Cell-penetrating peptide
[6] A recent discovery found that Papillomaviridae, such as the human papillomavirus, use CPPs to penetrate the intracellular membrane to trigger retrograde trafficking of the viral unit to the nucleus.
[7] Cell-penetrating peptides are of different sizes, amino acid sequences, and charges, but all CPPs have the ability to translocate the plasma membrane and facilitate the delivery of various molecular cargoes to the cytoplasm or an organelle.
[8][9] Cell-penetrating peptides (CPP) are able to transport different types of cargo molecules across plasma membrane; thus, they act as molecular delivery vehicles.
This mechanism explains how key ingredients, such as the cooperation among the peptides, the large positive charge, and specifically the guanidinium groups, contribute to the uptake.
According to this model, a penetratin dimer combines with the negatively charged phospholipids, thus generating the formation of an inverted micelle inside of the lipid bilayer.
Increasing the amount of peptide on the outer leaflets causes the electric field to reach a critical value that can generate an electroporation-like event.
Two similar models have been proposed based on physicochemical studies, consisting of circular dichroism, Fourier transform infrared, and nuclear magnetic resonance spectroscopy.
[29] Nucleic acid-based macromolecules such as siRNA, antisense oligonucleotide, decoy DNA, and plasmid are promising biological and pharmacological therapeutics in regulation of gene expression.
[30][31][32] However, unlike other small-molecular drugs, their development and applications are limited by high molecular weight and negative charges, which results in poor uptake efficiency and low cellular traffic.
[35] Thus, a new non-covalent strategy requiring no chemical modification with short amphipathic CPPs, like MPG and Pep-1 as carriers has been successfully applied for delivery of cargoes.
In one study, MPG/siRNA complexes formed through stable non-covalent strategy showed successful introduction of siRNA into cultured cells and induced robust regulation of target mRNA.
[41] MPG forms stable complexes with siRNA with a low degradation rate and can be easily functionalized for specific targeting, which are major advantages compared with the covalent CPP technology.
Secondary amphipathic peptides based on aromatic tryptophan and arginine residues linked with lysine as spacer have been reported under the name of CADY.
[45] CPP-PMO conjugates have also been successfully used to inhibit the replication of several viruses such as SARS[46] and influenza[47] and attachment of CPPs has improved the efficacy of splice-modifying Morpholinos in development for treatment of Duchenne muscular dystrophy[48] Decoy DNA is an exogenous double-strand DNA (dsDNA), which can mimic a promoter sequence that can inhibit the activity of a specific transcription factor.
A method using macro-branched TAT has been proposed for plasmid DNA delivery into various cell lines and showed significant transfection capabilities.
[55][56] Recently, several methods using CPPs as vehicles to deliver biologically active, full-length proteins into living cells and animals have been reported.
In one study, TAT-fused proteins are rapidly internalized by lipid raft−dependent macropinocytosis using a transducible TAT−Cre recombinase reporter assay on live cells.
Moreover, cR10, a cyclic poly-arginine CPP, enabled the endocytose independent transduction of antigen binding proteins through the cellular membrane with immediate bioavailability.
These contrast agents are able to label the tumor cells, making the compounds important tools in cancer diagnosis; they are also used in in vivo and in vitro cellular experiments.
A new strategy to enhance the cellular up-take capacity of CPP is based on association of polycationic and polyanionic domains that are separated by a linker.
In vivo tests have shown that several positively charged peptides (based on guanidine residues) are able to cross cell membranes and to promote cellular uptake of attached molecules including quantum dots.
QD properties can be easily modified by changing the organic substrates linked to them, offering a versatile biological tool as cell markers.
[71][72][73][74][75] Quantum dots are colloidal nanocrystals, based on a cadmium-selenium (CdSe) core covered with a zinc-sulfur (ZnS) layer.
Colloidal QD emission can be modulated from UV-Vis to the infrared by using different types of coating agents, such as ZnS, CdS, ZnSe, CdTe and PbSe.
High-quality QD contrast agents are obtained at elevated temperatures; however, because they have lower water solubility, their usage as cell markers is limited.
[77] Magnetic resonance imaging (MRI) is a powerful tool for disease diagnosis such as cancer metastasis and inflammation, using different metal chelates.
Metal chelates increase the contrast signal between normal and diseased tissues by catalyzing the relaxation of water protons in their proximities.
Applications of SPIO includes cell labeling in vivo; due to low toxicity, they are clinically approved for use in liver, spleen, and gastrointestinal imaging.
[81] The presence of octamer arginine residues allows cell membrane transduction of various cargo molecules including peptides, DNA, siRNA, and contrast agents.
To solve this problem, contrast agents with disulfide, reversible bond between metal chelate and transduction moiety enhance the cell-associated retention.
