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. Equipment preparation
NOTE: Carry out all the procedures in an operating fume hood with suitable personal protective equipment.
<ol>
	<li>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.</li>
	<li>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.</li>
</ol>
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&#39;,4&#39;-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.
<ol>
	<li>Prepare the resin for peptide synthesis.
	<ol>
		<li>Calculate the mass of resin needed for the synthesis:Mass of resin (mg) = scale (mmol) / resin loading capacity (mmol/g) &#215; 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 &#215; 1,000 mg/g = 350 mg.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>Dissolve Fmoc-Arg (Pbf)-OH (5 equiv., 648.8 mg) and 2-(7-Azobenzotriazole)-N, N, N&#39;, N&#39;-tetramethyluronium hexafluorophosphate (HATU, 4.9 equiv., 372.6 mg) in 5 mL of DMF in a centrifuge tube.</li>
		<li>Add DIPEA (10 equiv., 348.4 &#181;L) 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.</li>
		<li>Repeat the coupling reaction (step 2.2.1 and step 2.2.2) once.</li>
		<li>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.</li>
		<li>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&#945;-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 &#176;C for a few days (or at &#8722;20&#176;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.</li>
	</ol>
	</li>
	<li>Label the peptides with fluorescein isothiocyanate (FITC) as described below.
	<ol>
		<li>Couple the beta-alanine as a spacer for FITC labeling using the same process as used for amino acid coupling in step 2.2.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>For the synthesis of FITC-sR8-4, perform cyclization of the linear peptide as described below.
	<ol>
		<li>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.</li>
		<li>Subsequently, perform sequential washes of the resin with DMF and DCM at least 3x. Afterward, dissolve 4,4&#39;-bis(bromomethyl)biphenyl (2 equiv.) in DMF with DIPEA (4 equiv.), add it to the column, and react for 4 h.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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 &#215; g for 4 min. Carefully discard the supernatant and air-dry the peptide for 3 min in an effective fume hood.</li>
	<li>Analysis of the peptides: Dissolve a small-scale crude peptide (cleaved from approximately 10 mg of peptide-bound resin) in 800 &#181;L 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).</li>
	<li>Purify the peptides using RP-HPLC and LC-MS.
	<ol>
		<li>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 &#197;, particle size: 4 &#181;m; 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.</li>
		<li>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 &#8722;20 &#176;C. 
		&#8203;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): &#8722;20 V; CDL temperature: 250 &#176;C; block temperature: 400 &#176;C; flow rate: 0.2 mL/min; mobile phase: 50% H2O/50% ACN.</li>
	</ol>
	</li>
</ol>
3. Quantification of the FITC-labeled peptides
<ol>
	<li>Dissolve a small amount of purified peptide in DMSO as stock solution (e.g., 40 &#181;mol/mL).</li>
	<li>Measure the absorbance at 494 nm (A494) of 2 &#181;L of the stock solution in 498 &#181;L 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 &#181;L/ 2 &#181;L = 250, and the path length of the microtiter plate is 0.5 mm.</li>
	<li>Calculate the concentration of the stock solution using the following formula: 
	Concentration (mM) = A494 &#215; dilution factor / 0.05 (cm) / 77,000 (cm&#8722;1&#183;M&#8722;1) &#215; 1,000 (mM&#183;M&#8722;1)</li>
	<li>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&#8722;1&#183;M&#8722;1 arises from the FITC group.</li>
</ol>
4. Stability of peptides in fetal bovine serum (FBS)
<ol>
	<li>Incubate the peptide at a concentration of 100 &#181;M with 250 &#181;L of 25% FBS/H2O (v/v) at 37 &#176;C. After incubating for 0 h, 1 h, 2 h, and 4 h, take 10 &#181;L aliquots and then add 150 &#181;l of 12% trichloroacetic acid dissolved in H2O/ACN (1/3, v/v) to precipitate the serum proteins.</li>
	<li>Centrifuge the samples at 10,000 &#215; g for 5 min and analyze the supernatant using HPLC (as described in step 2.8) to determine the extent of peptide degradation.</li>
	<li>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.</li>
</ol>
5. Cellular uptake of the peptides
<ol>
	<li>Fluorescence microscopic imaging
	<ol>
		<li>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&#39;s Modified Eagle Medium (DMEM, see Table of Materials) supplemented with 10% FBS in a 37 &#176;C humidified incubator containing 5% CO2.</li>
		<li>Incubate the cells with 1 mL of 3 &#181;M FITC-labeled peptides in FBS-free DMEM for 1 h at 37 &#176;C. Afterward, remove the peptide-containing medium and wash the cells 3x with 1 mL of PBS.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Flow cytometry analysis
	<ol>
		<li>Inoculate 5 x 105 HeLa cells uniformly in 12-well plates and culture in DMEM for 24 h at 37 &#176;C. Afterward, remove the medium and wash the cells 3x with 1 mL of PBS.</li>
		<li>Incubate the cells with 1 mL of 3 &#181;M FITC-labeled peptides in FBS-free DMEM for 1 h at 37 &#176;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 &#215; g for 4 min. Wash the cell pellet with PBS.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
6. Exploration of the cell-to-cell penetration of the peptides using transwell models
<ol>
	<li>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 &#176;C humidified incubator containing 5% CO2. Afterward, remove the medium and incubate the cells in the chambers with 1 mL of 10 &#181;M FITC-R8 or FITC-sR8-4 (purified using HPLC) in FBS-free DMEM for 1 h.</li>
	<li>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.</li>
	<li>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.</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solid-phase+synthesis+of+short+&#945;-helices+stabilized+by+the+hydrogen+bond+surrogate+approach.">Solid-phase synthesis of short &#945;-helices stabilized by the hydrogen bond surrogate approach.</a> Nature Protocols. 5 (11), 1857-1865 (2010).</li><li>Baek, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+stapled+p53+peptide+bound+to+Mdm2.">Structure of the stapled p53 peptide bound to Mdm2.</a> Journal of the American Chemical Society. 134 (1), 103-106 (2012).</li><li>Traboulsi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrocyclic+cell+penetrating+peptides:+A+study+of+structure-penetration+properties.">Macrocyclic cell penetrating peptides: A study of structure-penetration properties.</a> Bioconjugate Chemistry. 26 (3), 405-411 (2015).</li><li>Tian, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+proline-derived+transannular+N-cap+for+nucleation+of+short+&#945;-helical+peptides.">A proline-derived transannular N-cap for nucleation of short &#945;-helical peptides.</a> Chemical Communications. 52 (59), 9275-9278 (2016).</li><li>Muppidi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+of+proteolytically+stable,+cell-permeable+peptide-based+selective+Mcl-1+inhibitors.">Rational design of proteolytically stable, cell-permeable peptide-based selective Mcl-1 inhibitors.</a> Journal of the American Chemical Society. 134 (36), 14734-14737 (2012).</li><li>Wiradharma, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+cationic+amphiphilic+&#945;-helical+peptides+as+antimicrobial+agents.">Synthetic cationic amphiphilic &#945;-helical peptides as antimicrobial agents.</a> Biomaterials. 32 (8), 2204-2212 (2011).</li><li>Jones, A. T., Sayers, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+entry+of+cell+penetrating+peptides:+Tales+of+tails+wagging+dogs.">Cell entry of cell penetrating peptides: Tales of tails wagging dogs.</a> Journal of Controlled Release. 161 (2), 582-591 (2012).</li></ol>

The authors have nothing to disclose.

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 ...

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/&#946;-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.

<table><tbody><tr><td>1,2-ethanedithiol</td><td>Aladdin</td><td>K1722093</td><td>stench</td></tr><tr><td>2-(7-Azobenzotriazole)-N,N,N&#039;,N&#039;-tetramethyluronium hexafluorophosphate (HATU)</td><td>HEOWNS</td><td>A-0443697</td><td></td></tr><tr><td>4,4&#039;-bis(bromomethyl)biphenyl</td><td>TCI</td><td>B1921</td><td></td></tr><tr><td>4T1 cells</td><td>ATCC</td><td></td><td>4T1 cells were cultured in DMEM medium supplemented with 10% FBS (Hyclone) in a 37 &amp;deg;C humidified incubator containing 5% CO&lt;sub&gt;2&lt;/sub&gt;.</td></tr><tr><td>Acetonitrile&amp;nbsp;</td><td>Adamas</td><td>1484971</td><td>toxicity</td></tr><tr><td>Dichloromethane</td><td>Energy</td><td>W330229</td><td>skin harmful</td></tr><tr><td>Diethyl ether</td><td>Aldrich</td><td>673811</td><td>flammable</td></tr><tr><td>Dimethyl sulfoxide</td><td>Beyotime</td><td>ST038</td><td>skin harmful</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM)</td><td>Gibco</td><td></td><td></td></tr><tr><td>Electrospray Ionization Mass Spectrometer</td><td>Waters</td><td>G2-S Tof</td><td></td></tr><tr><td>Ethylene Diamine Tetraacetic Acid (EDTA)</td><td>BioFroxx</td><td>1340</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>HyClone</td><td></td><td></td></tr><tr><td>Flow cytometer</td><td>Beckman Coulter</td><td>CytoFLEX</td><td></td></tr><tr><td>Fluorescein isothiocyanate isomer (FITC)</td><td>Energy</td><td>E0801812500</td><td></td></tr><tr><td>Fluorescent microscope</td><td>Carl Zeiss</td><td>Axio Observer 7</td><td></td></tr><tr><td>Fmoc-Arg(Pbf)-OH</td><td>HEOWNS</td><td>F-81070</td><td></td></tr><tr><td>Fmoc-Cys(Trt)-OH</td><td>GL Biochem</td><td>GLS201115-35202</td><td></td></tr><tr><td>Fmoc-&amp;beta;Ala-OH</td><td>Adamas</td><td>51341C</td><td></td></tr><tr><td>HeLa cells</td><td>ATCC</td><td></td><td>HeLa cells were cultured in DMEM supplemented with 10% FBS (Hyclone) in a 37 &amp;deg;C humidified incubator containing 5% CO&lt;sub&gt;2&lt;/sub&gt;.</td></tr><tr><td>High-Performance Liquid Chromatography</td><td>Agilent</td><td>Agilent 1260</td><td></td></tr><tr><td>High-Performance Liquid Chromatography column</td><td>Agilent</td><td>Poroshell EC-C18 120, 4.6 &amp;times; 150 mm (pore size 120 &amp;Aring;, particle size 4 &amp;mu;m)</td><td></td></tr><tr><td>Lyophilizer</td><td>SP Scientific</td><td>Vir Tis</td><td></td></tr><tr><td>Methanol</td><td>Aldrich</td><td>9758</td><td>toxicity</td></tr><tr><td>Microtiter plate</td><td>Thermo &amp;mu;drop plate</td><td>N12391</td><td></td></tr><tr><td>Morpholine</td><td>HEOWNS</td><td>M99040</td><td>irritant</td></tr><tr><td>Multi-technology microplate reader</td><td>Thermo</td><td>VARIOSKAN LUX</td><td></td></tr><tr><td>N,N-Diisopropylethylamine</td><td>HEOWNS</td><td>E-81416</td><td>irritant</td></tr><tr><td>N,N-Dimethyl formamide</td><td>Energy</td><td>B020051</td><td>harmful to skin</td></tr><tr><td>Poly-Prep column</td><td>Bio-Rad</td><td>7321010</td><td>polypropylene chromatography columns</td></tr><tr><td>Rink Amide MBHA resin (0.572 mmol/g)</td><td>GL Biochem</td><td>GLS180301-49101</td><td></td></tr><tr><td>Three-way stopcocks</td><td>Bio-Rad</td><td>7328107</td><td></td></tr><tr><td>Tissue culture plate insert</td><td>LABSELECT</td><td>14211</td><td></td></tr><tr><td>Trifluoroacetic acid</td><td>HEOWNS</td><td>T63278</td><td>corrosive</td></tr><tr><td>Triisopropylsilane</td><td>HEOWNS</td><td>T-0284475</td><td></td></tr><tr><td>Trypsin</td><td>BioFroxx</td><td>1004</td><td></td></tr><tr><td>Vacuum manifold</td><td>Promega</td><td>A7231</td><td></td></tr></tbody></table>

construction of cyclic cell-penetrating peptides for enhanced penetration of 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.

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&#945;-Fmoc protecting group. Then, the N&#945;-Fmoc-protected amino acid was anchored on the resin unt...

Watch this Scientific Journal Video about Construction of Cyclic Cell-Penetrating Peptides for Enhanced Penetration of Biological Barriers at JoVE.com

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

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 ...

construction-cyclic-cell-penetrating-peptides-for-enhanced

Research

JoVE Journal

Chemistry

2.0K Views.  Southwest Jiaotong University. Een ernstig obstakel voor de behandeling van kanker is de beperkte toegang van therapeutica tot diepere tumorcellen als gevolg van fysiologische barrières. De ontwikkeling van nieuwe moleculaire transporters die in staat zijn om biologische barrières te behandelen, is dus sterk gewenst om de efficiëntie van de medicijnafgifte te verbeteren. Het doel van deze procedure is om de cyclische celdoordringende peptiden te synthetiseren voor verbeterde cel-tot-cel penetratie.Het voordeel va...

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

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The modification presented includes the combination of this technique with cell-penetrating sequences. We propose to design cell-penetrating baits that can be incubated with living cells instead of cell lysates and therefore the interactions found will reflect those that occur within the intracellular context. Connexin43 (Cx43), a protein that forms gap junction channels and hemichannels is down-regulated in high-grade gliomas. The Cx43 region comprising amino acids 266-283 is responsible for the inhibition of the oncogenic activity of c-Src in glioma cells. Here we use TAT as the cell-penetrating sequence, biotin as the pull-down tag and the region of Cx43 comprised between amino acids 266-283 as the target to find intracellular interactions in the hard-to-transfect human glioma stem cells. One of the limitations of the proposed method is that the molecule used as bait could fail to fold properly and, consequently, the interactions found could not be associated with the effect. However, this method can be especially interesting for the interactions involved in signal transduction pathways because they are usually carried out by intrinsically disordered regions and, therefore, they do not require an ordered folding. In addition, one of the advantages of the proposed method is that the relevance of each residue on the interaction can be easily studied. This is a modular system; therefore, other cell-penetrating sequences, other tags, and other intracellular targets can be employed. Finally, the scope of this protocol is far beyond protein-protein interaction because this system can be applied to other bioactive cargoes such as RNA sequences, nanoparticles, viruses or any molecule that can be transduced with cell-penetrating sequences and fused to pull-down tags to study their intracellular mechanism of action.

This is a protocol to study intracellular protein-protein interactions based on the biotin-avidin pull-down system with the novelty of combining cell-penetrating sequences. The main advantage is that the target sequence is incubated with living cells instead of cell lysates and therefore the interactions will occur within the cellular context.

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The ...

Biochemistry

Development of a Backbone Cyclic Peptide Library as Potential Antiparasitic Therapeutics Using Microwave Irradiation

Protein-protein interactions (PPIs) are intimately involved in almost all biological processes and are linked to many human diseases. Therefore, there is a major effort to target PPIs in basic research and in the pharmaceutical industry. Protein-protein interfaces are usually large, flat, and often lack pockets, complicating the discovery of small molecules that target such sites. Alternative targeting approaches using antibodies have limitations due to poor oral bioavailability, low cell-permeability, and production inefficiency.
Using peptides to target PPI interfaces has several advantages. Peptides have higher conformational flexibility, increased selectivity, and are generally inexpensive. However, peptides have their own limitations including poor stability and inefficiency crossing cell membranes. To overcome such limitations, peptide cyclization can be performed. Cyclization has been demonstrated to improve peptide selectivity, metabolic stability, and bioavailability. However, predicting the bioactive conformation of a cyclic peptide is not trivial. To overcome this challenge, one attractive approach it to screen a focused library to screen in which all backbone cyclic peptides have the same primary sequence, but differ in parameters that influence their conformation, such as ring size and position.
We describe a detailed protocol for synthesizing a library of backbone cyclic peptides targeting specific parasite PPIs. Using a rational design approach, we developed peptides derived from the scaffold protein Leishmania receptor for activated C-kinase (LACK). We hypothesized that sequences in LACK that are conserved in parasites, but not in the mammalian host homolog, may represent interaction sites for proteins that are critical for the parasites&#39; viability. The cyclic peptides were synthesized using microwave irradiation to reduce reaction times and increase efficiency. Developing a library of backbone cyclic peptides with different ring sizes facilitates a systematic screen for the most biological active conformation. This method provides a general, fast, and facile way to synthesize cyclic peptides.

A simple and general method for the synthesis of cyclic peptides using microwave irradiation is outlined. This procedure enables the synthesis of backbone cyclic peptides with a collection of different conformations while retaining the side chains and the pharmacophoric moieties., and therefore, allows to screen for the bioactive conformation.

Protein-protein interactions (PPIs) are intimately involved in almost all biological processes and are linked to many human diseases. Therefore, there is ...

Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide

Cell-penetrating peptides (CPPs) are defined as carriers that are able to cross the plasma membrane and to transfer a cargo into cells. One of the main common features required for this activity resulted from the interactions of CPPs with the plasma membrane (lipids) and more particularly with components of the extracellular matrix of the membrane itself (heparan sulphate). Indeed, independent of the direct translocation or the endocytosis-dependent internalization, lipid bilayers are involved in the internalization process both at the level of the plasma membrane and at the level of intracellular traffic (endosomal vesicles). In this article, we present a detailed protocol describing the different steps of a large unilamellar vesicles (LUVs) formulation, purification, characterization, and application in fluorescence leakage assay in order to detect possible CPP-membrane destabilization/interaction and to address their role in the internalization mechanism. LUVs with a lipid composition reflecting the plasma membrane content are generated in order to encapsulate both a fluorescent dye and a quencher. The addition of peptides in the extravesicular medium and the induction of peptide-membrane interactions on the LUVs might thus induce in a dose-dependent manner a significant increase in fluorescence revealing a leakage. Examples are provided here with the recently developed tryptophan (W)- and arginine (R)-rich Amphipathic Peptides (WRAPs), which showed a rapid and efficient siRNA delivery in various cell lines. Finally, the nature of these interactions and the affinity for lipids are discussed to understand and to improve the membrane translocation and/or the endosomal escape.

The fluorescence leakage assay is a simple method that enables the investigation of peptide/membrane interactions in order to understand their involvement in several biological processes and especially the ability of cell-penetrating peptides to disturb phospholipids bilayers during a direct cellular translocation process.

Cell-penetrating peptides (CPPs) are defined as carriers that are able to cross the plasma membrane and to transfer a cargo into cells. One of the main ...

Engineering Cell-permeable Protein

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can make use of the translocating ability of so-called cell penetrating peptides (CPPs), also designated as protein transduction domains (PTDs). The TAT-CPP derived from the human immunodeficiency virus type 1 (HIV-1) Tat (trans-activator of transcription) protein has been widely used. The positively charged TAT promotes cell permeability thereby overcoming the barriers of the cellular membrane by endocytosis or/and direct membrane penetration2. In combination with a nuclear localization signal (NLS) fusion proteins are able to enter the nucleus exhibiting functionality. Our video presentation demonstrates, as an exemplification for the engineering of cell-permeable proteins, the construction, production and application of a cell-permeable version of the DNA-modifying enzyme Cre.
Cre is a site-specific recombinase that is able to recognize and recombine 34 base pair loxP sites in mammalian cells in vitro and in vivo. Therefore the Cre/loxP system is widely used to conditionally induce mutations in the genome of living cells3,4. The delivery of active Cre recombinase to cells, however, represents a limitation.
We describe the pSESAME vector system, which allows a direct insertion of the gene-of-interest and provides a platform to rapidly clone different domains and tags used within the vector in a convenient and standardized manner. Rearranging of the different tags has been shown to modify the biochemical properties of the fusion proteins providing a possibility to achieve higher yield and better solubility. We demonstrate how to express and purify recombinant cell-permeant proteins in and from E. coli. The functionality of the recombinant Cre protein is finally validated in cell culture by assessing its intracellular recombinase activity.

Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can ...

Biology

Constructing Cyclic Peptides Using an On-Tether Sulfonium Center

In recent years, cyclic peptides have attracted increasing attention in the field of drug discovery due to their excellent biological activities, and, as a consequence, they are now used clinically. It is, therefore, critical to seek effective strategies for synthesizing cyclic peptides to promote their application in the field of drug discovery. This paper reports a detailed protocol for the efficient synthesis of cyclic peptides using on-resin or intramolecular (intermolecular) bisalkylation. Using this protocol, linear peptides were synthesized by taking advantage of solid-phase peptide synthesis with cysteine (Cys) and methionine (Met) coupled simultaneously on the resin. Further, cyclic peptides were synthesized via bisalkylation between Met and Cys using a tunable tether and an on-tether sulfonium center. The whole synthetic route can be divided into three major processes: the deprotection of Cys on the resin, the coupling of the linker, and the cyclization between Cys and Met in a trifluoroacetic acid (TFA) cleavage solution. Furthermore, inspired by the reactivity of the sulfonium center, a propargyl group was attached to the Met to trigger thiol-yne addition and form a cyclic peptide. After that, the crude peptides were dried and dissolved in acetonitrile, separated, and then purified by high-performance liquid chromatography (HPLC). The molecular weight of the cyclic peptide was confirmed by liquid chromatography-mass spectrometry (LC-MS), and the stability of the cyclic peptide combination with the reductant was further confirmed using HPLC. In addition, the chemical shift in the cyclic peptide was analyzed by 1H nuclear magnetic resonance (1H NMR) spectra. Overall, this protocol aimed to establish an effective strategy for synthesizing cyclic peptides.

This protocol presents the synthesis of cyclic peptides via bisalkylation between cysteine and methionine and the facile thiol-yne reaction triggered by the propargyl sulfonium center.

In recent years, cyclic peptides have attracted increasing attention in the field of drug discovery due to their excellent biological activities, and, as ...

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an attractive alternative to liposomes or fatty acid-based vesicles. Externally or within the vesicles, peptides can be easily expressed and simplify the synthesis of membrane precursors. Provided here is a protocol for the creation of vesicles with diameters of ~200 nm based on the amphiphilic elastin-like polypeptides (ELP) utilizing dehydration-rehydration from glass beads. Also presented are protocols for bacterial ELP expression and purification via inverse temperature cycling, as well as their covalent functionalization with fluorescent dyes. Furthermore, this report describes a protocol to enable the transcription of RNA aptamer dBroccoli inside ELP vesicles as a less complex example for a biochemical reaction. Finally, a protocol is provided, which allows in vesiculo expression of fluorescent proteins and the membrane peptide, whereas synthesis of the latter results in vesicle growth.

Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...

Bioengineering

Measuring Peptide Translocation into Large Unilamellar Vesicles

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1. Many of these are referred to as cell-penetrating peptides, which are frequently noted for their potential as drug delivery vectors1-3. Moreover, there is increasing interest in antimicrobial peptides that operate via non-membrane lytic mechanisms4,5, particularly those that cross bacterial membranes without causing cell lysis and kill cells by interfering with intracellular processes6,7. In fact, authors have increasingly pointed out the relationship between cell-penetrating and antimicrobial peptides1,8. A firm understanding of the process of membrane translocation and the relationship between peptide structure and its ability to translocate requires effective, reproducible assays for translocation. Several groups have proposed methods to measure translocation into large unilamellar lipid vesicles (LUVs)9-13. LUVs serve as useful models for bacterial and eukaryotic cell membranes and are frequently used in peptide fluorescent studies14,15. Here, we describe our application of the method first developed by Matsuzaki and co-workers to consider antimicrobial peptides, such as magainin and buforin II16,17. In addition to providing our protocol for this method, we also present a straightforward approach to data analysis that quantifies translocation ability using this assay. The advantages of this translocation assay compared to others are that it has the potential to provide information about the rate of membrane translocation and does not require the addition of a fluorescent label, which can alter peptide properties18, to tryptophan-containing peptides. Briefly, translocation ability into lipid vesicles is measured as a function of the Foster Resonance Energy Transfer (FRET) between native tryptophan residues and dansyl phosphatidylethanolamine when proteins are associated with the external LUV membrane (Figure 1). Cell-penetrating peptides are cleaved as they encounter uninhibited trypsin encapsulated with the LUVs, leading to disassociation from the LUV membrane and a drop in FRET signal. The drop in FRET signal observed for a translocating peptide is significantly greater than that observed for the same peptide when the LUVs contain both trypsin and trypsin inhibitor, or when a peptide that does not spontaneously cross lipid membranes is exposed to trypsin-containing LUVs. This change in fluorescence provides a direct quantification of peptide translocation over time.

This protocol details a method for the quantitative measure of peptide translocation into large unilamellar lipid vesicles. This method also provides information about the rate of membrane translocation and can be used to identify peptides that efficiently and spontaneously cross lipid bilayers.

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1.  Many of these are referred ...

Evaluating Biofunctional Self-Assembling Peptides for Cell Adhesion and Morphology

Fabrication and Characterization of Colorectal Cancer Organoids from SW1222 Cell Line in Ultrashort Self&#45;Assembling Peptide Matrix

Ultrashort self-assembling peptides (SAPs) can spontaneously form nanofibers that resemble the extracellular matrix. These fibers allow the formation of hydrogels that are biocompatible, biodegradable, and non-immunogenic. We have previously proven that SAPs, when biofunctionalized with protein-derived motifs, can mimic the extracellular matrix characteristics that support colorectal organoid formation. These biofunctional peptide hydrogels retain the original parent peptide's mechanical properties, tunability, and printability while incorporating cues that allow cell-matrix interactions to increase cell adhesion. This paper presents the protocols needed to evaluate and characterize the effects of various biofunctional peptide hydrogels on cell adhesion and lumen formation using an adenocarcinoma cancer cell line able to form colorectal cancer organoids cost-effectively. These protocols will help evaluate biofunctional peptide hydrogel effects on cell adhesion and luminal formation using immunostaining and fluorescence image analysis. The cell line used in this study has been previously utilized for generating organoids in animal-derived matrices.

Author Spotlight: Optimization of Ultrashort Peptide Matrices for Colorectal Cancer Organoids

This protocol aims to evaluate biofunctional self-assembling peptides for cell adhesion, organoid morphology, and gene expression by immunostaining. We will use a colorectal cancer cell line to provide a cost-effective way of obtaining organoids for intensive testing.

Ultrashort self-assembling peptides (SAPs) can spontaneously form nanofibers that resemble the extracellular matrix. These fibers allow the formation of ...

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...

In Vivo Imaging of Transduction Efficiencies of Cardiac Targeting Peptide

Since the initial description of protein transduction domains, also known as cell penetrating peptides, over 25 years ago, there has been intense interest in developing these peptides, especially cell-specific ones, as novel vectors for delivering diagnostic and therapeutic materials. Our past work involving phage display identified a novel, nonnaturally occurring, 12 amino acid-long peptide that we named cardiac targeting peptide (CTP) due to its ability to transduce normal heart tissue in vivo with peak uptake seen in as little as 15 min after an intravenous injection. We have undertaken detailed biodistribution studies by injecting CTP labeled with fluorophore cyanine5.5, allowing it to circulate for various periods of time, and euthanizing, fixing, and sectioning multiple organs followed by fluorescent microscopy imaging. In this publication, we describe these processes as well as ex vivo imaging of harvested organs using an in vivo imaging system in detail. We provide detailed methodologies and practices for undertaking transduction as well as biodistribution studies using CTP as an example.

We describe protocols for assessing the degree of transduction by cell-penetrating peptides utilizing ex vivo imaging systems followed by paraffin embedding, sectioning, and confocal fluorescent microscopy using cardiac targeting peptide as an example. In our protocol, a single animal can be used to acquire both types of imaging assessment of the same organs, thereby cutting the number of animals needed for studies by half.

Since the initial description of protein transduction domains, also known as cell penetrating peptides, over 25 years ago, there has been intense interest ...

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

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