Tuning self-assembled nanostructures through enzymatic degradation of a peptide amphiphile.
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PROVIDER: S-EPMC3678293 | BioStudies |
REPOSITORIES: biostudies
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Project description:Peptide amphiphiles (PAs) are promising tools for the intracellular delivery of numerous drugs. PAs are known to be biodegradable systems. Here, four PA derivatives containing arginine and lysine conjugated with fatty acyl groups with different chain lengths, namely, PA1: R-K(C14)-R, PA2: R-K(C16)-R, PA3: K(C14)-R-K(C14), and PA4: K(C16)-R-K(C16), where C16 = palmitic acid and C14 = myristic acid, were synthesized through Fmoc chemistry. Flow cytometry studies showed that, among all synthesized PAs, only K(C16)-R-K(C16), PA4 was able to enhance the cellular uptake of a fluorescence-labeled anti-HIV drug 2',3'-dideoxy-3'-thiacythidine (F'-3TC, F' = fluorescein) and a biologically important phosphopeptide (F'-PEpYLGLD) in human leukemia cells (CCRF-CEM) after 2 h incubation. For example, the cellular uptake of F'-3TC and F'-PEpYLGLD was enhanced approximately 7.1- and 12.6-fold in the presence of the PA4 compared to those of the drugs alone. Confocal microscopy of F'-3TC and F'-PEpYLGLD loaded PA4 in live cells showed significantly higher intracellular localization than the drug alone in human ovarian cells (SK-OV-3) after 2 h incubation. The high-performance liquid chromatography (HPLC) results showed that loading of Dox by the peptide amphiphile was 56% after 24 h. The loaded Dox was released (34%) within 48 h intracellularly. The circular dichrosim (CD) results exhibited that the secondary structure of the peptide was changed upon interactions with Dox. Mechanistic studies revealed that endocytosis is the major pathway of the internalization. These studies suggest that PAs containing the appropriate sequence of amino acids, chain length, charge, and hydrophobicity can be used as cellular delivery tools for transporting drugs and biomolecules.
| S-EPMC3873380 | BioStudies
Project description:Peripheral nerve injuries can result in lifelong disability. Primary coaptation is the treatment of choice when the gap between transected nerve ends is short. Long nerve gaps seen in more complex injuries often require autologous nerve grafts or nerve conduits implemented into the repair. Nerve grafts, however, cause morbidity and functional loss at donor sites, which are limited in number. Nerve conduits, in turn, lack an internal scaffold to support and guide axonal regeneration, resulting in decreased efficacy over longer nerve gap lengths. By comparison, peptide amphiphiles (PAs) are molecules that can self-assemble into nanofibers, which can be aligned to mimic the native architecture of peripheral nerve. As such, they represent a potential substrate for use in a bioengineered nerve graft substitute. To examine this, we cultured Schwann cells with bioactive PAs (RGDS-PA, IKVAV-PA) to determine their ability to attach to and proliferate within the biomaterial. Next, we devised a PA construct for use in a peripheral nerve critical sized defect model. Rat sciatic nerve defects were created and reconstructed with autologous nerve, PLGA conduits filled with various forms of aligned PAs, or left unrepaired. Motor and sensory recovery were determined and compared among groups. Our results demonstrate that Schwann cells are able to adhere to and proliferate in aligned PA gels, with greater efficacy in bioactive PAs compared to the backbone-PA alone. In vivo testing revealed recovery of motor and sensory function in animals treated with conduit/PA constructs comparable to animals treated with autologous nerve grafts. Functional recovery in conduit/PA and autologous graft groups was significantly faster than in animals treated with empty PLGA conduits. Histological examinations also demonstrated increased axonal and Schwann cell regeneration within the reconstructed nerve gap in animals treated with conduit/PA constructs. These results indicate that PA nanofibers may represent a promising biomaterial for use in bioengineered peripheral nerve repair.
| S-EPMC4289709 | BioStudies
Project description:There has been recent interest in designing smart diagnostic or therapeutic self-assembling peptide or polymeric materials that can selectively undergo morphological transitions to accumulate at a disease site in response to specific stimuli. Developing approaches to probe these self-assembly transitions in environments that accurately amalgamate the diverse plethora of proteins, biomolecules, and salts of blood is essential for creating systems that function in vivo. Here, we have developed a fluorescence anisotropy approach to probe the pH-dependent self-assembly transition of peptide amphiphile (PA) molecules that transform from spherical micelles at pH 7.4 to nanofibers under more acidic pH's in blood serum. By mixing small concentrations of a Ru(bipy)3(2+)-tagged PA with a Gd(DO3A)-tagged PA having the same lipid-peptide sequence, we showed that the pH dependence of self-assembly is minimally affected and can be monitored in mouse blood serum. These PA vehicles can be designed to transition from spherical micelles to nanofibers in the pH range 7.0-7.4 in pure serum. In contrast to the typical notion of serum albumin absorbing isolated surfactant molecules and disrupting self-assembly, our experiments showed that albumin does not bind these anionic PAs and instead promotes nanofibers due to a molecular crowding effect. Finally, we created a medium that replicates the transition pH in serum to within 0.08 pH units and allows probing self-assembly behavior using conventional spectroscopic techniques without conflicting protein signals, thus simplifying the development pathway from test tube to in vivo experimentation for stimuli-responsive materials.
| S-EPMC5877399 | BioStudies
Project description:Peptides synthesized in the likeness of their native interaction domain(s) are natural choices to target protein-protein interactions (PPIs) due to their fidelity of orthostatic contact points between binding partners. Despite therapeutic promise, intracellular delivery of biofunctional peptides at concentrations necessary for efficacy remains a formidable challenge. Peptide amphiphiles (PAs) provide a facile method of intracellular delivery and stabilization of bioactive peptides. PAs consisting of biofunctional peptide headgroups linked to hydrophobic alkyl lipid-like tails prevent peptide hydrolysis and proteolysis in circulation, and PA monomers are internalized via endocytosis. However, endocytotic sequestration and steric hindrance from the lipid tail are two major mechanisms that limit PA efficacy to target intracellular PPIs. To address these problems, we have constructed a PA platform consisting of cathepsin-B cleavable PAs in which a selective p53-based inhibitory peptide is cleaved from its lipid tail within endosomes, allowing for intracellular peptide accumulation and extracellular recycling of the lipid moiety. We monitor for cleavage and follow individual PA components in real time using a Förster resonance energy transfer (FRET)-based tracking system. Using this platform, we provide a better understanding and quantification of cellular internalization, trafficking, and endosomal cleavage of PAs and of the ultimate fates of each component.
| S-EPMC5937119 | BioStudies
Project description:The coupling of electronic and biological functionality through self-assembly is an interesting target in supramolecular chemistry. We report here on a set of diacetylene-derivatized peptide amphiphiles (PAs) that react to form conjugated polydiacetylene backbones following self-assembly into cylindrical nanofibers. The polymerization reaction yields highly conjugated backbones when the peptidic segment of the PAs has a linear, as opposed to a branched, architecture. Given the topotactic nature of the polymerization, these results suggest that a high degree of internal order exists in the supramolecular nanofibers formed by the linear PA. On the basis of microscopy, the formation of a polydiacetylene backbone to covalently connect the beta-sheets that help form the fibers does not disrupt the fiber shape. Interestingly, we observe the appearance of a polydiacetylene (PDA) circular dichroism band at 547 nm in linear PA nanofibers suggesting the conjugated backbone in the core of the nanostructures is twisted. We believe this CD signal is due to chiral induction by the beta-sheets, which are normally twisted in helical fashion. Heating and cooling shows simultaneous changes in beta-sheet and conjugated backbone structure, indicating they are both correlated. At the same time, poor polymerization in nanofibers formed by branched PAs indicates that less internal order exists in these nanostructures and, as expected, then a circular dichroism signal is not observed for the conjugated backbone. The general variety of materials investigated here has the obvious potential to couple electronic properties and in vitro bioactivity. Furthermore, the polymerization of monomers in peptide amphiphile assemblies by a rigid conjugated backbone also leads to mechanical robustness and insolubility, two properties that may be important for the patterning of these materials at the cellular scale.
| S-EPMC2547985 | BioStudies
Project description:C16-YEALRVANEVTLN, a peptide amphiphile (PA) incorporating a biologically active amino acid sequence found in lumican, has been examined for its influence upon collagen synthesis by human corneal fibroblasts in vitro, and the roles of supra-molecular assembly and activin receptor-like kinase ALK receptor signaling in this effect were assessed. Cell viability was monitored using the Alamar blue assay, and collagen synthesis was assessed using Sirius red. The role of ALK signaling was studied by receptor inhibition. Cultured human corneal fibroblasts synthesized significantly greater amounts of collagen in the presence of the PA over both 7-day and 21-day periods. The aggregation of the PA to form nanotapes resulted in a notable enhancement in this activity, with an approximately two-fold increase in collagen production per cell. This increase was reduced by the addition of an ALK inhibitor. The data presented reveal a stimulatory effect upon collagen synthesis by the primary cells of the corneal stroma, and demonstrate a direct influence of supra-molecular assembly of the PA upon the cellular response observed. The effects of PA upon fibroblasts were dependent upon ALK receptor function. These findings elucidate the role of self-assembled nanostructures in the biological activity of peptide amphiphiles, and support the potential use of a self-assembling lumican derived PA as a novel biomaterial, intended to promote collagen deposition for wound repair and tissue engineering purposes.
| S-EPMC4743677 | BioStudies
Project description:High aspect ratio peptide nanofibers have potential as biodegradable vehicles for drug delivery. We report here the synthesis of four self-assembling peptide amphiphiles (PAs) containing a lysine ?-amine-derivatized hydrazide that was systematically placed at different positions along the backbone of the peptide sequence C(16)V(2)A(2)E(2) (where C(16) = palmitic acid). Hydrazones were formed from each hydrazide by condensation with the solvatochromic dye 6-propionyl-2-dimethylaminonaphthalene (Prodan), which is typically used to probe cell membranes. All four compounds were found to self-assemble into nanofibers, and Prodan release was measured from filamentous gels prepared by screening PA charges with divalent cations. Near zero-order release kinetics were observed for all nanofibers, but release half-lives differed depending on the position of the fluorophore in the PA sequence. Dye release kinetics were rationalized through the use of cryogenic transmission electron microscopy, small-angle X-ray scattering, fluorescence spectroscopy, fluorescence anisotropy, circular dichroism, and partition coefficient calculations. Relative release rates were found to correlate directly with fluorophore mobility, which varied inversely with packing density, degree of order in the hydrophobic PA core, and the ?-sheet character of the peptide.
| S-EPMC3487392 | BioStudies
Project description:This study investigated the ability of nanoscale, biomimetic peptide amphiphile (PA) scaffolds inscribed with specific cellular adhesive ligands to direct the osteogenic differentiation of human mesenchymal stem cells (hMSCs) without osteogenic supplements. PA sequences were synthesized to mimic the native bone extracellular matrix (ECM), expressing different isolated ligands (i.e., RGDS, DGEA, KRSR). All PAs were presented as self-assembled two-dimensional coatings for the seeded hMSCs. Initial attachment results demonstrated that the different PAs could be individually recognized based on the incorporated adhesive ligands. Long-term studies assessed osteogenic differentiation up to 35 days. The RGDS-containing PA nanomatrix expressed significantly greater alkaline phosphatase activity, indicating the early promotion of osteogenic differentiation. A progressive shift toward osteogenic morphology and positive staining for mineral deposition provided further confirmation of the RGDS-containing PA nanomatrix. Overall, the PA nanomatrix clearly has great promise for directing the osteogenic differentiation of hMSCs without the aid of supplements by mimicking the native ECM, providing an adaptable environment that allows for different adhesive ligands to control cellular behaviors. This research model establishes the beginnings of a new versatile approach to regenerate bone tissues by closely following the principles of natural tissue formation.
| S-EPMC2760643 | BioStudies
Project description:Formation of the native bone extracellular matrix (ECM) provides an attractive template for bone tissue engineering. The structural support and biological complexity of bone ECM are provided within a composite microenvironment that consists of an organic fibrous network reinforced by inorganic hydroxyapatite (HA) nanoparticles. Recreating this biphasic assembly, a bone ECM analogous scaffold comprising self-assembling peptide amphiphile (PA) nanofibers and interspersed HA nanoparticles was investigated. PAs were endowed with biomolecular ligand signaling using a synthetically inscribed peptide sequence (i.e., RGDS) and integrated with HA nanoparticles to form a biphasic nanomatrix hydrogel. It was hypothesized the biphasic hydrogel would induce osteogenic differentiation of human mesenchymal stem cells (hMSCs) and improve bone healing as mediated by RGDS ligand signaling within PA nanofibers and embedded HA mineralization source. Viscoelastic stability of the biphasic PA hydrogels was evaluated with different weight concentrations of HA for improved gelation. After demonstrating initial viability, long-term cellularity and osteoinduction of encapsulated hMSCs in different PA hydrogels were studied in vitro. Temporal progression of osteogenic maturation was assessed by gene expression of key markers. A preliminary animal study demonstrated bone healing capacity of the biphasic PA nanomatrix under physiological conditions using a critical size femoral defect rat model. The combination of RGDS ligand signaling and HA nanoparticles within the biphasic PA nanomatrix hydrogel demonstrated the most effective osteoinduction and comparative bone healing response. Therefore, the biphasic PA nanomatrix establishes a well-organized scaffold with increased similarity to natural bone ECM with the prospect for improved bone tissue regeneration.
| S-EPMC3691849 | BioStudies
Project description:Proanthocyanidins (PAs) are fundamental nutritional metabolites in different types of grape products consumed by human beings. Although the biosynthesis of PAs in berry of Vitis vinifera has gained intensive investigations, the understanding of PAs in other Vitis species is limited. In this study, we report PA formation and characterization of gene expression involved in PA biosynthesis in leaves of V. bellula, a wild edible grape species native to south and south-west China. Leaves are collected at five developmental stages defined by sizes ranging from 0.5 to 5 cm in length. Analyses of thin layer chromatography (TLC) and high performance liquid chromatography-photodiode array detector (HPLC-PAD) show the formation of (+)-catechin, (-)-epicatechin, (+)-gallocatechin and (-)-epigallocatechin during the entire development of leaves. Analyses of butanol-HCl boiling cleavage coupled with spectrometry measurement at 550 nm show a temporal trend of extractable PA levels, which is characterized by an increase from 0.5 cm to 1.5 cm long leaves followed by a decrease in late stages. TLC and HPLC-PAD analyses identify cyanidin, delphinidin and pelargonidin produced from the cleavage of PAs in the butanol-HCl boiling, showing that the foliage PAs of V. bellula include three different types of extension units. Four cDNAs, which encode VbANR, VbDFR, VbLAR1 and VbLAR2, respectively, are cloned from young leaves. The expression patterns of VbANR and VbLAR2 but not VbLAR1 and VbDFR follow a similar trend as the accumulation patterns of PAs. Two cDNAs encoding VbMYBPA1 and VbMYB5a, the homologs of which have been demonstrated to regulate the expression of both ANR and LAR in V. vinifera, are also cloned and their expression profiles are similar to those of VbANR and VbLAR2. In contrast, the expression profiles of MYBA1 and 2 homologs involved in anthocyanin biosynthesis are different from those of VbANR and VbLAR2. Our data show that both ANR and LAR branches are involved in PA biosynthesis in leaves of V. bellula.
| S-EPMC3901255 | BioStudies