An Offset Patterned Cross-ß Structure in Assemblies of C3 -Symmetric Peptide Amphiphiles.

Developing peptide-based materials with controlled morphology is a critical theme of soft matter research. Herein, we report the formation of a novel, patterned cross-ß structure formed by self-assembled C3 -symmetric peptide amphiphiles based on diphenylalanine and benzene-1,3,5-tricarboxamide (BTA). The cross-ß motif is an abundant structural element in amyloid fibrils and aggregates of fibril-forming peptides, including diphenylalanine. The incorporation of topological constraints on one edge of the diphenylalanine fragment limits the number of ß-strands in ß-sheets and leads to the creation of an unconventional offset-patterned cross-ß structure consisting of short 3×2 parallel ß-sheets stabilized by phenylalanine zippers. In the reported assembly, two patterned cross-ß structures bind parallel arrays of BTA stacks in a superstructure within a single-molecule-thick nanoribbon. In addition to a threefold network of hydrogen bonds in the BTA stack, each molecule becomes simultaneously bound by hydrogen bonds from three ß-sheets and four phenylalanine zippers. The diffuse layer of alkyl chains with terminal polar groups prevents the nanoribbons from merging and stabilizes cross-ß-structure in water. Our results provide a simple approach to the incorporation of novel patterned cross-ß motifs into supramolecular superstructures and shed light on the general mechanism of ß-sheet formation in C3 -symmetric peptide amphiphiles.

Utility of Triethyloxonium Tetrafluoroborate for Chloride Removal during Sarcosine N-Carboxyanhydride Synthesis: Improving NCA Purity.

The clinical translation of polysarcosine (pSar) as polyethylene glycol (PEG) replacement in the development of novel nanomedicines creates a broad demand of polymeric material in high-quality making high-purity sarcosine N-carboxyanhydride (Sar-NCA) as monomer for its production inevitable. Within this report, we present the use of triethyloxonium tetrafluoroborate in Sar-NCA synthesis with focus on amino acid and chloride impurities to avoid the sublimation of Sar-NCAs. With a view towards upscaling into kilogram or ton scale, a new methodology of monomer purification is introduced by utilizing the Meerwein's Salt triethyloxonium tetrafluoroborate to remove chloride impurities by covalent binding and converting chloride ions into volatile products within a single step. The novel straightforward technique enables access to monomers with significantly reduced chloride content (<100 ppm) compared to Sar-NCA derived by synthesis or sublimation. The derived monomers enable the controlled-living polymerization in DMF and provide access to pSar polymers with Poisson-like molecular weight distribution within a high range of chain lengths (Xn 25-200). In conclusion, the reported method can be easily applied to Sar-NCA synthesis or purification of commercially available pSar-NCAs and eases access to well-defined hetero-telechelic pSar polymers.

Rational design of heat-set and specific-ion-responsive supramolecular hydrogels based on the Hofmeister effect.

Smart supramolecular hydrogels have been prepared from a bolaamphiphilic L-valine derivative in aqueous solutions of different salts. The hydrogels respond selectively to different ions and are either reinforced or weakened. In one case, in contrast to conventional systems, the hydrogels are formed upon heating of the system. The use of the hydrogels in the controlled release of an entrapped dye is described as a proof of the potential applications of these systems. The responsive hydrogels were rationally designed by taking into account the noticeable effect of different ions from the Hofmeister series in the solubility of the hydrogelator, which was assessed by using NMR experiments. On the one hand, kosmotropic anions such as sulfate produce a remarkable solubility decrease in the gelator, which is associated with gel reinforcement, as measured by rheological experiments. On the other hand, chaotropic species such as perchlorate weaken the gel. A dramatic effect was observed in the presence of guanidinium chloride, which boosted the solubility of the gelator, in accordance with its chaotropic behaviour reported in protein science. In this case, a direct interaction of the guanidinium species with the carbonyl groups of the hydrogelator is detected by (13) C NMR spectroscopy. The weakening of this interaction upon a temperature increase allows for the preparation of heat-set hydrogelating systems.

Freezing capture of polymorphic aggregates of bolaamphiphilic (L)-valine-based molecular hydrogelators.

Nanostructured xerogels have been prepared by the freeze-drying of hydrogels and aggregates formed by bolaamphiphilic L-valine derivatives after aging under different environmental conditions. A wide variety of shapes and sizes has been achieved by a simple methodology. These nanostructures have been studied by SEM and WAXD and a dramatic influence of structural flexibility on the kinetics of aggregation has been observed. Such flexibility and a modulation of the hydrophobic effect have shown a profound influence in the packing of these compounds and revealed a high degree of polymorphism.

Control of molecular gelation by chemical stimuli.

Molecular gels are formed by the self-assembly of low-molecular weight compounds by weak non-covalent interactions and thus, they may be easily disassembled in response to external stimuli. Chemically sensitive gels can be prepared by introducing in the molecular design functional groups that may interact either by covalent or non-covalent forces with other molecules present in the medium. Functional molecular gels have been reported that are sensitive to acids, bases, ions, redox-active compounds, neutral species, reactive compounds and enzymes. Here we present a broad revision of the different chemical inputs that can be used to tune gel properties through some appealing application-based selected examples.

Interplay of molecular hydrogelators and SDS affords responsive soft matter systems with tunable properties.

The gelation efficiency of low molecular weight bolaamphiphilic hydrogelators 1 and 2 is influenced by the presence of SDS micelles. Similarly, the critical micellar concentration value of SDS is reduced in the presence of the studied molecular hydrogelators. Rheological measurements indicate that the strength of the hydrogels can be modulated with SDS, the gels becoming weaker in the presence of micelles. This behavior has been rationalized with the help of NMR studies using diffusion measurements and NOE correlations. The results obtained clearly point to the formation of mixed micelles composed of SDS and the hydrogelators. In the case of 1, the gelator:SDS ratio in the mixed micelles has been estimated from solubility studies to be ca. 1:2.5. Electron microscopy reveals that when SDS is present, the morphology of the xerogels is modified in its appearance at the micrometer scale but fibers with diameter in the nanometer range are observed in all the cases. The interplay between the surfactant and the gelators provides with new possibilities for the modulation of both gel and micelle formation. Examples are shown to highlight the potential usefulness of this type of interconnected system. In one case the release of a gel entrapped dye is modulated by the presence of SDS and sodium chloride. In another example, an intricate system that responds to a temperature excursion by irreversible micelle disassembly is shown.

Molecular hydrogels from bolaform amino acid derivatives: a structure-properties study based on the thermodynamics of gel solubilization.

Insight is provided into the aggregation thermodynamics associated to hydrogel formation by molecular gelators derived from L-valine and L-isoleucine. Solubility data from NMR measurements are used to extract thermodynamic parameters for the aggregation in water. It is concluded that at room temperature and up to 55 °C, these systems form self-assembled fibrillar networks in water with quite low or zero enthalpic component, whereas the entropy of the aggregation is favorable. These results are explained by considering that the hydrophobic effect is dominant in the self-assembly. However, studies by NMR and IR spectroscopy reveal that intermolecular hydrogen bonding is also a key issue in the aggregation process of these molecules in water. The low enthalpy values measured for the self-assembly process are ascribed to the result of a compensation of the favorable intermolecular hydrogen-bond formation and the unfavorable enthalpy component of the hydrophobic effect. Additionally, it is shown that by using the hydrophobic character as a design parameter, enthalpy-controlled hydrogel formation, as opposed to entropy-controlled hydrogel formation, can be achieved in water if the gelator is polar enough. It is noteworthy that these two types of hydrogels, enthalpy-versus entropy-driven hydrogels, present quite different response to temperature changes in properties such as the minimum gelator concentration (mgc) or the rheological moduli. Finally, the presence of a polymorphic transition in a hydrogel upon heating above 70 °C is reported and ascribed to the weakening of the hydrophobic effect upon heating. The new soft polymorphic materials present dramatically different solubility and rheological properties. Altogether these results are aimed to contribute to the rational design of molecular hydrogelators, which could be used for the tailored preparation of this type of soft materials. The reported results could also provide ground for the rationale of different self-assembly processes in aqueous media.

Higher-order interfiber interactions in the self-assembly of benzene-1,3,5-tricarboxamide-based peptides in water.

Mimicking the complexity of biological systems with synthetic supramolecular materials requires a deep understanding of the relationship between the structure of the molecule and its self-assembly pattern. Herein, we report a series of water-soluble benzene-1,3,5-tricarboxamide-based di- and tripeptide derivatives modified with small non-bulky terminal amine salt to induce self-assembly into twisted one-dimensional higher-order nanofibers. The morphology of nanofibers strongly depends on the nature, order, and quantity of amino acids in the short peptide fragments and vary from simple cylindrical to complex helical. From observations of several fiber-splitting events, we detected interfiber interactions that always occur in a pairwise manner, which implies that the C3 symmetry of benzene-1,3,5-tricarboxamide-based molecules in higher-order fibers becomes gradually distorted, thus facilitating hydrophobic contact interactions between fibrils. The proposed mechanism of self-assembly through hydrophobic contact allowed the successful design of a compound with pH-responsive morphology, and may find use in the future development of complex hierarchical architectures with controlled functionality.

Polymer Conjugation of Docosahexaenoic Acid Potentiates Cardioprotective Therapy in Preclinical Models of Myocardial Ischemia/Reperfusion Injury.

While coronary angioplasty represents an effective treatment option following acute myocardial infarction, the reperfusion of the occluded coronary artery can prompt ischemia-reperfusion (I/R) injury that significantly impacts patient outcomes. As ω-3 polyunsaturated fatty acids (PUFAs) have proven, yet limited cardioprotective abilities, an optimized polymer-conjugation approach is reported that improves PUFAs bioavailability to enhance cardioprotection and recovery in animal models of I/R-induced injury. Poly-l-glutamic acid (PGA) conjugation improves the solubility and stability of di-docosahexaenoic acid (diDHA) under physiological conditions and protects rat neonatal ventricular myocytes from I/R injury by reducing apoptosis, attenuating autophagy, inhibiting reactive oxygen species generation, and restoring mitochondrial membrane potential. Enhanced protective abilities are associated with optimized diDHA loading and evidence is provided for the inherent cardioprotective potential of PGA itself. Pretreatment with PGA-diDHA before reperfusion in a small animal I/R model provides for cardioprotection and limits area at risk (AAR). Furthermore, the preliminary findings suggest that PGA-diDHA administration in a swine I/R model may provide cardioprotection, limit edema and decrease AAR. Overall, the evaluation of PGA-diDHA in relevant preclinical models provides evidence for the potential of polymer-conjugated PUFAs in the mitigation of I/R injury associated with coronary angioplasty.

Polypeptide-corticosteroid conjugates as a topical treatment approach to psoriasis.

Topical treatment of mild-to-moderate psoriasis with corticosteroids suffers from challenges that include reduced drug bioavailability at the desired site of action. The retention of therapeutics within the epidermis can safely treat skin inflammation, scaling, and erythema associated with psoriasis while avoiding possible side effects associated with systemic treatments. We successfully synthesized and characterized a pH-responsive biodegradable poly-L-glutamic acid (PGA)-fluocinolone acetonide (FLUO) conjugate that allows the controlled release of the FLUO to reduce skin inflammation. Additionally, the application of a hyaluronic acid (HA)-poly-L-glutamate cross polymer (HA-CP) vehicle boosted skin permeation. During in vitro and ex vivo analyses, we discovered that PGA-FLUO inhibited pro-inflammatory cytokine release, suggesting that polypeptidic conjugation fails to affect the anti-inflammatory activity of FLUO. Additionally, ex vivo human skin permeation studies using confocal microscopy revealed the presence of PGA-FLUO within the epidermis, but a minimal presence in the dermis, thereby reducing the likelihood of FLUO entering the systemic circulation. Finally, we demonstrated that PGA-FLUO applied within HA-CP effectively reduced psoriasis-associated phenotypes in an in vivo mouse model of human psoriasis while also lowering levels of pro-inflammatory cytokines in tissue and serum. Overall, our experimental results demonstrate that PGA-FLUO within an HA-CP penetration enhancer represents an effective topical treatment for psoriasis.

Polypeptide-Based Conjugates as Therapeutics: Opportunities and Challenges.

Synthetic polypeptides or polyamino acids have become a useful and multifunctional platform in advanced drug delivery studies. Nonetheless, the full potential of these systems has yet to be achieved. The final structure of polypeptide conjugates and their in vivo behavior are dependent on an extraordinarily complex pattern of interconnected physico-chemical and structural parameters, making sophisticated directional design of such systems difficult and often unachievable. In this review, the authors aim to discuss the role of these parameters in the successful design of different drug delivery architectures and to delineate some basic correlations between structure, properties, and the biological behavior of polypeptide-based conjugates.

Capturing "Extraordinary" Soft-Assembled Charge-Like Polypeptides as a Strategy for Nanocarrier Design.

The rational design of nanomedicines is a challenging task given the complex architectures required for the construction of nanosized carriers with embedded therapeutic properties and the complex interface of these materials with the biological environment. Herein, an unexpected charge-like attraction mechanism of self-assembly for star-shaped polyglutamates in nonsalty aqueous solutions is identified, which matches the ubiquitous "ordinary-extraordinary" phenomenon previously described by physicists. For the first time, a bottom-up methodology for the stabilization of these nanosized soft-assembled star-shaped polyglutamates is also described, enabling the translation of theoretical research into nanomaterials with applicability within the drug-delivery field. Covalent capture of these labile assemblies provides access to unprecedented architectures to be used as nanocarriers. The enhanced in vitro and in vivo properties of these novel nanoconstructs as drug-delivery systems highlight the potential of this approach for tumor-localized as well as lymphotropic delivery.

Relevant Physicochemical Descriptors of "Soft Nanomedicines" to Bypass Biological Barriers.

Herein, we present an overview on the current status of the characterization techniques and methodologies used to study the physicochemical descriptors that influence the final clinical performance of a given nanomedicine. The described techniques were selected based on their suitability to operate under relevant "native" conditions that mimic the physiological environment. Special emphasis is placed on those techniques that hold a greater potential to unravel dynamic, structural, and compositional features of soft organic nanomedicines relevant to the ability to bypass biological barriers, and hence allow for the rational design of drug delivery platforms with improved biological output.

Vibrational Circular Dichroism Shows Reversible Helical Handedness Switching in Peptidomimetic l-Valine Fibrils.

We elucidate the supramolecular organization in the form of microsize fibrils of gels formed by a l-Valine peptidomimetic compound. Analysis was based on circular dichroism spectroscopies, vibrational (VCD) and electronic (CD), supported by microscopy (atomic force and scanning electron). We show how the VCD spectra give account of the micrometric structure of the fibrils formed by the helicoidal arrangement of simpler proto-fibrils, which are organized in a lower hierarchical level. This ability is used to monitorize a fully reversible change in the handedness of the helix by modulating different external stimuli as pH or ionic strength, thus providing the first observation by VCD of such a phenomenon in a short peptide.