Cyclic Peptide Nanotubes: Definition, Structure, Classifications and Applications
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What are the cyclic peptide nanotubes?
Cyclic peptide nanotubes (cPNTs) are tubular entities formed by cyclic peptides that autonomously organize into stable nanotube configurations. These structures have demonstrated potential in several domains, including biomedicine and materials science, owing to their stability, adaptability, and capacity to enhance transport across biological membranes. The fundamental constituents of cPNTs are cyclopeptides composed of linear polypeptides with alternating chirality, linked at the head and tail. The rings assume a flat-ring conformation, with the C=O and N-H groups oriented almost perpendicular to the ring plane, so promoting ring stacking through inter-ring hydrogen bonding and self-assembly into elongated hollow tubular structures.
The sizes of nanotubes vary with the quantity of amino acid residues used. Research suggests that cyclic peptides comprising six, eight, ten, and twelve residues are optimal for the design and application of cyclic peptide nanotubes, as cyclic D, L-peptides with fewer than six residues exhibit excessive ring strain, while those exceeding twelve residues are excessively large to adopt a flat, ring-shaped conformation.
Schematic representation of nanotube formation from cyclic peptides. (Brea R J., et al., 2010)
Self-assembled nanotube from cyclic peptides. (Zhu J., et al., 2008)
Types of cyclic peptide nanotubes
Up to now, three distinct kinds of cPNT structures have been introduced: cyclic D,L-α-peptides, cyclic β-peptides, and cyclic α-γ-peptides. It has been demonstrated that these structures may be stacked into tubular configurations. The initial discovery was made in 1993 by Ghadiri's team of cyclic D,L-α-peptides. The amino acid side chain in these peptides splits apart under acidic circumstances, and the amide and carbonyl groups run perpendicular to the ring axis. Eventually, by self-assembly, they form an antiparallel β-sheet tubular structure. It was later demonstrated that cyclic β-peptides' self-assembling characteristics are comparable to those of cyclic D,L-α-peptides, and that the amino acid side chain extends equatorially from the peptide ring. In contrast, the carbonyl and amide groups are oriented in the opposite way along the tube's longitudinal axis, creating parallel stacking. The cyclic α-γ-peptide-based cPNTs have a distinct structure compared to the cyclic D,L-α-peptides and cyclic β-peptides. Its three types of hydrogen bonding patterns include γ-γ and α-α contacts, and its nonequivalent facing rings stack flat.
Categories of cyclic peptide nanotubes (cPNTs) by β-sheet stacking. (Hsieh W H., et al., 2019)
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Advantages of cyclic peptide nanotubes
Stability: Incorporating d-amino acids and cyclizing cPNTs were discovered to shield inclusion molecules from proteolytic degradation and biological pH through conformational regulation and steric hindrance, resulting in stable shuttles. For instance, in contrast to its linear analogue, the anticoagulative action of a cyclic analog of RGD peptide may be maintained for more than three hours. Similarly, the earlier research showed that cyclic D-Trp-Tyr-constructed plasmid DNA containing stable cPNTs could withstand acid, bile, and DNase I digestion for 50, 60, and 180 minutes, respectively, longer than unloaded plasmids. Bioavailability may be improved by controlling the amount of N-methylation on cyclic peptides, which in turn affects the stability of the metabolic enzymes on cPNTs. One possible explanation for the increased stability caused by N-methylation is that it prevents cleavage at the N-terminal D-Trp, Lys, and Phe sites. Another aspect that could affect stability is the vehicle's size. The reason why high-AR vehicles with lengths longer than 8 μm can stay in circulation for up to one week following intravenous injection in mice is because to the delayed cellular absorption under fluid flow circumstances. This was discovered by Geng's team. In a follow-up study on mice, researchers found that injecting paclitaxel (a hydrophobic anticancer drug) into a single tail vein along with 8 μm-long filomicelles improved the drug's circulation stability, which in turn reduced tumor size and increased drug exposure to cancer cells.
Enhancement of permeability: Another characteristic that allows cPNTs to transport molecules and attain high bioavailability is the improvement of permeability. Since active and passive transport are only two of many uptake processes that contribute to the absorption process, there are currently no standard criteria for the development of highly permeable cPNTs. The physiological process of cell absorption, surface characteristics, and molecule size, shape, weight, and rules of thumb are all worth considering. To explain the passive transport of cPNTs, Fernandez-Lopez's team put up a number of interesting processes, including the intramolecular pore, barrel stave, and carpet models. With its polyvalent display of surface-exposed hydrophilic side chains for potential interactions with various membrane constituents, the carpet model was believed to have the greatest potential for participation in membrane permeation among these three models. This characteristic has been confirmed by coarse-grained molecular dynamics (MD) simulation. In contrast, endocytosis and phagocytosis are energy-dependent entry mechanisms for bigger particles inside cells. In a similar vein, the earlier research shown that plasmid DNA could be made more permeable for in vitro duodenal penetration by an energy-dependent process when combined with high-AR cyclo-(D-Trp-Tyr) PNTs that ranged in width from 100 to 800 nm and length from 1 to 20 μm. Hence, efficient high-AR cPNT vehicles offer a flexible option to boost membrane absorption through active or passive mechanisms.
Sustained release: Additionally, cPNT carriers include guest molecules by electrostatic contact, hydrogen binding, or hydrophobic interaction, allowing for their continuous release. This property is appealing for biological applications. To illustrate the point, when small-molecule RhB was combined with diphenylalanine PNTs, the release occurred more slowly according to first-order kinetics than when RhB was used alone. The sustained-release features of diphenylalanine PNT complexation have been demonstrated using flufenamic acid, another small-molecule model. When cPNTs were combined with big molecules like DNA, the same outcomes were seen. As an illustration, our earlier research shown that, when complexed with cyclo-(D-Trp-Tyr) PNT, the DNA release rate was 3.57 × 1011copies DNA/t1/2, but when unformulated DNA was used, the rate was 5.92 × 1011 copies DNA/t1/2.
Applications of cyclic peptide nanotubes
Drug delivery
As a medication delivery agent, cPNTs have demonstrated promise. Their tubular shape allows them to encapsulate pharmaceuticals within their hollow centers, which increases their bioavailability and protects the drug molecules from degradation. Alterations in pH or temperature, for example, can trigger their programmed release of the medicine at a precise location. Due to their large surface area and unique hollow core and exterior surface characteristics, cPNTs have been shown in multiple studies to physically co-treat with anticancer drugs or to complex with anticancer drugs and modify their surface with polymers, both of which can lower the IC50 in different cancer cell lines when compared to the drug alone. The inclusion of a highly hydrophobic cyclic peptide, namely cyclo-[Gln-(D-Leu-Trp)4-D-Leu, improved the dose-dependent liposomal membrane release of the anticancer medication 5-FU, according to Chen's group. Additionally, the cytotoxic impact was amplified in BEL7402, HeLa, and S180 cells cotreated with 5-FU and these cPNTs, as demonstrated by the reduction in IC50. Physical cotreatment with cyclo-[Gln-(D-Leu-Trp)4-D-Leu] did not damage the cell membrane, according to additional mechanistic investigations and research. Drugs with a smaller internal diameter than cPNTs, as 5-FU (0.44 nm), may undergo dose-dependent penetration across the hydrophilic channel of cPNTs. The idea was put forth, based on steered MD simulations and conventional MD computation, that cPNTs move along cPNT subunits by converting 5-FU's hydrophobic interaction with the cPNTs' interior wall into hydrogen bonding interactions with the cPNTs' backbone carbonyl groups and amide groups.
Gene delivery
New evidence suggests that cyclic octa- and di-cPNTs can transport genes. As an example, cyclic octa-cPNTs composed of four l-lysines and four d-alanines with one lysine residue functionalized by a guanidiniocarbonyl pyrrole moiety, measuring 10-400 nm in width and micrometers in length, were discovered to interact with negatively charged calf thymus DNA through aggregates of positively charged lysine residues. These findings suggest that cyclic octa-cPNTs could potentially transport genes into cells bypassing the endocytotic pathway. The DNA-cPNT complex showed similar transfection outcomes to the positive controlled PEI group after 24 hours of incubation with HeLa cells, however it was less cytotoxic than PEI. Oral administration of cyclo-(D-Trp-Tyr) cPNTs with widths ranging from 100 to 800 nm and lengths ranging from 1 to 20 μm effectively delivered plasmid DNA in vivo in an energy-dependent fashion. The binding constant of cyclo-(D-Trp-Tyr) cPNTs with DNA was determined to be 3.2 × 108 M−1. The villous epithelium of duodenal villi, lobules, hepatocytes, sinusoidal endothelial cells near the portal vein of the liver, endothelial cells of the proximal tubular, and endothelial cells of the renal cortex were observed to express β-Gal or Renilla luciferase protein after receiving these cPNTs with either pCMV-LacZ or pCMV-hRluc plasmids through oral administration, respectively. The protein expression that was successful might have been due to the following: increasing stability at an acidic pH for 60 minutes, in bile for 180 minutes, and with enzymes present for 50 minutes. It could have also involved increasing permeability in the gastrointestinal tract from 49.2 ± 21.6 × 10−10 cm/s for naked DNA to 395.6 ± 142.2 × 10−10 cm/s for DNA/cPNT complexes. This pathway, which required energy, and the sustained release of DNA from 5.92 × 1011 copies of DNA/t1/2 for naked DNA to 3.57 × 1011 copies of DNA/t1/2 for the cPNT formulated DNA. In addition, a corneal epithelial debridement model also demonstrated the capacity to considerably reduce caspase 3 activity when plasmid-encoded caspase 3 silencing shRNA and CAP3 pRFP-C-RS with cyclo-(D-Trp-Tyr) cPNTs, with an average width of 290 nm and length of 1.8 μm, were administered by eye drops.
Biosensors
Biosensors that can detect biomolecules have been developed using cPNTs. It is possible to add target analyte-specific binding sites to the cyclic peptide structure. Biomolecules can be detected with great sensitivity and selectivity when analytes bind to nanotubes, which alter their electrical or optical characteristics in a way that can be detected. The electric characteristics of carbon nanotubes (cPNTs) with lengths over 100 μm and widths exceeding several microns, stacked by cyclo-(Ala)3 with tetrathiafulvalene as a side chain, were examined by Uji's group using a current-sensing atomic force microscopy (CS-AFM) equipped with a conducting Pt/Ir cantilever on a gold mica substrate. Cyclo-(Ala)3 modified with tetrathiafulvalene showed p-type semiconductor characteristics, according to the results. Detailed analyses of the current fluctuation conducted by these cPNTs further demonstrated that their dielectric properties are potential-dependent. Researchers led by Lee found that vapor-phase self-assembled cyclo-(Phe-Phe) nanotubes have semiconductor characteristics as well. In particular, while maintaining a constant voltage, the current grew as the temperature rose from 273 to 387 K. Based on these results, cPNTs could be used as an organic electronic material in the creation of nanoelectronic sensors that can detect viral particles and proteins with extreme sensitivity, and that can record, stimulate, and inhibit neural signals in hybrid nanowire-neuron structures.
Antiviral effects
It has been proven that cPNTs built with an equal amount of alternating D- and l-α-amino acids exhibit antiviral properties. Peptide sequences largely dictate the antiviral range, which includes both envelope and nonenvelope viruses. Generally speaking, it was believed that cPNT hydrophobicity influenced cPNT affiliation with the cell membrane, which in turn influenced the final activity. Hore's group discovered that a 5 μM IC50 cyclo-(Ser-D-His-Lys-D-Arg-Lys-D-Trp-Leu-D-Trp) (code 1), an eight-residue cyclic D,L-α-peptide with antitype A influenza virus activity, inhibits viral escape from endosomes and stops HeLa cells from generating low-pH endocytic vehicles. Its antiviral actions might not be mediated by a receptor or ligand, as the IC50 values of the reverse (code 2) and enantiomeric sequences (code 3) were identical when compared to the template cyclo-(Ser-D-His-Lys-D-Arg-Lys-D-Trp-Leu-D-Trp) (code 1). In addition, the manner of action was preserved by the code 1 sequence's analogues with a single alanine substitution (codes 4-11).
Antibacterial effects
In recent decades, there has been a lot of focus on superbacteria that have developed resistance to antibiotics as a result of their overuse. In order to fight these germs, there is an increasing demand for antimicrobial drugs that work in new ways. In this regard, Fernandez-Lopez's group proved in 2001 that gram-positive and gram-negative bacteria could be targeted by six- and eight-residue amphipathic cyclic D,L-α-peptides that had the right amino acid sequences for stacking into hollow and β-sheet-like cPNT structures with appropriate outer surface properties. A carpet-like mechanism was suggested to be responsible for this, as it increases membrane permeability and leads to quick cell death. As an illustration, the methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus coli (E. coli) were both effectively inhibited by the sequence cyclo-(D-Ser-Lys-D-Ser-Trp-D-Leu-Trp-D-Leu-Trp) (code 28) in vitro, with MIC values of 8 μM for both bacteria.
Applications of cPNTs. (Hsieh W H., et al., 2019)
References
- Brea R J., et al., Towards functional bionanomaterials based on self-assembling cyclic peptide nanotubes, Chemical Society Reviews, 2010, 39(5): 1448-1456.
- Zhu J., et al., Investigation of structures and properties of cyclic peptide nanotubes by experiment and molecular dynamics, Journal of computer-aided molecular design, 2008, 22: 773-781.
- Hsieh W H., et al., Applications of cyclic peptide nanotubes (cPNTs), journal of food and drug analysis, 2019, 27(1): 32-47.
- Sun L., et al., Tunable synthesis of self-assembled cyclic peptide nanotubes and nanoparticles, Soft Matter, 2015, 11(19): 3822-3832.
