Jing-Xiao Chen

Construction of cell penetrating peptide vectors with N-terminal stearylated nuclear localization signal for targeted delivery of DNA into the cell nuclei

Cellular uptake and nuclear localization are two barriers to gene delivery. Here, we designed new gene delivery carriers with an N-terminal stearylated (STR) nuclear localization signal (NLS), PKKKRKV, present in the Simian Virus 40 large T antigen with the aim to overcome limitations, such as cell membrane and nuclear pores, offering attractive possibilities to enhance gene delivery. Four vectors with different structures of N-stearylated nuclear localization signal-octaarginine peptide (STR-PKKKRKV-R(8) or STR-NLS-R(8), STR-VKRKKKP-R(8) or STR-reverse NLS-R(8), PKKKRKV-R(8) or NLS-R(8), and VKRKKKP-R(8) or reverse NLS-R(8)) were compared. The gene expression mediated by these vectors in dividing and non-dividing cells (both in 293T and HeLa cell lines) was investigated. The most efficient N/P ratio was 4 for STR-PKKKRKV-R(8,) STR-VKRKKKP-R(8,) and 0.25 for PKKKRKV-R(8), VKRKKKP-R(8.) The maximum transfection activity of these vehicles (VKRKKKP-R(8)) was up to 80% as effective as jetPEI™ and the vehicles did not exhibit cytotoxicity. Interestingly, N-stearylated peptides presented lower transfection activity compared to peptides without N-stearylation at lower N/P ratios (0.25 to 1). Confocal study showed that the vectors could effectively promote the nuclear translocation.

Construction of surfactant-like tetra-tail amphiphilic peptide with RGD ligand for encapsulation of porphyrin for photodynamic therapy

A surfactant-like tetra-tail amphiphilic peptide, [(C(18))(2)K](2)KR(8)GRGDS was designed and synthesized for targeted drug delivery. The resulting peptide-amphiphile, consisting of four hydrophobic aliphatic tails and a hydrophilic peptide head group, was able to self-assemble into nanosized micelles in aqueous medium at low concentration. Ibuprofen and doxorubicin (DOX) was loaded into peptide micelles as model hydrophobic drugs respectively, and the sustained release behavior was observed. Due to the incorporation of targeted arginine-glycine-aspartic acid (RGD) sequences and cell penetrating peptide (CPP) residue octaarginine (R(8)), the micelles could be recognized specifically by cancer cells, as well as transport through the cell membrane efficiently. The observation of laser-scanning confocal microscopy confirmed effective cellular uptaking of porphyrin-loaded peptide micelles. Furthermore, the porphyrin-loaded micelles exhibited low dark toxicity and high phototoxicity against cancer cells, indicating the powerful potential for effective photodynamic therapy. Combined with the low cytotoxicity of the peptide against both HeLa and 293T cell lines, the surfactant-like peptide developed in this study may be promising in clinical application for targeted drug delivery.

Multi-functional envelope-type nanoparticles assembled from amphiphilic peptidic prodrug with improved anti-tumor activity.

A novel multifunctional amphiphilic peptidic prodrug was reported here by conjugating the antitumor drug of doxorubicin (DOX) to the hydrophobic tail of a designed peptide-amphiphile (PA), in which the hydrophilic peptide headgroup comprises a glycine-arginine-glycine-aspartic acid-serine (GRGDS) sequence and octaarginine (R8) sequence. Because of the amphiphilic nature, this peptidic prodrug can spontaneously self-assemble into spherical multifunctional envelop-type nanoparticles (MENPs) with the functional peptide sequences gathered on surface. By means of the multifunctions of RGD-mediated tumor targeting, R8-mediated membrane penetration and intracellular protease-mediated hydrolyzing peptide bonds, the MENPs could targeted deliver doxorubicin (DOX) to tumor cells, showing improved antitumor activity both in vitro and in vivo with much reduced side effects.

Self-Assembled BolA-like Amphiphilic Peptides as Viral-Mimetic Gene Vectors for Cancer Cell Targeted Gene Delivery

In this study, two types of BolA-like amphiphilic peptides with dual ligands comprising a tumor-targeting moiety of RGD sequence and a cell-penetrating moiety of R8 sequence are designed and synthesized as gene vectors. The BolA-structural peptide carriers can self-assemble into spherical nanoparticles with a hydrophilic core and shell, which are similar to the viral capsid and can bind plasmid DNA in an aqueous medium to form viral-mimetic complexes. It is found that the BolA-like dual ligands system exhibits significantly enhanced gene expression in both HeLa and 293T cell lines, as compared with poly(ethylenimine) PEI. These BolA-like amphiphilic peptides are promising in clinical trials of gene therapy.

Facile Construction of Nanofibers as a Functional Template for Surface Boron Coordination Reaction

A facile strategy to perform the boron coordination reaction on a template of nanofibers is developed. Peptides with phenylboronic acid tails (peptidyl boronic acids) are designed and prepared as building blocks that can self-assemble into nanofibers. After the addition of vicinal diol structural motifs to the self-assembling system, matrix-assisted laser desorption-ionization time-of-flight mass spectrometry indicates that the boron coordination reaction occurs on the template of nanofibers, which results in the increase of the width and roughness of the nanofibers as demonstrated by transmission electron microscopy and atomic force microscopy measurements. Because the surface-bound vicinal diol structural motifs have an ability to form hydrogen bonds with the peptide segments on the nanofibers, which restrain and disturb the hydrogen-bonding interaction among the nanofibers, the network structure formed based on the entanglement of nanofibers via hydrogen-bonding interaction is destroyed, which leads to a gel-sol transition. The novel concept of post-self-assembly modification demonstrated here could lead to a new technique for using self-assembled nanostructures in the emerging fields of nanoscience and nanotechnology.

Supramolecular architectures self-assembled from asymmetrical hetero cyclopeptides.

In this study, two asymmetrical cyclopeptides (CP1 and CP2) were designed and synthesized. The self-assembly behaviors of the asymmetrical cyclopeptides at varying pHs were investigated in terms of transmission electron microscopy (TEM), circular dichroism (CD), and Fourier transform infrared (FT-IR) spectroscopy. It was found that the self-assembly of CP1 resulted in the formation of nanofibers with α-helix conformation, while CP2 self-assembled into well-ordered nanorods with anti-parallel β-sheet conformation. The strategy demonstrated here presents great potential for preparation of well-defined nanostructures via rationally designing the molecular structures of cyclopeptides.

Novel gene transfer vectors based on artificial recombinant multi-functional oligopeptides

Viral vectors, except for their safety concern, have shown high efficiency in both delivery and expression of gene. Here, a series of new gene carriers, comprised of short peptide subunits with special functions to imitate viral vectors, were designed and three vectors, (C(18))(2)KH(4)R(8)GDS, AcKH(4)R(8)GDS and (C(18))(2)KH(4)R(8), designated as ARM1, ARM2, ARM3, respectively, were synthesized and evaluated. The transfection efficiency in vitro was studied in terms of 293T, HepG2 and HeLa cell lines. It was found that the transfection efficiency was enhanced significantly for the vectors (ARM1 and ARM3) with double hydrophobic aliphatic tails. Interestingly, the conjugation of RGDS sequence in vectors displayed no obvious difference in cell adhesion for all of the three cell lines. Moreover, confocal laser scanning microscope results indicated that the peptide/pDNA complexes can enter the cell and nuclei successfully. On the other hand, all the vectors displayed low cytotoxicity. The artificial recombinant multi-block oligopeptides (ARMs) demonstrated here might give a promising potential of the peptide-based vectors in gene therapy.