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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#39;,N&#39;-tetramethyluronium hexafluorophosphate (HATU)</td>
			<td>HEOWNS</td>
			<td>A-0443697</td>
			<td></td>
		</tr>
		<tr>
			<td>4,4&#39;-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 &#176;C humidified incubator containing 5% CO2.</td>
		</tr>
		<tr>
			<td>Acetonitrile&#160;</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&#8217;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-&#946;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 &#176;C humidified incubator containing 5% CO2.</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 &#215; 150 mm (pore size 120 &#197;, particle size 4 &#956;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 &#956;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 ...

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1.8K Views.  Southwest Jiaotong University. This protocol describes the synthesis of cyclic cell-penetrating peptides with aromatic cross-links and the evaluation of their permeability across biological barriers.

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

Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young&#8217;s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.&#160;primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.

Microwave-assisted Functionalization of Polyethylene glycol and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

This video will illustrate a rapid, efficient method to methacrylate poly(ethylene glycol), enabling chain polymerizations and hydrogel synthesis. It will demonstrate how to similarly introduce methacrylamide functionalities into peptides, detail common analytical methods to assess functionalization efficiency, provide suggestions for troubleshooting and advanced modifications, and demonstrate typical hydrogel characterization techniques.

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large ...

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control self-assembly and mimicking this process in vitro will bring about major advances in the areas of materials science and nanotechnology. Among the available biological building blocks, peptides have several advantages as they present substantial diversity, their synthesis in large scale is straightforward, and they can easily be modified with biological and chemical entities1,2. Several classes of designed peptides such as cyclic peptides, amphiphile peptides and peptide-conjugates self-assemble into ordered structures in solution. Homoaromatic dipeptides, are a class of short self-assembled peptides that contain all the molecular information needed to form ordered structures such as nanotubes, spheres and fibrils3-8. A large variety of these peptides is commercially available.
This paper presents a procedure that leads to the formation of ordered structures by the self-assembly of homoaromatic peptides. The protocol requires only commercial reagents and basic laboratory equipment. In addition, the paper describes some of the methods available for the characterization of peptide-based assemblies. These methods include electron and atomic force microscopy and Fourier-Transform Infrared Spectroscopy (FT-IR). Moreover, the manuscript demonstrates the blending of peptides (coassembly) and the formation of a &#34;beads on a string&#34;-like structure by this process.9 The protocols presented here can be adapted to other classes of peptides or biological building blocks and can potentially lead to the discovery of new peptide-based structures and to better control of their assembly.

This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based assemblies.

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

Preparation of Hydrophobic Metal-Organic Frameworks via Plasma Enhanced Chemical Vapor Deposition of Perfluoroalkanes for the Removal of Ammonia

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high surface area microporous materials, such as metal-organic frameworks (MOFs), unique challenges present themselves for PECVD treatments. Herein the protocol for development of a MOF that was previously unstable to humid conditions is presented. The protocol describes the synthesis of Cu-BTC (also known as HKUST-1), the treatment of Cu-BTC with PECVD of perfluoroalkanes, the aging of materials under humid conditions, and the subsequent ammonia microbreakthrough experiments on milligram quantities of microporous materials. Cu-BTC has an extremely high surface area (~1,800 m2/g) when compared to most materials or surfaces that have been previously treated by PECVD methods. Parameters such as chamber pressure and treatment time are extremely important to ensure the perfluoroalkane plasma penetrates to and reacts with the inner MOF surfaces. Furthermore, the protocol for ammonia microbreakthrough experiments set forth here can be utilized for a variety of test gases and microporous materials.

Herein the procedures for plasma enhanced chemical vapor deposition of perfluoroalkanes on microporous materials such as metal-organic frameworks to enhance their stability and hydrophobicity are described. Furthermore, breakthrough testing of milligram quantities of samples is described in detail.

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high ...

Preparation of Highly Porous Coordination Polymer Coatings on Macroporous Polymer Monoliths for Enhanced Enrichment of Phosphopeptides

We describe a protocol for the preparation of hybrid materials based on highly porous coordination polymer coatings on the internal surface of macroporous polymer monoliths. The developed approach is based on the preparation of a macroporous polymer containing carboxylic acid functional groups and the subsequent step-by-step solution-based controlled growth of a layer of a porous coordination polymer on the surface of the pores of the polymer monolith. The prepared metal-organic polymer hybrid has a high specific micropore surface area. The amount of iron(III) sites is enhanced through metal-organic coordination on the surface of the pores of the functional polymer support. The increase of metal sites is related to the number of iterations of the coating process.
The developed preparation scheme is easily adapted to a capillary column format. The functional porous polymer is prepared as a self-contained single-block porous monolith within the capillary, yielding a flow-through separation device with excellent flow permeability and modest back-pressure. The metal-organic polymer hybrid column showed excellent performance for the enrichment of phosphopeptides from digested proteins and their subsequent detection using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The presented experimental protocol is highly versatile, and can be easily implemented to different organic polymer supports and coatings with a plethora of porous coordination polymers and metal-organic frameworks for multiple purification and/or separation applications.

A procedure for the preparation of porous hybrid separation media composed of a macroporous polymer monolith internally coated by a high surface area microporous coordination polymer is presented.

We describe a protocol for the preparation of hybrid materials based on highly porous coordination polymer coatings on the internal surface of macroporous ...

A Filter-based Surface Enhanced Raman Spectroscopic Assay for Rapid Detection of Chemical Contaminants

We demonstrate a method to fabricate highly sensitive surface-enhanced Raman spectroscopic (SERS) substrates using a filter syringe system that can be applied to the detection of various chemical contaminants. Silver nanoparticles (Ag NPs) are synthesized via reduction of silver nitrate by sodium citrate. Then the NPs are aggregated by sodium chloride to form nanoclusters that could be trapped in the pores of the filter membrane. A syringe is connected to the filter holder, with a filter membrane inside. By loading the nanoclusters into the syringe and passing through the membrane, the liquid goes through the membrane but not the nanoclusters, forming a SERS-active membrane. When testing the analyte, the liquid sample is loaded into the syringe and flowed through the Ag NPs coated membrane. The analyte binds and concentrates on the Ag NPs coated membrane. Then the membrane is detached from the filter holder, air dried and measured by a Raman instrument. Here we present the study of the volume effect of Ag NPs and sample on the detection sensitivity as well as the detection of 10 ppb ferbam and 1 ppm ampicillin using the developed assay.

A procedure for fabrication and performing the filter-based surface enhanced Raman spectroscopic (SERS) assay for the detection of chemical contaminants (i.e., pesticide ferbam and antibiotic ampicillin) is presented.

We demonstrate a method to fabricate highly sensitive surface-enhanced Raman spectroscopic (SERS) substrates using a filter syringe system that can be ...

Construction of Models for Nondestructive Prediction of Ingredient Contents in Blueberries by Near-infrared Spectroscopy Based on HPLC Measurements

Nondestructive prediction of ingredient contents of farm products is useful to ship and sell the products with guaranteed qualities. Here, near-infrared spectroscopy is used to predict nondestructively total sugar, total organic acid, and total anthocyanin content in each blueberry. The technique is expected to enable the selection of only delicious blueberries from all harvested ones. The near-infrared absorption spectra of blueberries are measured with the diffuse reflectance mode at the positions not on the calyx. The ingredient contents of a blueberry determined by high-performance liquid chromatography are used to construct models to predict the ingredient contents from observed spectra. Partial least squares regression is used for the construction of the models. It is necessary to properly select the pretreatments for the observed spectra and the wavelength regions of the spectra used for analyses. Validations are necessary for the constructed models to confirm that the ingredient contents are predicted with practical accuracies. Here we present a protocol to construct and validate the models for nondestructive prediction of ingredient contents in blueberries by near-infrared spectroscopy.

We present here a protocol to construct and validate models for nondestructive prediction of total sugar, total organic acid, and total anthocyanin content in individual blueberries by near-infrared spectroscopy.

Nondestructive prediction of ingredient contents of farm products is useful to ship and sell the products with guaranteed qualities. Here, near-infrared ...

Synthesis of Cyclic Polymers and Characterization of Their Diffusive Motion in the Melt State at the Single Molecule Level

We demonstrate a method for the synthesis of cyclic polymers and a protocol for characterizing their diffusive motion in a melt state at the single molecule level. An electrostatic self-assembly and covalent fixation (ESA-CF) process is used for the synthesis of the cyclic poly(tetrahydrofuran) (poly(THF)). The diffusive motion of individual cyclic polymer chains in a melt state is visualized using single molecule fluorescence imaging by incorporating a fluorophore unit in the cyclic chains. The diffusive motion of the chains is quantitatively characterized by means of a combination of mean-squared displacement (MSD) analysis and a cumulative distribution function (CDF) analysis. The cyclic polymer exhibits multiple-mode diffusion which is distinct from its linear counterpart. The results demonstrate that the diffusional heterogeneity of polymers that is often hidden behind ensemble averaging can be revealed by the efficient synthesis of the cyclic polymers using the ESA-CF process and the quantitative analysis of the diffusive motion at the single molecule level using the MSD and CDF analyses.

A protocol for the synthesis and characterization of diffusive motion of cyclic polymers at the single molecule level is presented.

We demonstrate a method for the synthesis of cyclic polymers and a protocol for characterizing their diffusive motion in a melt state at the single ...

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals leads to the formation of composite materials with unique properties. In addition, the undesirable process of biofouling is initiated by the adsorption of biomolecules, mainly proteins, on surfaces. This organic layer is an adhesion layer for bacteria and allows them to interact with the surface. Understanding the fundamental forces that govern the interactions at the organic-inorganic interface is therefore important for many areas of research and could lead to the design of new materials for optical, mechanical and biomedical applications. This paper demonstrates a single-molecule force spectroscopy technique that utilizes an AFM to measure the adhesion force between either peptides or amino acids and well-defined inorganic surfaces. This technique involves a protocol for attaching the biomolecule to the AFM tip through a covalent flexible linker and single-molecule force spectroscopy measurements by atomic force microscope. In addition, an analysis of these measurements is included.

Here we present a protocol to measure the force of interactions between a well-defined inorganic surface and either peptides or amino acids by single-molecule force spectroscopy measurements using an atomic force microscope (AFM). The information obtained from the measurement is important to better understand the peptide-inorganic material interphase.

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals ...

Observation and Analysis of Blinking Surface-enhanced Raman Scattering

From a single molecule at a silver nanoaggregate junction, blinking surface-enhanced Raman scattering (SERS) is observed. Here, a protocol is presented on how to prepare the SERS-active silver nanoaggregate, record a video of certain blinking spots in the microscopic image, and analyze the blinking statistics. In this analysis, a power law reproduces the probability distributions for bright events relative to their duration. The probability distributions for dark events are fitted by a power law with an exponential function. The parameters of the power law represent molecular behavior in both bright and dark states. The random walk model and the speed of the molecule across the entire silver surface can be estimated. It is difficult to estimate even when using averages, autocorrelation functions, and super-resolution SERS imaging. In the future, power law analyses should be combined with spectral imaging, because the origins of blinking cannot be confirmed by this analysis method alone.

This protocol describes the analysis of blinking surface-enhanced Raman scattering due to the random walk of a single molecule on a silver surface using power laws.

From a single molecule at a silver nanoaggregate junction, blinking surface-enhanced Raman scattering (SERS) is observed. Here, a protocol is presented on ...

Synthesis and Characterization of 1,2-Dithiolane Modified Self-Assembling Peptides

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting self-assembled supramolecular structures. The synthetic route takes advantage of solid-phase peptide synthesis with the on-resin coupling of the dithiolane precursor molecule, 3-(acetylthio)-2-(acetylthiomethyl)propanoic acid, and the microwave-assisted thioacetate deprotection of the peptide N-terminus before final cleavage from the resin to yield the 1,2-dithiolane modified peptide. After the high-performance liquid chromatography (HPLC) purification of the 1,2-dithiolane peptide, derived from the nucleating core of the A&#946; peptide associated with Alzheimer&#39;s disease, the peptide is shown to self-assemble into cross-&#946; amyloid fibers. Protocols to characterize the amyloid fibers by Fourier-transform infrared spectroscopy (FT-IR), circular dichroism spectroscopy (CD) and transmission electron microscopy (TEM) are presented. The methods of N-terminal modification with a 1,2-dithiolane moiety to well-characterized self-assembling peptides can now be explored as model systems to develop post-assembly modification strategies and explore dynamic covalent chemistry on supramolecular peptide nanofiber surfaces.

A protocol for the synthesis of a 1,2-dithiolane modified peptide and the characterization of the supramolecular structures resulting from the peptide self-assembly.