biological barriers. Subsequently, the trityl protecting groups of cysteines were selectively deprotected on the resin, followed by peptide cyclization with 4,4'-bis(bromomethyl)biphenyl cross-link (Figure 3A). The HPLC and MS spectra of FITC-R8 and FITC-sR8-4 are shown in Figure 3B. The retention time of FITC-sR8-4 was substantially longer than that of the linear analog, indicating enhanced overall hydrophobicity of the peptide after cyclization with the hydrophobic cross-link. Furthermore, as shown in Figure 3C, the cyclic R8 remained 77.3% intact after incubation with 25% FBS for 4 h, while its linear counterpart was mostly degraded, suggesting enhanced proteolytic stability of the cyclic R8 peptide. In the subsequent cell-based studies, cells treated with cyclic R8 with aromatic crosslink exhibited higher intracellular fluorescence than those treated with its linear counterpart, as demonstrated by live-cell fluorescence microscopy imaging (Figure 4A). Similar results were obtained with flow cytometry analysis (Figure 4B). 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 the intracellular fluorescence (Figure 4C). To sum up, the cyclic R8 peptide exhibited superior permeability across biological barriers over its linear counterpart.
Figure 1: Equipment setup for the manual peptide synthesis apparatus. A 10 mL polypropylene column is set up on the vacuum manifold using a three-way stop valve. N2 is used for agitation. Please click here to view a larger version of this figure.
Figure 2: General procedure of Fmoc solid-phase peptide synthesis (SPPS). An Nα-Fmoc-protected amino acid is anchored to the 4-(2',4'-Dimethoxypheyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucyl-MBHA resin (rink amide MBHA resin) (step 1), followed by deprotection of the Nα-Fmoc protecting groups of the amino acids (step 2) and subsequent amino acid coupling (step 3). Step 2 and step 3 are repeated several times to synthesize the desired peptide (step 4). After the completion of synthesis, a cleavage cocktail is added to remove the side chain protecting groups and cleave the desired peptide from the resin (step 5). Abbreviations: DMF = N, N-dimethylformamide; DCM = dichloromethane; HATU = 2-(7-Azobenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate; DIPEA = N, N-diisopropylethylamine; TFA = trifluoroacetic acid. Please click here to view a larger version of this figure.
Figure 3: Synthesis of FITC-labeled linear R8 peptide (FITC-R8) and FITC-labeled stapled R8 peptide (FITC-sR8-4) using solid-phase peptide synthesis (SPPS). (A) Schematic diagram of the synthesis of FITC-R8 and FITC-sR8-4. (B) HPLC and MS spectra (inset) of FITC-R8 and FITC-sR8-4. (C) Stability of FITC-R8 and FITC-sR8-4 in the presence of 25% FBS. Intact peptide (%) refers to the fraction of undegraded peptide. This figure has been modified from Shi et al.15. Please click here to view a larger version of this figure.
Figure 4: Penetration of FITC-labeled linear R8 peptide (FITC-R8) and FITC-labeled stapled R8 peptide (FITC-sR8-4). (A) Live-cell fluorescence microscopy images of HeLa cells and 4T1 cells after 1 h incubation with 3 µM FITC-R8 and FITC-sR8-4. FITC (green), Hoechst (blue). Scale bar = 20 µm. (B) Relative mean fluorescence (with respect to the linear R8 peptide), mean ± s.d., and n = 3; (C) Cell-to-cell penetration of FITC-R8 and FITC-sR8-4 in a transwell model using HeLa cells. Live-cell fluorescence microscopy images (scale bar = 20 µm), and relative mean fluorescence (with respect to the linear R8 peptide), mean ± s.d., and n = 3. ** P < 0.01, *** P < 0.001. This figure has been modified from Shi et al.15. Please click here to view a larger version of this figure.
The chemical stabilization of peptides by incorporating conformational constraints has proven to be an effective strategy for improving the stability and cell permeability of the peptide26. In this protocol, a step-by-step procedure is described for the synthesis of cyclic CPPs with aromatic cross-links and the evaluation of their permeability across biological barriers. Compared to the hydrophilic lactam or triazole cross-links22,27, the incorporation of aromatic cross-links (used in this study) improves the overall hydrophobicity of the CPPs, thereby significantly increasing their cell permeability. On the other hand, peptide cyclization can be easily achieved through substitution reactions with cysteines without requiring any metal catalysts. In this protocol, the cyclization of the CPPs was conducted on resin; however, the cyclization efficiency also depends on the specific sequences and lengths of the peptides due to steric effects, which may result in the formation of a dimeric byproduct28. In such a case, using a resin with a lower loading capacity would be helpful. In addition, it is also recommended to cyclize these specific peptides under dilute concentrations in the solution phase29.
There are a few critical points in this protocol. First, the cleavage cocktail TFA/TIS/EDT/H2O (92.5/2.5/2.5/2.5, v/v/v/v) is used for the cleavage of cysteine-containing peptides to prevent oxidation of the sulfhydryl group. Second, it is suggested to perform a small-scale preliminary study to obtain the appropriate cleavage condition. The optimal time required to cleave the peptides from the resin is 2-3 h, with a longer cleavage time (more than 5 h) tending to produce more unidentified byproducts. The peptide synthesis could be monitored by LC-MS to optimize the cleavage time. Third, FITC labeling should be done in the dark to avoid fluorescence quenching.
Furthermore, trypan blue should be used to quench the surface-bound fluorescence as flow cytometry analysis cannot distinguish intracellular or surface-bound fluorescence. This will help to specifically quantify the peptide internalized by the cancer cells27. In addition, as cationic peptides may also cause non-specific membrane lysis30, hemolytic activity and cell viability could also be conducted to evaluate the toxicity of the cyclic CPPs.
Cyclic CPPs constitute one of the effective drug delivery vehicles for conquering biological barriers. However, the membrane interaction and perturbation of cationic CPPs generally lead to potential non-specific cytotoxicity31. Further efforts will be devoted to understanding the detailed penetration mechanism, which should aid the discovery of the next generation of cyclic CPPs to penetrate biological barriers with minimal cytotoxicity. These highly active and stable CPPs hold great promise for improving the treatment of important life-threatening diseases.
The authors have nothing to disclose.
This work is supported by the Natural Science Foundation of China (21708031), China Postdoctoral Science Foundation (BX20180264, 2018M643519), and the Fundamental Research Funds for the Central Universities (2682021ZTPY075).
1,2-ethanedithiol | Aladdin | K1722093 | stench |
2-(7-Azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) | HEOWNS | A-0443697 | |
4,4'-bis(bromomethyl)biphenyl | TCI | B1921 | |
4T1 cells | ATCC | 4T1 cells were cultured in DMEM medium supplemented with 10% FBS (Hyclone) in a 37 °C humidified incubator containing 5% CO2. | |
Acetonitrile | Adamas | 1484971 | toxicity |
Dichloromethane | Energy | W330229 | skin harmful |
Diethyl ether | Aldrich | 673811 | flammable |
Dimethyl sulfoxide | Beyotime | ST038 | skin harmful |
Dulbecco’s Modified Eagle Medium (DMEM) | Gibco | ||
Electrospray Ionization Mass Spectrometer | Waters | G2-S Tof | |
Ethylene Diamine Tetraacetic Acid (EDTA) | BioFroxx | 1340 | |
Fetal bovine serum (FBS) | HyClone | ||
Flow cytometer | Beckman Coulter | CytoFLEX | |
Fluorescein isothiocyanate isomer (FITC) | Energy | E0801812500 | |
Fluorescent microscope | Carl Zeiss | Axio Observer 7 | |
Fmoc-Arg(Pbf)-OH | HEOWNS | F-81070 | |
Fmoc-Cys(Trt)-OH | GL Biochem | GLS201115-35202 | |
Fmoc-βAla-OH | Adamas | 51341C | |
HeLa cells | ATCC | HeLa cells were cultured in DMEM supplemented with 10% FBS (Hyclone) in a 37 °C humidified incubator containing 5% CO2. | |
High-Performance Liquid Chromatography | Agilent | Agilent 1260 | |
High-Performance Liquid Chromatography column | Agilent | Poroshell EC-C18 120, 4.6 × 150 mm (pore size 120 Å, particle size 4 μm) | |
Lyophilizer | SP Scientific | Vir Tis | |
Methanol | Aldrich | 9758 | toxicity |
Microtiter plate | Thermo μdrop plate | N12391 | |
Morpholine | HEOWNS | M99040 | irritant |
Multi-technology microplate reader | Thermo | VARIOSKAN LUX | |
N,N-Diisopropylethylamine | HEOWNS | E-81416 | irritant |
N,N-Dimethyl formamide | Energy | B020051 | harmful to skin |
Poly-Prep column | Bio-Rad | 7321010 | polypropylene chromatography columns |
Rink Amide MBHA resin (0.572 mmol/g) | GL Biochem | GLS180301-49101 | |
Three-way stopcocks | Bio-Rad | 7328107 | |
Tissue culture plate insert | LABSELECT | 14211 | |
Trifluoroacetic acid | HEOWNS | T63278 | corrosive |
Triisopropylsilane | HEOWNS | T-0284475 | |
Trypsin | BioFroxx | 1004 | |
Vacuum manifold | Promega | A7231 |