Anisotropic formation mechanism and nanomechanics for the self-assembly process of cross-β peptides

Nanostructures self-assembled by cross-β peptides with ordered structures and advantageous mechanical properties have many potential applications in biomaterials and nanotechnologies. Quantifying the intra-and inter-molecular driving forces for peptide self-assembly at the atomistic level is essential for understanding the formation mechanism and nanomechanics of various morphologies of self-assembled peptides. We investigate the thermodynamics of the intra-and inter-sheet structure formations in the self-assembly process of cross-β peptide KⅢIK by means of steered molecular dynamics simulation combined with umbrella sampling. It is found that the mechanical properties of the intra-and inter-sheet structures are highly anisotropic with their intermolecular bond stiffness at the temperature of 300 K being 5.58 N/m and 0.32 N/m, respectively. This mechanical anisotropy comes from the fact that the intra-sheet structure is stabilized by enthalpy but the inter-sheet structure is stabilized by entropy. Moreover, the formation process of KⅢIK intra-sheet structure is cooperatively driven by the van der Waals （VDW） interaction between the hydrophobic side chains and the electrostatic interaction between the hydrophilic backbones, but that of the inter-sheet structure is primarily driven by the VDW interaction between the hydrophobic side chains. Although only peptide KⅢIK is studied, the qualitative conclusions on the formation mechanism should also apply to other cross-β peptides.

Experimental Study on Self-assembly of KLD-12 Peptide Hydrogel and 3-D Culture of MSC Encapsulated within Hydrogel In Vitro

To synthesize KLD-12 peptide with sequence of AcN-KLDLKLDLKLDL-CNH2 and trigger its self-assembly in vitro, to encapsulate rabbit MSCs within peptide hydrogel for 3-D culture and to evaluate the feasibility of using it as injectable scaffold for tissue engineering of IVD. KLD-12 peptide was purified and tested with high performance liquid chromatography （HPLC） and mass spectroscopy （MS）. KLD-12 peptide solutions with concentrations of 5 g/L, 2.5 g/L and 1 g/L were triggered to self-assembly with 1 xPBS in vitro, and the self-assembled peptide hydrogel was morphologically observed. Atomic force microscope （AFM） was employed to examine the inner structure of self-assembled peptide hydrogel. Mesenchymal stem cells （MSCs） were encapsulated within peptide hydrogel for 3-D culture for 2 weeks. Calcein-AM/PI fluorescence staining was used to detect living and dead cells. Cell viability was observed to evaluate the bioactivity of MSCs in KLD-12 peptide hydrogel. The results of HPLC and MS showed that the relative molecular mass of KLD-12 peptide was 1467.83, with a purity quotient of 95.36%. KLD-12 peptide at 5 g/L could self-assemble to produce a hydrogel, which was structurally integral and homogeneous and was able to provide sufficient cohesion to retain the shape of hydrogel. AFM demonstrated that the self-assembly of KLD-12 peptide hydrogel was successful and the assembled material was composed of a kind of nano-fiber with a diameter of 3040 nm and a length of hundreds of nm. Calcein-AM/PI fluorescence staining revealed that MSCs in KLD-12 peptide hydrogel grew well. Cell activity detection exhibited that the A value increased over the culture time. It is concluded that KLD-12 peptide was synthesized successfully and was able to self-assemble to produce nano-fiber hydrogel in vitro. MSCs in KLD-12 peptide hydrogel grew well and proliferated with the culture time. KLD-12 peptide hydrogel can serve as an excellent injectable material of biological scaffolds in tissue engineering of IVD.

Hierarchical processes in β-sheet peptide self-assembly from the microscopic to the mesoscopic level

Under appropriate physicochemical conditions, short peptide fragments and their synthetic mimics have been shown to form elongated cross-fl nanostructures through self-assembly. The self-assembly process and the resultant peptide nanos- tructures are not only related to neurodegenerative diseases but also provide inspiration for the development of novel bionanomaterials. Both experimental and theoretical studies on peptide self-assembly have shown that the self-assembly process spans multiple time and length scales and is hierarchical, β-sheet self-assembly consists of three sub-processes from the microscopic to the mesoscopic level： β-sheet locking, lateral stacking, and morphological transformation. De- tailed atomistic simulation studies have provided insight into the early stages of peptide nanostructure formation and the interplay between different non-covalent interactions at the microscopic level. This review gives a brief introduction of the hierarchical peptide self-assembly process and focuses on the roles of various non-covalent interactions in the sub-processes based on recent simulation, experimental, and theoretical studies.

A matrix metalloproteinase-responsive hydrogel system controls angiogenic peptide release for repair of cerebral ischemia/reperfusion injury

Vascular endothelial growth factor and its mimic peptide KLTWQELYQLKYKGI(QK)are widely used as the most potent angiogenic factors for the treatment of multiple ischemic diseases.However,conventional topical drug delivery often results in a burst release of the drug,leading to transient retention(inefficacy)and undesirable diffusion(toxicity)in vivo.Therefore,a drug delivery system that responds to changes in the microenvironment of tissue regeneration and controls vascular endothelial growth factor release is crucial to improve the treatment of ischemic stroke.Matrix metalloproteinase-2(MMP-2)is gradually upregulated after cerebral ischemia.Herein,vascular endothelial growth factor mimic peptide QK was self-assembled with MMP-2-cleaved peptide PLGLAG(TIMP)and customizable peptide amphiphilic(PA)molecules to construct nanofiber hydrogel PA-TIMP-QK.PA-TIMP-QK was found to control the delivery of QK by MMP-2 upregulation after cerebral ischemia/reperfusion and had a similar biological activity with vascular endothelial growth factor in vitro.The results indicated that PA-TIMP-QK promoted neuronal survival,restored local blood circulation,reduced blood-brain barrier permeability,and restored motor function.These findings suggest that the self-assembling nanofiber hydrogel PA-TIMP-QK may provide an intelligent drug delivery system that responds to the microenvironment and promotes regeneration and repair after cerebral ischemia/reperfusion injury.

Modulation of intra- and inter-sheet interactions in short peptide self-assembly by acetonitrile in aqueous solution

Besides our previous experimental discovery （Zhao Y R, et al. 2015 Langmuir, 31, 12975） that acetonitrile （ACN） can tune the morphological features of nanostructures self-assembled by short peptides KIIIIK （KI4K） in aqueous solution, further experiments reported in this work demonstrate that ACN can also tune the mass of the self-assembled nanostructures. To understand the microscopic mechanism how ACN molecules interfere peptide self-assembly process, we conducted a series of molecular dynamics simulations on a monomer, a cross-β sheet structure, and a proto-fibril of KI4K in pure water, pure ACN, and ACN-water mixtures, respectively. The simulation results indicate that ACN enhances the intra-sheet interaction dominated by the hydrogen bonding （H-bonding） interactions between peptide backbones, but weakens the inter-sheet interaction dominated by the interactions between hydrophobic side chains. Through analyzing the correlations between different groups of solvent and peptides and the solvent behaviors around the proto-fibril, we have found that both the polar and nonpolar groups of ACN play significant roles in causing the opposite effects on intermolecular interactions among peptides. The weaker correlation of the polar group of ACN than water molecule with the peptide backbone enhances H-bonding interactions between peptides in the proto-fibril. The stronger correlation of the nonpolar group of ACN than water molecule with the peptide side chain leads to the accumulation of ACN molecules around the proto-fibril with their hydrophilic groups exposed to water, which in turn allows more water molecules close to the proto-fibril surface and weakens the inter-sheet interactions. The two opposite effects caused by ACN form a microscopic mechanism clearly explaining our experimental observations.

Recent advances in design and applications of biomimetic self-assembled peptide hydrogels for hard tissue regeneration

The development of natural biomaterials applied for hard tissue repair and regeneration is of great importance,especially in societies with a large elderly population.Self-assembled peptide hydrogels are a new generation of biomaterials that provide excellent biocompatibility,tunable mechanical stability,injectability,trigger capability,lack of immunogenic reactions,and the ability to load cells and active pharmaceutical agents for tissue regeneration.Peptide-based hydrogels are ideal templates for the deposition of hydroxyapatite crystals,which can mimic the extracellular matrix.Thus,peptide-based hydrogels enhance hard tissue repair and regeneration compared to conventional methods.This review presents three major self-assembled peptide hydrogels with potential application for bone and dental tissue regeneration,including ionic self-complementary peptides,amphiphilic(surfactant-like)peptides,and triple-helix(collagen-like)peptides.Special attention is given to the main bioactive peptides,the role and importance of self-assembled peptide hydrogels,and a brief overview on molecular simulation of self-assembled peptide hydrogels applied for bone and dental tissue engineering and regeneration.

A novel artificial nerve graft for repairing longdistance sciatic nerve defects:a self-assembling peptide nanofiber scaffold-containing poly (lactic-co-glycolic acid) conduit

In this study, we developed a novel artificial nerve graft termed self-assembling peptide nanofiber scaffold （SAPNS）-containing poly（lactic-co-glycolic acid） （PLGA） conduit （SPC） and used it to bridge a 10-mm-long sciatic nerve defect in the rat. Retrograde tracing, behavioral testing and histomorphometric analyses showed that compared with the empty PLGA conduit implantation group, the SPC implantation group had a larger number of growing and extending axons, a markedly increased diameter of regenerated axons and a greater thickness of the myelin sheath in the conduit. Furthermore, there was an increase in the size of the neuromuscular junction and myofiber diameter in the target muscle. These findings suggest that the novel artificial SPC nerve graft can promote axonal regeneration and remyelination in the transected peripheral nerve and can be used for repairing peripheral nerve injury.

Self-assembled IKVAV Peptide Nanofibers Promote Adherence of PC12 Cells

Lack of biocompatibility and bioactivity is a big problem for the synthetic materials that have been generated for neural tissue engineering. To get around the problem and generate better scaffold for neural tissue repair, we intended to generate nano-fibers by self-assembly of polypeptide IKVAV. Bioactive IKVAV Peptide-Amphiphile （IKVAV-PA） was first synthesized and purified, the property of which was analyzed and determined by high-performance liquid chromatography （HPLC） and mass spectrometry （MS）. Then, by addition of hydrogen chloride （HC1）, self-assembly of IKVAV-PA was induced in vitro and nano-fibers formed as shown by transmission electron microscopy （TEM）. The effect of IKVAV nanofibers on adherence of PCI2 cells was assayed in cell culture and the results showed that the rates of adherence of PC12 increased significantly when the density of IKVAV was within a certain range （0.58 μg/cm^2 to 15.6 μg/cm^2）. However, its effect on the rates of adherence did not significantly alter with time, whether after 1 hour or 3 hours of culture. In general, we showed that IKVAV-PA can successfully self-assemble to form nanofiber, and promote rapid and stable adherence of PC12 cells, and the effect of the self-assembled IKVAV to promote PCI2 cells adherence is dosage-dependent within a certain range of densities.

Chondrogenesis of Precartilaginous Stem Cells in KLD-12 Self-assembling Peptide Nanofiber Scaffold Loading TGF-β3 Gene

The effect of culture in KLD-12 self-assembling peptide nanofiber scaffold containing TGF-β3 gene on differentiation of precartilaginous stem cells （PSCs） into chondrocytes was studied. KLD-12 was synthesized by solid-state method. After TGF-β3 plasmid was loaded into KLD-12 self-assembling peptide nanofiber scaffold, DNA release ability was investigated. PSCs and hTGF-β3 gene were loaded into KLD-12 3-D scaffold, and MTT assay was performed to investigate the cell proliferation, and ELASA assay was used to investigate the expression of TGF-β3. Specific cartilage matrix was examined by quantitative real-time PCR, immunohistochemistry and Alcian Blue staining. Compared with control group, DNA synthesis level of PSCs reached the peak within 3 days when PSCs were cultured in self-assembling peptide nanofiber scaffold loading TGF-β3 plasmid, and maintained this high level within 2 weeks. MTT results showed that the proliferation ability of experimental group was statistically higher than that in control group （P〈0.05）. Quantitative real-time PCR suggested that the percentage of TGF-β3 positive PSCs in experimental group was higher than that in control group （P〈0.01）. ELISA assay showed that the TGF-β3 protein level increased in supernatant of experimental group＇s PSCs, reached the peak after 72 h and then declined a little to the plateau phase. Compared with the control group, the specific gene of chondrocyte typical extracellular matrix significantly up-regulated （P〈0.01）. The results showed that PSCs differentiated into chondrocytes in self-assembling peptide nanofiber scaffold loading TGF-β3 plasmid, which provided a fresh approach to cartilage tissue engineering.

Self-assembling peptide nanofibrous hydrogel as a promising strategy in nerve repair after traumatic injury in the nervous system

Following injury in central nervous system（CNS）,there are pathological changes in the injured region,which include neuronal death,axonal damage and demyelination,inflammatory response and activation of glial cells.The proliferation of a large number of astrocytes results in the formation of glial scar.

Toward Artificial Peptide Nanocapsules

HIGHLIGHTS The formation of peptide nanocapsules is facilitated by a gradient interface,where the differential solvent concentration drives the peptides to preferentially localize and assemble.The peptide nanocapsules,characterized by their hollow structures,demonstrated potential as carriers for targeted drug delivery.1 Introduction Peptide nanocapsules are a type of nanoscale delivery system that encapsulates active substances within a shell composed of peptides,leveraging the unique properties of peptides such as biocompatibility and biodegradability[1].Historically,the development of peptide nanocapsules was inspired primordially by the natural biological processes.

Revolutionizing Antimicrobial Biomaterials:Integrating an Enzyme Degradation‑Resistant Sequence into Self‑Assembled Nanosystems to Overcome Stability Limitations of Peptide‑Based Drugs

Incorporating enzyme-resistant peptide sequences into self-assembled nanosystems is a promising strategy to enhance the stability and versatility of peptide-based antibacterial drugs,aiming to replace ineffective antibiotics.By combining newly designed enzymatic-resistant sequences with synthetically derived compounds bearing single,double,triple,or quadruple aromatic rings.A series of nanoscale antimicrobial self-assembled short peptides for the purpose of combating bacterial infections are generated.Nap^(*)(Nap–DNal–Nal–Dab–Dab–NH2,where Nap represents the 1-naphthylacetyl group)possesses the greatest clinical potential(GMSI=23.96)among the peptides in this series.At high concentrations in an aqueous environment,Nap^(*)spontaneously generates nanofibers to capture bacteria and prevent their evasion,exhibiting broad-spectrum antimicrobial effects and exceptional biocompatibility.In the presence of physiological salt ions and serum,the antimicrobial agent exhibits strong effectiveness and retains impressive resistance even when exposed to high levels of proteases(trypsin,chymotrypsin,pepsin).Nap^(*)exhibits negligible in vivo toxicity and effectively alleviates systemic bacterial infections in mice.Mechanistically,Nap^(*)initially captures bacteria and induces bacterial cell death primarily through membrane dissolution,achieved by multiple synergistic mechanisms.In summary,these advances have the potential to greatly expedite the clinical evolution of nanomaterials based on short peptides combined with naphthyl groups and foster the development of peptides integrated with self-assembled systems in this domain.

Self‑Assembling Peptide Nanofibers Anchored Parathyroid Hormone Derivative for Bone Tissue Engineering

Parathyroid hormone(PTH)has been used for bone regeneration through intermittent subcutaneous injection;however,the topical administration of PTH for bone repair remains challenging because of the overactivation of osteoclasts.Here,a PTH derivative,i.e.,PTHrP-1,which exhibits enhanced osteogenesis and relatively reduced osteoclastogenesis,is anchored to RADA16-I to fabricate a novel self-assembling peptide,called P1R16.Firstly,P1R16 self-assembles into long nanofibers with PTHrP-1 exposed to the side end,which interacts with Type I collagen(Col)to form P1R16-Col composites.The RADA16 segment in P1R16 helps the sustained release of P1R16 from the composites.Secondly,the P1R16 self-assembling peptide nanofibers exhibit multiple functions.The nanofibers promote stem cell proliferation and recruitment,and then direct stem cell fate towards osteogenic differentiation but not adpipogenic differentiation,improving the quality of the regenerated bone.The nanofibers further promote bone regeneration through bone remodeling between osteoblasts and osteoclasts.Thirdly,the P1R16 self-assembling peptide nanofibers also promote the proliferation and recruitment of endothelial cells,which facilitate the vascularization of implants to support bone regeneration further.Overall,the P1R16 self-assembling peptide nanofibers maintain multiple functions,including pro-proliferation,direction of stem cell fate,bone remodeling and vascularization,showing considerable promise for bone tissue engineering to repair bone defects or fractures.

In situ aneuploidy assessment in human sperm:the use of primed in situ and peptide nucleic acid-fluorescence in situ hybridization techniques

Both the primed in situ(PRINS)and the peptide nucleic acid-fluorescence in situ hybridization(PNA-FISH) techniques constitute alternatives to the conventional(fluorescence in situ hybridization,FISH)procedure for chro- mosomal investigations.The PRINS reaction is based on the use of a DNA polymerase and labeled nucleotide in an in situ primer extension reaction.Peptide nucleic acid probes are synthetic DNA analogs with uncharged polyamide backbones.The two procedures present several advantages(specificity,rapidity and discriminating ability)that make them very attractive for cytogenetic purposes.Their adaptation to human spermatozoa has allowed the development of new and fast procedures for the chromosomal screening of male gametes and has provided efficient complements to FISH for in situ assessment of aneuploidy in male gametes.(Asian J Androl 2006 Jul;8:387-392)

Multistep Desolvation as a Fundamental Principle Governing Peptide Self-Assembly Through Liquid-Liquid Phase Separation

Biomolecular self-assembly based on peptides and proteins is a general phenomenon encountered in natural and synthetic systems.Liquid–liquid phase separation(LLPS)is intimately involved in biomolecular self-assembly,yet the key factors at a molecular scale activating or modulating such a process remain largely elusive.Herein,we discovered in our experiments that multistep desolvation is fundamental to the formation and evolution of peptide-rich droplets:The first step was partial desolvation of peptides to form peptide clusters,and the second step was selective desolvation of hydrophobic groups within clusters to trigger LLPS and the formation of peptiderich droplets,followed by complete desolvation of droplets,initiating the nucleation of peptide selfassembly.Manipulation of the degree of desolvation at different stages was an effective strategy to control the self-assembly pathways and polymorphisms.This study sheds light on the molecular origin of LLPS-mediated self-assembly distinct from classical one-step self-assembly and paves the way for the precise control of supramolecular self-assembly.

Tailoring supramolecular antimicrobial peptides:from self-assembled nanoarchitectures to activities

The emergence of antibiotic-resistant bacteria has become a major threat to global public health and has prompted the discovery of antibiotic alternatives.Natural antimicrobial peptides(AMPs)confer a unique non-specific membrane rupture mechanism,showing great potential in killing drug-resistant bacteria.However,natural AMPs have certain weaknesses,including stability and toxicity issues,which seriously hinder their in vivo applications.Synthetic AMPs possess similar characteristics to natural AMPs,including positive charges,amphiphilicity,and the ability to fold into diverse secondary structures.These properties are essential for AMPs penetration into membranes,allowing them to exhibit antimicrobial effects.Moreover,supramolecular self-assembly,facilitated by hydrophobic interaction,hydrogen bonding,π-πstacking,and electrostatic interaction,can generate nanoparticles,nanotubes,nanofibers,and hydrogels with well-defined nanoarchitectures.Utilizing peptide self-assembly to form various nanoarchitectures is an effective approach for generating antibacterial nanomaterials,offering potential advantages such as enhanced antibacterial properties,improved stability,and reduced cytotoxicity.This review highlights recent advancements in tailoring supramolecular AMPs to create diverse nano-architectures for combating infectious diseases.

Biocompatibility of KLD-12 Peptide Hydrogel as a Scaffold in Tissue Engineering of Intervertebral Discs in Rabbits

KLD-12 peptide with a sequence of AcN-KLDLKLDLKLDL-CNH2 was synthesized and its biocompatibility was assessed in animals. Rabbit MSCs were cultured in the hydrogel for 2 weeks. Live cells were counted by using Calcein-AM/P1 fluorescence staining. MTT was employed to assess the viability of MSCs cultured in KLD-12 peptide solution of 0.01%, 0.03%, and 0.05%. Hemolysis test, skin irritation test and implantation test were conducted to evaluate its biocompatibility with host tissues. Our results demonstrated that the MSCs in hydrogel grew well and maintained round shape. Cell survival rate was 92.15% （mean： 92.15%±1.17%） at the 7th day and there was no difference in survival rate between day 7 and day 14. Cell proliferation test showed that the A value of the KLD-12 solutions was not significantly different from that of control groups （complete culture media） （P〉0.05） at the 24th and 48th h. The hemolysis rate of KLD-12 solution was 0.112%. Skin irritation test showed that the skin injected with KLD-12 solution remained normal and the score of skin irritation was 0. The histological examination with HE staining exhibited that the skin layers were clear and there was no infiltration with neutrophilic granulocytes and lymphocytes. It is concluded that KLD-12 peptide hydrogel bad a good biocompatibility with host rabbit and MSCs, and KLD-12 pep- tide hydrogel can provide an appropriate microenvironment for MSCs.

Self-assembly of amphiphilic peptides to construct activatable nanophotosensitizers for theranostic photodynamic therapy

A variety of nano-engineered photosensitizers have been developed for photodynamic therapy(PDT)of cancer diseases. However, traditional nano-engineering methods usually cannot avoid drug leakage and premature release, and have disadvantages such as low drug load and inaccurate release.The self-assembly strategy based on amphiphilic peptides has been considered to be more attractive nano-engineering method. Here we developed novel acid-activatable self-assembled nanophotosensitizers based on an amphiphilic peptide derivative. The peptide derivative was synthesized from a fluorescein molecule with thermally activated delayed fluorescence(TADF). The self-assembled nanophotosensitizers can specifically enter the tumor cells and disassemble inside lysosomes companied with “turn-on” fluorescence and photodynamic therapy effect. Such smart nanophotosensitizers will open new opportunities for cancer theranostics.

Self-assembly of surfactant-like peptides and their applications

Numerous peptides derived from naturally occurring proteins or de novo designed have been found to self-assemble into various nanostructures.These well-defined nanostructures have shown great potential for a variety of biomedical and biotechnological applications.In particular,surfactant-like peptides(SLPs)have distinctive advantages in their length,aggregating ability,and water solubility.In this article,we report recent advances in the mechanistic understanding of the self-assembly principles of SLPs and in their applications,most of which have been made in our laboratory.Hydrogen bonding between peptide backbones,hydrophobic interaction between hydrophobic side chains,and electrostatic repulsion between charged head groups all have roles in mediating the self-assembly of SLPs;the final self-assembled nanostructures are therefore dependent on their interplay.SLPs have shown diverse applications ranging from membrane protein stabilization and antimicrobial/anticancer agents to nanofabrication and biomineralization.Future advances in the self-assembly of SLPs will hinge on their large-scale production,the design of new functional SLPs with targeted properties,and the exploitation of new or improved applications.

Role of Thermolysin in Catalytic-Controlled Self-Assembly of Fmoc-Dipeptides

In recent years,short peptide self-assembled materials,prepared under the control of the thermolysin catalyst,have been investigated extensively and shown to acquire various morphologies and functions as building blocks for a wide range of biomaterials and device applications.However,the role played by thermolysin in this enzymatically triggered peptide self-assembly is still ambiguous.Herein,we designed a series of Fmoc-dipeptide amphiphiles to explore the catalytic role of thermolysin.