Self-assembling Peptide Nanofiber Research Articles

Parallel β-Sheet Structure and Structural Heterogeneity Detected within Q11 Self-Assembling Peptide Nanofibers.

Q11 peptide nanofibers are used as a biomaterial for applications such as antigen presentation and tissue engineering, yet detailed knowledge of molecular-level structure has not been reported. The Q11 peptide sequence was designed using heuristics-based patterning of hydrophobic and polar amino acids with oppositely charged amino acids placed at opposite ends of the sequence to promote antiparallel β-sheet formation. In this work, we employed solid-state nuclear magnetic resonance spectroscopy (NMR) to evaluate whether the molecular organization within Q11 self-assembled peptide nanofibers is consistent with the expectations of the peptide designers. We discovered that Q11 forms a distribution of molecular structures. NMR data from two-dimensional (2D) 13C-13C dipolar-assisted rotational resonance indicate that the K3 and E9 residues between Q11 β-strands are spatially proximate (within ∼0.6 nm). Frequency-selective rotational echo double resonance (fsREDOR) on K3 Nζ and E9 Cδ-labeled sites showed that approximately 9% of the sites are close enough for salt bridge formation to occur. Surprisingly, dipolar recoupling measurements revealed that Q11 peptides do not assemble into antiparallel β-sheets as expected, and structural analysis using Fourier-transform infrared spectroscopy and 2D NMR alone can be misleading. 13C PITHIRDS-CT dipolar recoupling measurements showed that the most abundant structure consists of parallel β-sheets, in contrast to the expected antiparallel β-sheet structure. Structural heterogeneity was detected from 15N{13C} REDOR measurements, with approximately 22% of β-strands having antiparallel nearest neighbors. We cannot propose a complete structural model of Q11 nanofibers because of the complexity involved when examining structurally heterogeneous samples using NMR. Altogether, our results show that while heuristics-based patterning is effective in promoting β-sheet formation, designing a peptide sequence to form a targeted β-strand arrangement remains challenging.

Tunable Macroscopic Alignment of Self-Assembling Peptide Nanofibers.

Progress in the design and synthesis of nanostructured self-assembling systems has facilitated the realization of numerous nanoscale geometries, including fibers, ribbons, and sheets. A key challenge has been achieving control across multiple length scales and creating macroscopic structures with nanoscale organization. Here, we present a facile extrusion-based fabrication method to produce anisotropic, nanofibrous hydrogels using self-assembling peptides. The application of shear force coinciding with ion-triggered gelation is used to kinetically trap supramolecular nanofibers into aligned, hierarchical macrostructures. Further, we demonstrate the ability to tune the nanostructure of macroscopic hydrogels through modulating phosphate buffer concentration during peptide self-assembly. In addition, increases in the nanostructural anisotropy of fabricated hydrogels are found to enhance their strength and stiffness under hydrated conditions. To demonstrate their utility as an extracellular matrix-mimetic biomaterial, aligned nanofibrous hydrogels are used to guide directional spreading of multiple cell types, but strikingly, increased matrix alignment is not always correlated with increased cellular alignment. Nanoscale observations reveal differences in cell-matrix interactions between variably aligned scaffolds and implicate the need for mechanical coupling for cells to understand nanofibrous alignment cues. In total, innovations in the supramolecular engineering of self-assembling peptides allow us to decouple nanostructure from macrostructure and generate a gradient of anisotropic nanofibrous hydrogels. We anticipate that control of architecture at multiple length scales will be critical for a variety of applications, including the bottom-up tissue engineering explored here.

Supramolecular Peptide Self-Assemblies Facilitate Oral Immunization.

Oral immunization is a promising strategy for preventing and treating gastrointestinal (GI) infections and diseases, as it allows for direct access to the disease site. To elicit immune responses within the GI tract, however, there are many obstacles that oral vaccines must surmount, including proteolytic degradation and thick mucus barriers. Here, we employed a modular self-assembling peptide nanofiber platform to facilitate oral immunization against both peptide and small molecule epitopes. Synthesizing nanofibers with d-amino acids rendered them resistant to proteases in vitro, whereas l-amino acid nanofibers were rapidly degraded. Additionally, the inclusion of peptide sequences rich in proline, alanine, and serine (PAS), increased nanofiber muco-penetration, and accelerated nanofiber transport through the GI tract. Oral immunization with PASylated nanofibers and mucosal adjuvant generated local and systemic immune responses to a peptide epitope but only for l-amino acid nanofibers. Further, we were able to apply this design to also enable oral immunization against a small molecule epitope and illustrated the therapeutic and prophylactic effectiveness of these immunizations in mouse models of colitis. These findings demonstrate that supramolecular peptide self-assemblies have promise as oral vaccines and immunotherapies.

Active immunotherapy for C5a-mediated inflammation using adjuvant-free self-assembled peptide nanofibers

The terminal protein in the complement cascade C5a is a potent inflammatory molecule and chemoattractant that is involved in the pathology of multiple inflammatory diseases including sepsis and arthritis, making it a promising protein to target with immunotherapies. Active immunotherapies, in which patients are immunized against problematic self-molecules and generate therapeutic antibodies as a result, have received increasing interest as an alternative to traditional monoclonal antibody treatments. In previous work, we have designed supramolecular self-assembling peptide nanofibers as active immunotherapies with defined combinations of B- and T-cell epitopes. Herein, the self-assembling peptide Q11 platform was employed to generate a C5a-targeting active immunotherapy. Two of three predicted B-cell epitope peptides from C5a were found to be immunogenic when displayed within Q11 nanofibers, and the nanofibers were capable of reducing C5a serum concentrations following immunization. Contrastingly, C5a's precursor protein C5 maintained its original concentration, promising to minimize side effects heretofore associated with C5-targeted therapies. Immunization protected mice against an LPS-challenge model of sepsis, and it reduced clinical severity in a model of collagen-antibody induced arthritis. Together, this work indicates the potential for targeting terminal complement proteins with active immunotherapies by leveraging the immunogenicity of self-assembled peptide nanomaterials. Statement of significanceChronic inflammatory diseases such as rheumatoid arthritis, psoriasis, and inflammatory bowel disease are currently treated primarily with monoclonal antibodies against key inflammatory mediators. While helpful for many patients, they have high non-response rates, are costly, and commonly fail as anti-drug antibodies are raised by the patient. The approach we describe here explores a fundamentally different treatment paradigm: raising therapeutic antibody responses with an active immunotherapy.We employ innovative supramolecular peptide nanomaterials to elicit neutralizing antibody responses against complement component C5a and demonstrate therapeutic efficacy in preclinical mouse models of sepsis and rheumatoid arthritis. The strategy reported may represent a potential alternative to monoclonal antibody therapies.

Experimental Study on Rapid Hemostasis Using Peptide Hydrogels.

Uncontrolled hemorrhaging resulting from trauma, surgery, and disease-associated or drug-induced blood disorders can cause significant morbidities and mortalities in civilian and military populations. Self-assembling peptide nanofibers are particularly attractive due to their rapid and efficient hemostasis, biocompatibility, and wound-healing properties. In this study, we designed two types of 12-residue peptides by using a strong fishnet-like peptide sequence and a pro-cell adhesion sequence (Arg-Gly-Asp, RGD). The peptides are HN2-X-Ser-Phe-Cys-Phe-Lys-Phe-Glu-X-Arg-Gly-Asp-OH (where X is Pro or Tyr), which dissolve in deionized (DI) water and form stable and transparent functional hydrogels. Transmission electron microscopy and scanning electron microscopy demonstrated that the two peptides self-assemble into nanowebs and nanofibers, forming a fishnet-like and three-dimensional network structure. Circular dichroism and Fourier transform infrared spectroscopy analysis demonstrated that the self-assembled peptides mainly adopt a β-sheet structure with β-turn and α-helix as auxiliary assembly growth. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and SEM analysis showed that the cell survival rates were very good, delivering an obvious promotion of cell proliferation of fibroblasts and hepatocytes. Importantly, in vivo hemostasis delivered that the self-assembled peptide nanowebs and nanofibers had a good hemostatic effect on rat saphenous vein and liver bleeding, achieving 38 s faster hemostasis, which was better than commercial "Instantaneous" hemostatic powder. Accoupling the fast hemostasis and effective promotion of liver defect rapid repair, the peptide self-assembly strategy offers a clinically promising treatment option for life-threatening liver bleeding and serves as a renewed impetus for the development of peptide hydrogels as effective hemostatic agents.

RADA-16-based Self-assembled Peptide Nanofiber Scaffolds Loaded with TGF-β1 Enhance the Chondrogenic Differentiation Potential of BMSCs In vitro.

At present, cartilage repair does not offer ideal efficacy. Fortunately, recent studies have claimed that RADA-16 peptide is an attractive therapeutic strategy for repairing cartilage defects. Therefore, this study tried to explore the effect of RADA-16 loaded with transforming growth factor-beta (TGF-β) 1 on cartilage differentiation of bone marrow mesenchymal stem cells (BMSCs). First, the RADA-16 peptide was synthesized by solid phase peptide, and a well-defined hydrogel was formed by supramolecular peptide self-assembly. Then, TGF-β1 (loading concentration of 10 ng/mL) was loaded into RADA-16, with scanning electron microscopy to observe the morphology of the TGF-β1/RADA-16 hydrogel and detect its related properties. Next, BMSCs were isolated from bone marrow samples and identified. TGF-β1/RADA-16 was co-cultured with L929, BMSCs, and C28/I2 cells, respectively, and the survival and proliferation ability of the cells was determined by live/dead cell staining and MTT assay. Chondrogenic differentiation and sGAG production of BMSCs were determined by Alcian blue staining and Blyscan assay, the expression of cartilage-associated genes by qRT-PCR, and the levels of inflammatory factors by ELISA. As for mechanism investigation, the Smad and ERK/MAPK signaling pathways were detected by western blot. RADA-16 hydrogel exhibited a well-distributed and interconnected porous surface structure, with a loading rate of 91.9% for TGF-β1. The TGF-β1/RADA-16 hydrogel had good release and degradation properties, and had no negative effect on the survival and proliferation ability of BMSCs, L929, and C28/I2 cells. Importantly, TGF-β1/RADA-16 hydrogel significantly accelerated chondrogenic differentiation and sGAG generation in BMSCs, and decreased pro-inflammatory factor production. In addition, the hydrogel also significantly activated the Smad and ERK/MAPK pathways of BMSCs. RADA-16 loaded with TGF-β1 has good biological properties and can enhance the chondrogenic differentiation ability of BMSCs.

A self-assembling peptide nanofiber hydrogel for biomaterials with rapid stimulation response to naturally positively charged group substances

Peptides as unique supramolecular nanofibrous materials are favored choices to fabricate hydrogel for artificial extracellular matrices. Herein, we devised an amphiphilic polypeptide that could be motivated by multiple substances with positively charged group at physiological temperature and pH to assemble supramolecular nanofiber hydrogels since the electrostatic repulsion between monomers was attenuated. The hydrogelation phenomenon and spectroscopy were compatible with molecular dynamics simulations (MDs) results for revealing the assembly mechanism of this peptide, confirming that the behavior of assembling and the typical secondary structure of the assemblies was β-sheet. The impact of peptide concentration on the mechanical properties of hydrogels were described using rheology. We found that the peptide formed elastic gels from 0.3 wt% onwards in practical use and that these recovered by themselves after shear thinning that indicated they were injectable hydrogels. The successful establishment of cell spheres with high cell viability during 15 days culture in 3D space demonstrated the nanofiber hydrogels had potential in cell model construction and microtissue culture. Overall, the superior biologically friendly gel-forming conditions and excellent biological properties of this supramolecular assembly peptide material have invigorated the progress of biomedical fields such as tissue regeneration based on in vitro 3D culture.

A novel octa-arginine-modified injectable self-assembling peptide hydrogel for multidrug-resistant cancer therapy

Surgery combined with systemic chemotherapy is currently the main modality of cancer treatment. However, the development of cancer multidrug resistance and inevitable surgical residual lesions lead to high rates of cancer recurrence and mortality. Meanwhile, systemic drug toxic side effects and surgical bleeding complications seriously reduce the quality of life of patients. To avoid cancer multidrug resistance and comprehensively optimize cancer treatment, this work provides a novel injectable cell penetrating peptide octa-arginine (R8)-modified RADA16 (RR) self-assembling peptide nanofiber hydrogel as an anticancer drug carrier for multidrug-resistant cancer therapy. In RR hydrogel, highly compatible RADA16 functions as the β-sheet-based self-assembling backbone which tightly adheres to cancer tissue, prevents postoperative bleeding, and sustainedly releases anticancer drugs for long-term effects. The introduced R8 motifs endow RR hydrogel with the capacity to selectively kill cancer cells and reverse cancer multidrug resistance, which minimally impact normal tissue cells. Specifically, R8 motifs on the hydrogel scaffold selectively interfere with cancer cell membranes, increase drug uptake and penetration into cancer cells and tissues by enhancing multiple internalization pathways, and reduce drug efflux by inhibiting P-gp and BCRP multidrug-resistant transporters. Therefore, this study provides a multifunctional hydrogel material with strong potential for multidrug-resistant cancer clinical translation.

Mechanistic Insights into Hyaluronic Acid Induced Peptide Nanofiber Organization in Supramolecular Hydrogels.

Composite hydrogels composed of low-molecular-weight peptide self-assemblies and polysaccharides are gaining great interest as new types of biomaterials. Interactions between polysaccharides and peptide self-assemblies are well reported, but a molecular picture of their impact on the resulting material is still missing. Using the phosphorylated tripeptide precursor Fmoc-FFpY (Fmoc, fluorenylmethyloxycarbonyl; F, phenylalanine; Y, tyrosine; p, phosphate group), we investigated how hyaluronic acid (HA) influences the enzyme-assisted self-assembly of Fmoc-FFY generated in situ in the presence of alkaline phosphatase (AP). In the absence of HA, Fmoc-FFY peptides are known to self-assemble in nanometer thick and micrometer long fibers. The presence of HA leads to the spontaneous formation of bundles of several micrometers thickness. Using fluorescence recovery after photobleaching (FRAP), we find that in the bundles both (i) HA colocalizes with the peptide self-assemblies and (ii) its presence in the bundles is highly dynamic. The attractive interaction between negatively charged peptide fibers and negatively charged HA chains is explained through molecular dynamic simulations that show the existence of hydrogen bonds. Whereas the Fmoc-FFY peptide self-assembly itself is not affected by the presence of HA, this polysaccharide organizes the peptide nanofibers in a nematic phase visible by small-angle X-ray scattering (SAXS). The mean distance d between the nanofibers decreases by increasing the HA concentration c, but remains always larger than the diameter of the peptide nanofibers, indicating that they do not interact directly with each other. At a high enough HA concentration, the nematic organization transforms into an ordered 2D hexagonal columnar phase with a nanofiber distance d of 117 Å. Depletion interaction generated by the polysaccharides can explain the experimental power law variation and is responsible for the bundle formation and organization. Such behavior is thus suggested for the first time on nano-objects using polymers partially adsorbing on self-assembled peptide nanofibers.

Metal-Chelating Self-Assembling Peptide Nanofiber Scaffolds for Modulation of Neuronal Cell Behavior.

Synthetic peptides are promising structural and functional components of bioactive and tissue-engineering scaffolds. Here, we demonstrate the design of self-assembling nanofiber scaffolds based on peptide amphiphile (PA) molecules containing multi-functional histidine residues with trace metal (TM) coordination ability. The self-assembly of PAs and characteristics of PA nanofiber scaffolds along with their interaction with Zn, Cu, and Mn essential microelements were studied. The effects of TM-activated PA scaffolds on mammalian cell behavior, reactive oxygen species (ROS), and glutathione levels were shown. The study reveals the ability of these scaffolds to modulate adhesion, proliferation, and morphological differentiation of neuronal PC-12 cells, suggesting a particular role of Mn(II) in cell-matrix interaction and neuritogenesis. The results provide a proof-of-concept for the development of histidine-functionalized peptide nanofiber scaffolds activated with ROS- and cell-modulating TMs to induce regenerative responses.

Solvent-Evaporation-Induced Synthesis of Graphene Oxide/Peptide Nanofiber (GO/PNF) Hybrid Membranes Doped with Silver Nanoparticles for Antibacterial Application

Designing functional membranes through the collaboration of multi-dimensional nanomaterials is of particular interest in environmental and biomedical applications. Herein, we propose a facile and green synthetic strategy by collaborating with graphene oxide (GO), peptides, and silver nanoparticles (AgNPs) to synthesize functional hybrid membranes with favourable antibacterial effects. GO nanosheets are functionalized with self-assembled peptide nanofibers (PNFs) to form GO/PNFs nanohybrids, in which the PNFs not only improve the biocompatibility and dispersity of GO, but also provide more active sites for growing and anchoring AgNPs. As a result, multifunctional GO/PNFs/AgNP hybrid membranes with adjustable thickness and AgNP density are prepared via the solvent evaporation technique. The structural morphology of the as-prepared membranes is characterized using scanning electron microscopy, transmission electron microscopy, and X-ray photoelectron spectroscopy, and their properties are analyzed by spectral methods. The hybrid membranes are then subjected to antibacterial experiments and their excellent antibacterial performances are demonstrated.

Repair of critical-sized rat cranial defects with RADA16-W9 self-assembled peptide hydrogel

Bone defects are common in orthopaedics and there is an urgent need to explore effective bone repair materials with osteoinductive activity. Peptide self-assembled nanomaterials have a fibrous structure like that of the extracellular matrix and are ideal bionic scaffold materials. In this study, a short peptide WP9QY (W9) with strong osteoinductive effect was tagged to a self-assembled peptide RADA16 molecule through solid phase synthesis to design a RADA16-W9 peptide gel scaffold. A rat cranial defect was used as a research model to explore the effect of this peptide material on the repair of bone defects in vivo. The structure characteristic of the functional self-assembling peptide nanofiber hydrogel scaffold RADA16-W9 was evaluated by atomic force microscopy (AFM). Then adipose stem cells (ASCs) were isolated from Sprague-Dawley (SD) rat and cultured. the cellular compatibility of scaffold was evaluated through Live/Dead assay. Furthermore, we explore the effects of hydrogels in vivo with the critical-sized mouse calvarial defect model. Micro-CT analysis showed that the RADA16-W9 group had higher levels of bone volume/total volume (BV/TV) (P < 0.05)，Trabecular number(TB.N) (P < 0.05)，bone mineral density (BMD)(P < 0.05) and trabecular thickness (Tb. Th) (P < 0.05) compared with the RADA16 and PBS groups. Hematoxylin and eosin (H&E) staining showed that RADA16-W9 group had the highest bone regeneration level. Histochemical staining showed significantly higher expression levels of osteogenic factors such as alkaline phosphatase (ALP) and osteocalcin (OCN) in the RADA16-W9 group than in the other two groups (P < 0.05). Reverse transcription polymerase chain reaction (RT-PCR) quantification showed higher mRNA expression levels of osteogenic-related genes ALP, Runt-related transcription factor 2(Runx2), OCN, Osteopontin (OPN) in the RADA16-W9 group than in the RADA16 and PBS groups (P < 0.05). The live/dead staining results showed that RADA16-W9 is not toxic to rASCs and has good biocompatibility. In vivo experiments show that it accelerates the process of bone reconstruction, significantly promoting bone regeneration and can be used to develop a molecular drug for bone defect repair.

Supramolecular nanofibers co-loaded with dabrafenib and doxorubicin for targeted and synergistic therapy of differentiated thyroid carcinoma.

Rationale: Although surgery, radioiodine therapy, and thyroid hormone therapy are the primary clinical treatments for differentiated thyroid carcinoma (DTC), effective therapy for locally advanced or progressive DTC remains challenging. BRAF V600E, the most common BRAF mutation subtype, is highly related to DTC. Previous studies prove that combination of kinase inhibitors and chemotherapeutic drugs may be a potential approach for DTC treatment. In this study, a supramolecular peptide nanofiber (SPNs) co-loaded with dabrafenib (Da) and doxorubicin (Dox) was constructed for targeted and synergistic therapy with BRAF V600E+ DTC. Methods: A self-assembling peptide nanofiber (Biotin-GDFDFDYGRGD, termed SPNs) bearing biotin at the N-terminus and a cancer-targeting ligand RGD at the C-terminus was used as a carrier for co-loading Da and Dox. D-phenylalanine and D-tyrosine (DFDFDY) are used to improve the stability of peptides in vivo. Under multiple non-covalent interactions, SPNs/Da/Dox assembled into longer and denser nanofibers. RGD ligand endows self-assembled nanofibers with targeting cancer cells and co-delivery, thereby improving cellular uptake of payloads. Results: Both Da and Dox indicated decreased IC50 values upon encapsulation in SPNs. Co-delivery of Da and Dox by SPNs exhibited the strongest therapeutic effect in vitro and in vivo by inhibiting ERK phosphorylation in BRAF V600E mutant thyroid cancer cells. Moreover, SPNs enable efficient drug delivery and lower Dox dosage, thereby significantly reducing its side effects. Conclusion: This study proposes a promising paradigm for the synergistic treatment of DTC with Da and Dox using supramolecular self-assembled peptides as carriers.

Atomistic Pictures of Self-Assembled Helical Peptide Nanofibers.

Spontaneous self-assembly of peptides has been at the forefront of supramolecular chemistry and materials science research over the last two decades. Despite the wealth of information on the morphology of the assembled objects, atomic resolution details of molecular arrangements inside them are largely unknown. In this paper, we investigated non-covalent assemblies of zwitterionic l-phenylalanine tripeptides in water using all-atom explicit-solvent molecular dynamics computer simulations. Our studies produced atomistic pictures of spontaneously assembled nanofibers composed of hundreds of peptide molecules. The dimensions of the nanofibers varied from 10 to 18 nm, with irregular helical twists along the long axes. Previously published experimental data, acquired under similar conditions, provided direct validation of the fibrous morphology and indirect support for the non-trivial helicity observed in our simulations. Quantitative analyses of peptide-water and peptide-peptide interactions revealed heterogeneous local environments of molecules across the nanometer length scales. The combination of electrostatic, hydrogen bonding, van der Waals, and hydrophobic interactions, adopted by a single molecule, was dependent on its relative position inside the fiber. Despite the presence of three hydrophobic phenyl groups, very few molecules were found to be completely shielded from the surrounding water, indicating a subtle role of the hydrophobic effect. Limited conformational flexibility of the tripeptide, along with bare electrostatic interactions, appeared to play a crucial role in the emergence of fibrous morphology of the nanostructures. Our analyses led us to formulate plausible qualitative explanations of the assembly behavior in terms of thermodynamic driving forces and kinetic considerations. We established a clear relationship between details of chemical interactions operating within few molecules and characteristics of the self-assembled states at much longer length scales.

Repairing sciatic nerve injury with self-assembling peptide nanofiber scaffold-containing chitosan conduit

BackgroundAn increasing number of nerve guide scaffolds have been used to replace the “gold-standard” autologous nerve graft for repairing peripheral nerve defects, but nerve regeneration is usually far from complete.MethodsHere, we designed and prepared two functionalized self-assembling peptides (SAP) with the IKVAV and KLT sequences, which were derived from the combination of laminin and VEGF, respectively. Their mixtures were also obtained to combine the effects of neuroprotective and neurotrophic and proangiogenic factors.ResultsThe beneficial effect of peptide gels on nerve regeneration was evaluated in vitro using Schwann cells (SCs). As a useful intraluminal filling, a three-dimensional (3D) functionalized self-assembling peptide (SAP) nanofiber hydrogel was formed in the hollow lumen of chitosan conduits under physiological conditions. In vivo, the combination of the two functionalized SAP gels containing a chitosan nerve conduit significantly accelerated nerve healing and enhanced morphological repair.ConclusionBased on the current findings, the combined application of two functionalized SAP gels with chitosan nerve conduit is a promising therapy for the engineering of peripheral nerve regeneration.

Self-assembling peptide nanofiber HIV vaccine elicits robust vaccine-induced antibody functions and modulates Fc glycosylation.

To develop vaccines for certain key global pathogens such as HIV, it is crucial to elicit both neutralizing and non-neutralizing Fc-mediated effector antibody functions. Clinical evidence indicates that non-neutralizing antibody functions including antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) contribute to protection against several pathogens. In this study, we demonstrated that conjugation of HIV Envelope (Env) antigen gp120 to a self-assembling nanofiber material named Q11 induced antibodies with higher breadth and functionality when compared to soluble gp120. Immunization with Q11-conjugated gp120 vaccine (gp120-Q11) demonstrated higher tier 1 neutralization, ADCP, and ADCC as compared to soluble gp120. Moreover, Q11 conjugation altered the Fc N-glycosylation profile of antigen-specific antibodies, leading to a phenotype associated with increased ADCC in animals immunized with gp120-Q11. Thus, this nanomaterial vaccine strategy can enhance non-neutralizing antibody functions possibly through modulation of immunoglobulin G Fc N-glycosylation.

Increased Stiffness Downregulates Focal Adhesion Kinase Expression in Pancreatic Cancer Cells Cultured in 3D Self-Assembling Peptide Scaffolds.

The focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that participates in integrin-mediated signal transduction and contributes to different biological processes, such as cell migration, survival, proliferation and angiogenesis. Moreover, FAK can be activated by autophosphorylation at position Y397 and trigger different signaling pathways in response to increased extracellular matrix stiffness. In addition, FAK is overexpressed and/or hyperactivated in many epithelial cancers, and its expression correlates with tumor malignancy and invasion potential. One of the characteristics of solid tumors is an over deposition of ECM components, which generates a stiff microenvironment that promotes, among other features, sustained cell proliferation and survival. Researchers are, therefore, increasingly developing cell culture models to mimic the increased stiffness associated with these kinds of tumors. In the present work, we have developed a new 3D in vitro model to study the effect of matrix stiffness in pancreatic ductal adenocarcinoma (PDAC) cells as this kind of tumor is characterized by a desmoplastic stroma and an increased stiffness compared to its normal counterpart. For that, we have used a synthetic self-assembling peptide nanofiber matrix, RAD16-I, which does not suffer a significant degradation in vitro, thus allowing to maintain the same local stiffness along culture time. We show that increased matrix stiffness in synthetic 3D RAD16-I gels, but not in collagen type I scaffolds, promotes FAK downregulation at a protein level in all the cell lines analyzed. Moreover, even though it has classically been described that stiff 3D matrices promote an increase in pFAKY397/FAK proteins, we found that this ratio in soft and stiff RAD16-I gels is cell-type-dependent. This study highlights how cell response to increased matrix stiffness greatly depends on the nature of the matrix used for 3D culture.

Biomimetic Organic-Inorganic Hybrid Membranes for Removal of Fluoride Ions.

Carbon nanofibers (CaNFs) exhibit promising applications in the fields of environmental science and nanotechnology, and self-assembled peptide nanofibers (PNFs) are useful for the biomimetic synthesis of organic-inorganic hybrid nanomaterials and the fabrication of functional hybrid membranes for the removal of various pollutants from water. In this work, we report the biomimetic synthesis of hybrid nanomaterials by the interweaving of CaNFs and PNFs. Using the biomimetic mineralization properties of PNFs, ZrO2 nanoparticles were synthesized along the nanofiber surface, and then functional nanohybrid porous membranes were prepared by the vacuum filtration technology. For the fabrication of membranes, the amount of PNFs and ZrO2 precursors in the hybrid membrane were optimized. The designed organic-inorganic hybrid membranes exhibited high removal performance for fluorine ion (F−) from water, and the removal efficiency of the fabricated membranes towards F− ion-containing aqueous solution with a concentration of 50–100 mg/L reached more than 80%. In addition, the nanofiltration membranes revealed good adsorption capacity for F− ions. It is expected that the strategies shown in this study will be beneficial for the design, biomimetic synthesis, and fabrication of nanoporous membranes for economic, rapid, and efficient water purification.

Designer self-assembling peptide nanofibers induce biomineralization of lidocaine for slow-release and prolonged analgesia

The burst release of small molecular water-soluble drugs is a major problem when pursuing their long-acting formulations. Although various types of carrier materials have been developed for tackling this problem, it is still a big challenge to prevent water-soluble small molecules from fast release and diffusion. In this study, a biomineralization strategy based upon a self-assembling peptide is proposed for the slow release of lidocaine, a classic anesthetic with high solubility and a very small molecular weight. A bolaamphiphilic peptide was designed to self-assemble and produce negatively charged nanofibers, which were used as the template to absorb positively charged lidocaine molecules through an electrostatic interaction. The biomineralization of lidocaine was then induced by adjusting the pH, which lead to the formation of lidocaine microcrystals with a homogenous size. The microcrystals were incorporated into a hyaluronic acid hydrogel to form an injectable formulation. This formulation slowly released lidocaine and generate a prolonged anesthetic and analgesic effect in rodent models. Due to the constrained local and plasma lidocaine concentration, as well as the biocompatibility and biodegradability of the peptide materials, this formulation also showed considerable safety. These results suggest that nanofiber assisted biomineralization can provide a potential strategy for the fabrication of long-acting formulations for small molecular water-soluble drugs. Statement of significanceLong-acting formulations are highly pursued to achieve stronger therapeutic effect, or to avoid repeated administration of drugs, especially through painful injection. Using carrier materials to slow down the release of bioactive molecules is a common strategy to reach this goal. However, for many water-soluble small molecular drugs currently used in clinic, it is notoriously difficult to slow down their release and diffusion. This study proposes a novel strategy based on a controllable mineralization process using self-assembling peptide nanofibers as the template. Taking lidocaine as an example, we showed how peptide-drug microcrystals with well-controlled size and shape could be obtained, which exhibit significantly prolonged anesthetic and analgesic effect. As a proof-of-concept study, this work proposes a promising strategy to control the release of water-soluble small molecular drugs.

Stem cell-homing hydrogel-based miR-29b-5p delivery promotes cartilage regeneration by suppressing senescence in an osteoarthritis rat model.

Osteoarthritis (OA) is a common joint disease characterized by progressive loss of cartilage and reduction in lubricating synovial fluid, which lacks effective treatments currently. Here, we propose a hydrogel-based miRNA delivery strategy to rejuvenate impaired cartilage by creating a regenerative microenvironment to mitigate chondrocyte senescence that mainly contributes to cartilage breakdown during OA development. An aging-related miRNA, miR-29b-5p, was first found to be markedly down-regulated in OA cartilage, and their up-regulation suppressed the expression of matrix metalloproteinases and senescence-associated genes (P16INK4a/P21) via ten-eleven-translocation enzyme 1 (TET1). An injectable bioactive self-assembling peptide nanofiber hydrogel was applied to deliver agomir-29b-5p, which was functionalized by conjugating a stem cell–homing peptide SKPPGTSS for endogenous synovial stem cell recruitment simultaneously. Sustained miR-29b-5p delivery and recruitment of synovial stem cells and their subsequent differentiation into chondrocytes led to successful cartilage repair and chondrocyte rejuvenation. This strategy enables miRNA-based therapeutic modality to become a viable alternative for surgery in OA treatment.