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

Summary

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

Abstract

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.

Introduction

The past few decades have witnessed rapid advances in the development of cell-penetrating peptides (CPPs) for drug delivery. CPPs have been widely used as molecular transporters for the treatment of a range of life-threatening diseases, including neurological disorders^1,2, heart diseases³, diabetes⁴, dermatosis⁵, and cancer^6,7. Cancer remains a global health burden accompanied by a high rate of morbidity and mortality despite widespread research efforts⁸. A ....

Protocol

1. Equipment preparation

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

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

Results

In this protocol, a synthetic procedure to constrain the linear polyarginine R8 into its cyclic form was presented. The SPPS was conducted manually using a simple apparatus (Figure 1). The detailed synthetic process of SPPS is shown in Figure 2. Briefly, the resin was sufficiently swelled, followed by deprotection of the N^α-Fmoc protecting group. Then, the N^α-Fmoc-protected amino acid was anchored on the resin unt.......

Discussion

The chemical stabilization of peptides by incorporating conformational constraints has proven to be an effective strategy for improving the stability and cell permeability of the peptide²⁶. In this protocol, a step-by-step procedure is described for the synthesis of cyclic CPPs with aromatic cross-links and the evaluation of their permeability across biological barriers. Compared to the hydrophilic lactam or triazole cross-links^22,27, the .......

Materials

Name	Company	Catalog Number	Comments
1,2-ethanedithiol	Aladdin	K1722093	stench
2-(7-Azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU)	HEOWNS	A-0443697
4,4'-bis(bromomethyl)biphenyl	TCI	B1921
4T1 cells	ATCC		4T1 cells were cultured in DMEM medium supplemented with 10% FBS (Hyclone) in a 37 °C humidified incubator containing 5% CO2.
Acetonitrile	Adamas	1484971	toxicity
Dichloromethane	Energy	W330229	skin harmful
Diethyl ether	Aldrich	673811	flammable
Dimethyl sulfoxide	Beyotime	ST038	skin harmful
Dulbecco’s Modified Eagle Medium (DMEM)	Gibco
Electrospray Ionization Mass Spectrometer	Waters	G2-S Tof
Ethylene Diamine Tetraacetic Acid (EDTA)	BioFroxx	1340
Fetal bovine serum (FBS)	HyClone
Flow cytometer	Beckman Coulter	CytoFLEX
Fluorescein isothiocyanate isomer (FITC)	Energy	E0801812500
Fluorescent microscope	Carl Zeiss	Axio Observer 7
Fmoc-Arg(Pbf)-OH	HEOWNS	F-81070
Fmoc-Cys(Trt)-OH	GL Biochem	GLS201115-35202
Fmoc-βAla-OH	Adamas	51341C
HeLa cells	ATCC		HeLa cells were cultured in DMEM supplemented with 10% FBS (Hyclone) in a 37 °C humidified incubator containing 5% CO2.
High-Performance Liquid Chromatography	Agilent	Agilent 1260
High-Performance Liquid Chromatography column	Agilent	Poroshell EC-C18 120, 4.6 × 150 mm (pore size 120 Å, particle size 4 μm)
Lyophilizer	SP Scientific	Vir Tis
Methanol	Aldrich	9758	toxicity
Microtiter plate	Thermo μdrop plate	N12391
Morpholine	HEOWNS	M99040	irritant
Multi-technology microplate reader	Thermo	VARIOSKAN LUX
N,N-Diisopropylethylamine	HEOWNS	E-81416	irritant
N,N-Dimethyl formamide	Energy	B020051	harmful to skin
Poly-Prep column	Bio-Rad	7321010	polypropylene chromatography columns
Rink Amide MBHA resin (0.572 mmol/g)	GL Biochem	GLS180301-49101
Three-way stopcocks	Bio-Rad	7328107
Tissue culture plate insert	LABSELECT	14211
Trifluoroacetic acid	HEOWNS	T63278	corrosive
Triisopropylsilane	HEOWNS	T-0284475
Trypsin	BioFroxx	1004
Vacuum manifold	Promega	A7231