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

Podsumowanie

This protocol describes the synthesis of cyclic cell-penetrating peptides with aromatic cross-links and the evaluation of their permeability across biological barriers.

Streszczenie

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.

Wprowadzenie

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 disorders^1,2, heart diseases³, diabetes⁴, dermatosis⁵, and cancer^6,7. 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 treatment^10,11.

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 permeablity^18,19,20. 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-links^22,23. 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.

Protokół

1. Equipment preparation

NOTE: Carry out all the procedures in an operating fume hood with suitable personal protective equipment.

1. Assemble the manual peptide synthesis apparatus in the fume hood (Figure 1). Place the three-way stopcocks (see Table of Materials) onto the vacuum manifold (see Table of Materials) and connect to the nitrogen (N₂). Make sure to cap the unused inlets.
2. Attach a 10 mL polypropylene column (see Table of Materials) onto the three-way stopcocks. Drain the reaction mixture or solvents from the polypropylene column using a rubber pipette bulb or a vacuum via a waste trap.

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.

1. Prepare the resin for peptide synthesis.
   1. Calculate the mass of resin needed for the synthesis:Mass of resin (mg) = scale (mmol) / resin loading capacity (mmol/g) × 1,000 (mg/g)
      NOTE: For example, the mass of rink amide MBHA resin (0.572 mmol/g) for 0.2 mmol = 0.2 mmol / 0.572 mmol/g × 1,000 mg/g = 350 mg.
   2. Add 4-5 mL of DMF to the required amount of resin and transfer into the 10 mL polypropylene column (step 1.2) with gentle N₂ bubbling for 30 min to swell the resin adequately, and then drain the DMF.
   3. Add 4-5 mL of 50% morpholine/DMF (v/v) to the resin, gently bubble N₂ for 30 min 2x to remove the N-terminal Fmoc group, and then drain the mixture. Afterward, wash the resin thoroughly 3x by adding 4-5 mL of DMF to the column and bubbling with N₂ for at least 1 min each time. Continue to wash the resin with DCM (3x) and DMF (3x) in the same way.
2. Perform Fmoc-protected amino acid coupling as described below.
   NOTE: The coupling of arginine in a 0.2 mmol scale manual synthesis is described here as an example.
   1. Dissolve Fmoc-Arg (Pbf)-OH (5 equiv., 648.8 mg) and 2-(7-Azobenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (HATU, 4.9 equiv., 372.6 mg) in 5 mL of DMF in a centrifuge tube.
   2. Add DIPEA (10 equiv., 348.4 µL) to activate the coupling reaction and then transfer the reaction mixture to the 10 mL polypropylene column with resin (prepared in step 2.1.3). Then, gently agitate the mixture with N₂ bubbling for 1-2 h.
   3. Repeat the coupling reaction (step 2.2.1 and step 2.2.2) once.
   4. After completion of the coupling, drain the reaction mixture and wash the resin sequentially with DMF, DCM, and DMF 3x each for at least 1 min each time.
   5. Perform coupling of each amino acid in ordered steps: Add 4-5 mL of 50% morpholine/DMF (v/v) to the resin, gently bubble with N₂ for 30 min 2x to remove the N^α-Fmoc group, then wash the resin (as shown in step 2.2.4), and proceed to couple the next amino acid (as shown in step 2.2.1 and step 2.2.2). Proceed with several cycles of this step to achieve synthesis of the desired peptide.
      NOTE: This process can be paused here. Condense the resin with methanol and dry the resin with a continuous flow of N₂. Cap the polypropylene column and then store the resin at 4 °C for a few days (or at −20°C for longer storage). Swell the resin with 4-5 mL of DMF for 0.5-1 h before starting a new synthesis. If directly proceeding to the next step, there is no need to condense the resin.
3. Label the peptides with fluorescein isothiocyanate (FITC) as described below.
   1. Couple the beta-alanine as a spacer for FITC labeling using the same process as used for amino acid coupling in step 2.2.
   2. Perform FITC labeling of peptides on the resin by adding a mixture of FITC (5 equiv.), DIPEA (10 equiv.), and DMF to the polypropylene column and reacting in the dark for 8 h.
4. For the synthesis of FITC-sR8-4, perform cyclization of the linear peptide as described below.
   1. Add a mixture of TFA/triisopropylsilane (TIS)/DCM (3/5/92, v/v/v) to the polypropylene column for 2 min to selectively remove the Cys (Trt) protecting group, and then drain the mixture. Repeat the above procedure until the yellowish solution becomes colorless to completely remove the Trt protecting group.
   2. Subsequently, perform sequential washes of the resin with DMF and DCM at least 3x. Afterward, dissolve 4,4'-bis(bromomethyl)biphenyl (2 equiv.) in DMF with DIPEA (4 equiv.), add it to the column, and react for 4 h.
5. Cleave the peptides as described: After the completion of peptide synthesis, wash the resin with 4-5 mL of methanol twice for 5 min each and dry it with a continuous flow of N₂. Treat the resin with an effective cleavage cocktail TFA/TIS/H₂O (95/2.5/2.5, v/v/v), or TFA/TIS/EDT/H₂O (92.5/2.5/2.5/2.5, v/v/v/v) for peptides containing cysteines, using approximately 1 mL of cleavage cocktail for per 100 mg of resin. Treat the peptide-bound resin for 2-3 h to cleave the peptide and then remove the TFA carefully with a stream of N₂.
6. To obtain the crude peptides, add 4-5 mL of diethyl ether to the cleaved peptide preparation to precipitate the crude peptides and centrifuge at 10,000 × g for 4 min. Carefully discard the supernatant and air-dry the peptide for 3 min in an effective fume hood.
7. Analysis of the peptides: Dissolve a small-scale crude peptide (cleaved from approximately 10 mg of peptide-bound resin) in 800 µL of acetonitrile (ACN)/H₂O (1/1, v/v) and then analyze using reverse-phase high-performance liquid chromatography (RP-HPLC) and liquid chromatography-mass spectrometry (LC-MS) (see Table of Materials).
8. Purify the peptides using RP-HPLC and LC-MS.
   1. Dissolve 50 mg of crude peptide product in 4 mL of ACN/H₂O (1/1, v/v), and inject the solution into an RP-HPLC system equipped with a C18 column (4.6 mm x 150 mm, pore size: 120 Å, particle size: 4 µm; see Table of Materials). Elute the peptide using a mobile phase containing 0.1% TFA/H₂O (v/v) and ACN, with a gradient of 10% to 90% ACN over 30 min. Conventional peptides were detected at 220 nm and FITC-labeled peptides at 494 nm.
   2. Collect the fractions corresponding to the major peptide peak as identified by MS, and then lyophilize the desired peptide fractions. Store the purified peptide at −20 °C.
      ​NOTE: The found m/z of the purified FITC-R8 are as follows: [M + 3 H]³⁺: 576.63; [M + 4 H]⁴⁺: 432.72; [M + 5 H]⁵⁺: 346.39; [M + 6 H]⁶⁺: 288.85. The found m/z of the purified FITC-sR8-4 are as follows: [M + 3 H]³⁺: 704.74; [M + 4 H]⁴⁺: 528.77; [M + 5 H]⁵⁺: 423.34; [M + 6 H]⁶⁺: 352.91. MS analytical conditions: instrument: ESI (probe bias: +4.5 kV; detector: 1.2 kV); nebulizer gas flow: 1.5 L/min; curved desolvation line (CDL): −20 V; CDL temperature: 250 °C; block temperature: 400 °C; flow rate: 0.2 mL/min; mobile phase: 50% H₂O/50% ACN.

3. Quantification of the FITC-labeled peptides

1. Dissolve a small amount of purified peptide in DMSO as stock solution (e.g., 40 µmol/mL).
2. Measure the absorbance at 494 nm (A₄₉₄) of 2 µL of the stock solution in 498 µL of 10 mM phosphate buffered saline (1x PBS, pH 7.4) with a microtiter plate (see Table of Materials) using a multi-technology microplate reader (see Table of Materials). The dilution factor is 500 µL/ 2 µL = 250, and the path length of the microtiter plate is 0.5 mm.
3. Calculate the concentration of the stock solution using the following formula:
   Concentration (mM) = A₄₉₄ × dilution factor / 0.05 (cm) / 77,000 (cm⁻¹·M⁻¹) × 1,000 (mM·M⁻¹)
4. Adjust to a proper dilution so that the measured A₄₉₄ value is between 0.1 and 1.0.
   NOTE: The measurements should be repeated several times to ensure that the measured concentration is accurate. The extinction coefficient of 77,000 cm⁻¹·M⁻¹ arises from the FITC group.

4. Stability of peptides in fetal bovine serum (FBS)

1. Incubate the peptide at a concentration of 100 µM with 250 µL of 25% FBS/H₂O (v/v) at 37 °C. After incubating for 0 h, 1 h, 2 h, and 4 h, take 10 µL aliquots and then add 150 µl of 12% trichloroacetic acid dissolved in H₂O/ACN (1/3, v/v) to precipitate the serum proteins.
2. Centrifuge the samples at 10,000 × g for 5 min and analyze the supernatant using HPLC (as described in step 2.8) to determine the extent of peptide degradation.
3. Calculate the ratio of peak area at 1 h, 2 h, and 4 h to that at 0 h to obtain the fraction of undegraded peptide at the corresponding time. The result is the average of three parallel samples.

5. Cellular uptake of the peptides

1. Fluorescence microscopic imaging
   1. Place a round coverslip into a 12-well plate. Then, inoculate 1 x 10⁵ cells uniformly on coverslips and culture overnight with 2 mL of medium. Remove the medium and wash the cells 3x with 1 mL of PBS.
      NOTE: In this study, HeLa cells and 4T1 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, see Table of Materials) supplemented with 10% FBS in a 37 °C humidified incubator containing 5% CO₂.
   2. Incubate the cells with 1 mL of 3 µM FITC-labeled peptides in FBS-free DMEM for 1 h at 37 °C. Afterward, remove the peptide-containing medium and wash the cells 3x with 1 mL of PBS.
   3. Stain the cells with 1 mL of Hoechst 33258 for 15 min. Observe the internalization of each peptide using a fluorescence microscope (see Table of Materials) with the same fluorescence intensity and exposure time.
      NOTE: The following settings were used for fluorescence microscopy. Objective: Plan-Apochromat: 63 x/1.40 Oil DIC M27; Channel 1 for FITC: excitation filter: 450-490 nm, emission filter: 500-550 nm, exposure time: 230 ms; Channel 2 for Hoechst 33258: excitation filter: 335-383 nm, emission filter: 420-470 nm, exposure time: 27 ms.
2. Flow cytometry analysis
   1. Inoculate 5 x 10⁵ HeLa cells uniformly in 12-well plates and culture in DMEM for 24 h at 37 °C. Afterward, remove the medium and wash the cells 3x with 1 mL of PBS.
   2. Incubate the cells with 1 mL of 3 µM FITC-labeled peptides in FBS-free DMEM for 1 h at 37 °C. Remove the peptide-containing medium, dissociate the cells with 0.25% (w/v) trypsin and 0.53 mM EDTA in PBS for 5 min, and then collect the cells by centrifugation at 306 × g for 4 min. Wash the cell pellet with PBS.
   3. Incubate the cells with 1 mL of 0.05% (w/v) trypan blue in PBS for 3 min to quench the surface-bound fluorescence, and perform quantitative analysis of intracellular fluorescence using a flow cytometer (see Table of Materials).
      NOTE: Flow cytometry settings: excitation: 488 nm, emission: 530 nm. Trypan blue can also quench the fluorescence of dead cells and help distinguish live/dead cells during analysis of peptide uptake.
   4. Treat and assay 4T1 cells using flow cytometry following the same protocol as described for HeLa cells. Collect 5 x 10⁵ cells per sample and set up three parallel samples per group.

6. Exploration of the cell-to-cell penetration of the peptides using transwell models

1. Inoculate 1 x 10⁵ HeLa cells in 2 mL of DMEM in a 12-well chamber with a tissue culture plate insert (see Table of Materials) and incubate for 24 h in a 37 °C humidified incubator containing 5% CO₂. Afterward, remove the medium and incubate the cells in the chambers with 1 mL of 10 µM FITC-R8 or FITC-sR8-4 (purified using HPLC) in FBS-free DMEM for 1 h.
2. Remove the medium containing the peptides and wash the cells 3x with 1 mL of PBS. Add 1 mL 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 h.
3. Fix the HeLa cells on the round coverslips with 2.5% glutaraldehyde for 15 min and then stain the cells with DAPI for 15 min. Then, observe the HeLa cells on the coverslips under a fluorescence microscope. Treat and assay 4T1 cells using the same protocol as that used for HeLa cells.

Wyniki

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 N^α-Fmoc protecting group. Then, the N^α-Fmoc-protected amino acid was anchored on the resin unt...

Dyskusje

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-links^22,27, the ...

Materiały

Name	Company	Catalog Number	Comments
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