Despite the widely held belief that hydrophilic molecules cannot pass through the plasma membrane, the discovery of cell-entering proteins was first reported in the late 80s. The results show that the HIV Trans-Activator of Transcription (Tat) protein may effectively infiltrate cells grown in a lab and boost viral gene expression. It was also discovered that the Drosophilia melanogaster transcription factor Antennapedia homeodomain could access nerve cells and control the process of neural morphogenesis. Both proteins' intriguing spontaneous entrance prompted comprehensive structure-function analyses to determine the minimal amino acid sequence required for absorption. Currently, it can be stated that CPPs are short peptides (often not surpassing 40 residues) that possess the ability to traverse biological membranes universally with little toxicity, through energy-dependent and/or independent pathways, without requiring chiral recognition by particular receptors. The most cell-penetrating peptides (CPPs) are positively charged, however, a limited number of anionic or hydrophobic CPPs have also been identified. The involvement of a primary or secondary amphipathic component is suggested, albeit not absolutely necessary for internalization.
The first CPPs were identified as a consequence of this process: penetratin, which corresponds to the third helix of the Antennapedia homeodomain, and Tat peptide, which corresponds to the basic domain of the HIV-1 Tat protein. This led to the discovery or rational design of more peptides with similar penetrating capabilities. The capacity to effectively transport cargo across cell membranes is a shared advantage of all CPPs.
Creative Peptides offers a diverse portfolio of cell-penetrating peptides with stable and efficient transmembrane delivery capabilities, supporting a wide range of research applications. Backed by standardized production and quality control systems, we provide high-purity, customizable solutions to facilitate tool development and mechanistic studies. We currently offer the following categories of CPP products:
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Cell-penetrating peptides are able to traverse cell membranes primarily due to their unique structural characteristics. Despite wide variability in sequence and function, many CPPs share common features that enable membrane penetration, including cationic (positively charged) and amphipathic (having both hydrophilic and hydrophobic regions) properties, which facilitate interactions with the negatively charged cell membrane.
CPPs are predominantly composed of linear sequences of amino acids, though their specific structures can vary across different types. Their positive charge is largely attributed to a high content of basic amino acids such as arginine and lysine. For instance, arginine-rich CPPs like the HIV-1 TAT (Trans-Activator of Transcription) peptide form strong electrostatic interactions with negatively charged membrane components, including phospholipids and proteoglycans, via the guanidinium group. Lysine-rich CPPs also exploit their positive charge for membrane interactions, although their translocation mechanisms may differ slightly from arginine-rich CPPs.
The secondary structure of CPPs significantly influences their ability to engage with the lipid bilayer. Many amphipathic CPPs adopt an α-helical conformation, where one side is polar (hydrophilic) and the other is non-polar (hydrophobic). This arrangement allows the hydrophobic face to penetrate the lipid bilayer while the hydrophilic side interacts with the aqueous environment, facilitating membrane engagement. Some CPPs form β-sheet structures with amphipathic characteristics, further stabilizing interactions with the membrane.
Amphipathic CPPs rely on distinct hydrophilic and hydrophobic regions for effective translocation. The hydrophobic segment interacts with the fatty acyl chains of the lipid bilayer, while the hydrophilic segment, typically enriched in arginine or lysine residues, engages with the polar head groups of lipids. This dual affinity enhances the peptide's ability to enter and exit the membrane. For example, Transportan is a hybrid CPP combining the hydrophobic domain of mastoparan with the hydrophilic domain of galanin, whose amphipathic nature underlies its efficient membrane penetration.
The abundance of positively charged amino acids, such as arginine and lysine, is a hallmark of CPPs. Electrostatic interactions between these cationic residues and the negatively charged membrane components are critical for initiating translocation. In arginine-rich CPPs, guanidinium groups facilitate membrane bridging by forming bidentate hydrogen bonds with phosphate groups in lipid bilayers, strengthening peptide-membrane interactions and promoting efficient cellular uptake.
This categorization identifies three primary categories of CPPs: 1) peptides originating from proteins, 2) chimeric peptides formed from two natural sequences, and 3) synthetic peptides designed based on structural-activity analyses.
The first category contains peptides derived from the N-terminal region of the capsid protein (CaP) of a plant-infecting Brome mosaic virus (BMV), such as Tat from the human immunodeficiency virus (HIV), penetratin, VP22 from the herpes simplex virus, and a 22-residue peptide from the same plant. These protein-derived CPPs naturally exhibit efficient membrane translocation, enabling intracellular delivery of various biomolecules. Their conserved structural motifs, including cationic and amphipathic regions, facilitate interactions with negatively charged cell membranes, supporting broad applications in research and molecular transport.
Synthetic peptides, including MAPs and polyarginine peptides, make up the last category of CPPs. Pep-1, the first peptide with three domains and twenty-one amino acids, was synthesized. A spacer domain, a hydrophilic lysine-rich domain derived from the nuclear localization sequence (NLS) of the Simian virus 40 (SV40) big T antigen, and a hydrophobic domain with many tryptophan residues make up the three components. The third one strengthens the stability and adaptability of the first two.
Chimeric peptides make up the second class of CPPs; these peptides inherit two or more patterns from different peptides and are partially generated from proteins or peptides found in nature. Take transportan, a 27-amino-acid peptide that shares a lysine bridge with mastoparan, a peptide produced from the venom of the Vespula lewisii wasp, and 12 functional amino acids from the neuropeptide galanin at its amino terminus. Mastoparan is the source of TP10, a peptide of 21 residues. Wasp venom provides the 14-residue peptide that is fused with the 6-residue sequence of the neuropeptide galanin by means of an additional lysine residue. In order to drive proteins into an intracellular compartment, certain chimeric CPPs, like NLS-derivatives, contain signal sequences that are recognized by acceptor proteins on the membrane of the relevant intracellular organelles.
The majority of CPPs get access to cells by endocytosis, a process by which cells selectively absorb nutrients, ligands, hormones, and other compounds from the outside. Endosomes are vesicular structures that are produced by the endocytic process through the use of chemical energy and are able to budded from the plasma membrane. The vesicles are then brought into the cell's interior. Phagocytosis, macropinocytosis, receptor-mediated endocytosis, and receptor-independent endocytosis are all types of endocytosis that have been documented.
Table.1 Three mechanisms of action of cell-penetrating peptides have been reported.
| Phagocytosis | Phagocytosis often occurs in particular scavenger cells, including macrophages. They are tasked for absorbing substantial particles over 0.5 μm in size. Particles possessing positive surface charges aggregate significantly in physiological fluids and are swiftly eliminated from the circulation by mononuclear phagocytes of the reticuloendothelial system in the liver or spleen. This mechanism is the primary cause for the premature removal of positively charged CPPs from circulation before to their entry into the targeted cells. Nevertheless, the phagocytic route may remain advantageous for CPP-mediated transport of particular antigens to dendritic cells for immunization purposes. |
| Macropinocytosis | During macropinocytosis, the plasma membrane is stretched out to encircle a vast quantity of non-specific extracellular fluids and molecules, allowing them to be ingested. Microscopic vesicles enclosing the extracellular matrix are known as macropinosomes. Although reports of macropinosomes larger than a micrometer are infrequent, their typical size range is 200–500 nm. The aggressive polymerization of actin filaments is directly linked to the enormous remodeling of the plasma membrane. Aggregates of CPP or CPP-cargo conjugates may cause macropinocytosis and plasma membrane remodeling for extensive membrane extension, according to one theory. This process might take place as a result of interactions between CPP and CPP or within CPP and membranes. |
| Endocytosis | Only a tiny fraction of the plasma membrane is used for receptor-mediated and receptor-independent endocytosis. The membrane is lined with clathrin and caveolin proteins, which aid in the budding of the inner endosome to the cytosol. The process of endosome membrane severing is also facilitated by dynamin. In terms of diameter, endosomes coated with clathrin are around 50-150 nm while those coated with caveolin are 50-80 nm. Caveolin facilitates endocytosis from lipid rafts, which are specialized regions of the plasma membrane, whereas clathrins are involved in over half of the receptor-mediated endocytosis processes. There is a strong correlation between ligand-receptor binding and these CPP-induced endocytic processes. But CPP can nonetheless trigger endocytotic pathways in the absence of a particular ligand. A specific GAG called the heparan sulfate proteoglycan receptor (HSPG) is responsible for triggering endocytosis when it detects CPPs that have positive charges. |
While designing CPPs, it is important to prioritize their ability to traverse the cell membrane, as well as their target selectivity, biocompatibility, and cargo-binding capabilities. Many structural features are shared, including cationic residues, amphipathic structures, and the capacity to conjugate with many cargo types. The following criteria were used to design the peptide: (i) keeping the peptide sequence short so it could still penetrate cells and bind nucleic acids; (ii) including at least six arginine residues, which is the minimum number needed for cell uptake; (iii) adding tryptophan residues so it could interact hydrophobically with cell membranes; (iv) inserting histidine residues at the core so it could escape from endosomes depending on pH; and (v) adding cysteine residues at the ends to make the peptide more stable and facilitate cargo release once inside the cell.
One common method for synthesizing CPPs is solid-phase peptide synthesis (SPPS), which involves adding amino acids in a specific order to create the desired peptide sequence. The efficiency and variety of SPPS have completely changed the game when it comes to peptide synthesis. It's now possible to create complex peptides, even ones with chemical alterations or non-natural amino acids. After synthesis, peptides undergo purification, typically via high-performance liquid chromatography (HPLC), and are characterized using mass spectrometry to confirm sequence accuracy and purity. This ensures reliable performance in cellular delivery, preserves structural integrity, and allows for subsequent conjugation with cargo molecules or functional modifications.
The capacity of cell-penetrating peptides to cross biological membranes and introduce various compounds into cells gives them a wide variety of potential uses. Many other areas can benefit from these, such as molecular biology research tools, gene therapy, diagnostics, and medication delivery.
The utilization of CPPs as transporters for medicinal substances that are incapable of penetrating cell membranes is on the rise. Proteins, peptides, and smaller molecules are all part of this category, as are bigger biologics. Conjugated polypeptides and treatments that are well-developed provide a potential means to deliver less hazardous drug concentrations to vital tissues, including cancers, the heart, and so on. Additionally, CPPs have been used to transport small chemotherapeutic medicines such as doxorubicin, methotrexate, cyclosporine A, and paclitaxel.
Plasmid DNA, small interfering RNA (siRNA), or mRNA can all be delivered into cells by CPPs. The field of gene editing, silencing, and modulation stands to benefit greatly from this. The transport of siRNA-used to inhibit the expression of particular genes-can be facilitated by CPPs. By blocking genes that cause disease, this method has a lot of potential in cancer treatment, antiviral drugs, and genetic diseases. Researchers have used CPP-siRNA conjugates to inhibit tumor development by downregulating oncogenes in cancer cells.
The enormous size and hydrophilic nature of proteins and peptides make their delivery into cells a formidable hurdle. To get bioactive peptides and proteins into cells for study or therapy, CPPs are utilized, and they offer a way to get past this barrier. The enormous size and hydrophilic nature of proteins and peptides make their delivery into cells a formidable hurdle. To get bioactive peptides and proteins into cells for study or therapy, CPPs are utilized, and they offer a way to get past this barrier.
For the purpose of live-cell imaging, CPPs can transport fluorescent probes into cells. Biological researchers utilize this to see where proteins are located inside cells, observe intracellular activities, and keep tabs on how cells work. Non-invasive imaging of disorders like cancer or cardiovascular diseases is made possible by conjugating CPPs with diagnostic molecules, such as radioactive isotopes or magnetic resonance imaging contrast agents. Using these instruments, one may monitor the development of a disease or the success of a therapy in real time.
It is usual practice to transport medicinal substances in the form of gold nanoparticles, quantum dots, or liposomes. These nanoparticle delivery techniques exhibit enhanced tissue and cell penetration when coupled with CPPs. Conjugating CPPs to nanoparticles enhances cellular uptake and enables precise targeting, improving tissue penetration, bioavailability, and intracellular delivery. This combination facilitates efficient transport of therapeutic or diagnostic agents while minimizing off-target effects and maximizing functional efficacy.
Fig.1 Recent techniques based on CPPs1,2.
Cell penetrating peptides (CPPs) facilitate the transport of various molecular cargo across cellular membranes by directly penetrating or exploiting endocytic pathways, improving drug delivery.
Yes, certain peptides, known as cell penetrating peptides, possess the ability to translocate across cell membranes, enabling them to deliver therapeutic agents intracellularly.
CPPs can be divided into different categories based on their origin and structure, such as cationic, amphipathic, and hydrophobic peptides, each utilizing distinct mechanisms for membrane translocation.
Specific examples include TAT peptides, penetratins, and transportans. Each has unique properties that facilitate their uptake and internalization by cells.
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