Systematical mutational analysis of teriparatide on anti-osteoporosis activity by alanine scanning.

Osteoporosis is a progressive systemic skeletal disease that decreases bone density and bone quality, making them fragile and easy to break. In spite of effective anti-osteoporosis potency, teriparatide, the first anabolic medications approved for the treatment of osteoporosis, was proven to exhibit various side effects. And the relevant structure-activity relationship (SAR) of teriparatide was in need. In this work, we performed a systematical alanine scanning against teriparatide and synthesized 34 teriparatide derivatives. Their biological activities were evaluated and the importance of each residue for anti-osteoporosis activity was also revealed. A remarkable decrease in activity was observed for alanine replacement of the residue Gly12, His14, Ser17, Arg20 and Leu24, showcasing the important role of these residues in teriparatide on anti-osteoporosis activity. On contrary, when Gly13 and Gln30 were mutated to Ala, the peptide derivatives exhibited the significantly increased activities, demonstrating that these two residues could be readily replaced. Our research expanded the peptide library of teriparatide analogues and presented a potential opportunity for designing the more powerful anti-osteoporosis peptide agents.

Discovery of novel antibacterial agent for the infected wound treatment: all-hydrocarbon stapling optimization of LL-37.

Rationale: Antimicrobial peptide LL-37 has been recognized as a favorable alternative to antibiotics due to its broad antibacterial spectrum, low resistance development and diverse biological activities. However, its high manufactory cost, poor proteolytic stability, and unpredictable cytotoxicity seriously hindered its medical translation. Methods: To push the frontiers of its clinical application, all-hydrocarbon stapling strategy was exploited here for the structural modification of KR-12, the core and minimal fragment of LL-37. Results: Based on a library of KR-12 derivatives that designed and synthesized to be stapled at positions of either i, i+4 or i, i+7, structure to activity relationship was investigated. Among them, KR-12(Q5, D9) with the glutamine and aspartic acid residues stapled displayed increased helical content and positive charge. The reinforced α-helical conformation not only protected it from proteolytic hydrolysis but also improved its antibacterial efficacy via effective membrane perturbation and anti-inflammatory efficacy via compact LPS binding. Besides, the increased positive charge endowed it with an enhanced therapeutic index. On infected wound mouse model, it was demonstrated to eliminate bacteria and promote wound closure and regeneration effectively. Conclusion: Overall, the all-hydrocarbon stapling was proven to lay the foundation for the future development of antibacterial agents. KR-12(Q5, D9) could serve as a lead compound for the clinical treatment of bacterial infections.

Cyclic peptide conjugated photosensitizer for targeted phototheranostics of gram-negative bacterial infection.

Antimicrobial photodynamic therapy (PDT) is a promising alternative to antibiotics for eradicating pathogenic bacterial infections. It holds advantage of not inducing antimicrobial resistance but is limited for the treatment of gram-negative bacterial infection due to the lack of photosensitizer (PS) capable of targeted permeating the outer membrane (OM) of gram-negative bacteria. To facilitate the targeted permeability of PS, cyclic polymyxin b nonapeptide that can specifically bind to the lipopolysaccharide on OM, is conjugated to an FDA approved PS chlorin e6 via variable linkers. Based on structure to activity study, C6pCe6 with aminohexanoic linker and P2pCe6 with amino-3, 6-dioxaoctanoic linker are identified to preferentially image gram-negative bacteria. These two conjugates also exhibit improved aqueous dispersity and enhanced ROS generation, consequently enabled their selective bactericidal activities against gram-negative bacteria upon 660 nm light irradiation. The effective photobactericidal ability of P2pCe6 is further validated on P. aeruginosa infected G. mellonella. Moreover, it is demonstrated to effectively treat the P. aeruginosa infection and accelerate the healing process at the wound site of mouse. Owing to the light irradiation triggered targeted imaging and enhanced bactericidal capacities, P2pCe6 hold great potential to serve as a potent PS for mediating the phototheranostics of gram-negative bacterial infection.

Discovery of a novel antifungal agent: All-hydrocarbon stapling modification of peptide Aurein1.2.

Aurein1.2 is secreted by the Australian tree frog Litoria aurea and is active against a broad range of infectious microbes including bacteria, fungi, and viruses. Its antifungal potency has garnered considerable interest in developing novel classes of natural antifungal agents to fight pathogenic infection by fungi. However, serious pharmacological hurdles remain, hindering its clinical translation. To alleviate its susceptibility to proteolytic degradation and improve its antifungal activity, six conformationally locked peptides were synthesized through hydrocarbon stapling modification and evaluated for their physicochemical and antifungal parameters. Among them, SAU2-4 exhibited significant improvement in helicity levels, protease resistance, and antifungal activity compared to the template linear peptide Aurein1.2. These results confirmed the prominent role of hydrocarbon stapling modification in the manipulation of peptide pharmacological properties and enhanced the application potential of Aurein1.2 in the field of antifungal agent development.

pH-Activated Ce-Doped Molybdenum Oxide Nanoclusters for Tumor Microenvironment Responsive Photothermal and Chemodynamic Therapy.

Molybdenum-based nanomaterials have shown promise for anticancer treatment due to their strong photothermal and redox-activated capabilities. Herein, we have fabricated cerium-doped MoOx (Ce-MoOv) with tunable Mo/Ce molar ratios by a one-pot method and investigated their effect on chemodynamic therapy (CDT) and photothermal therapy (PTT). It is found that Ce-MoOv can self-assemble into nanoclusters in acidic conditions and the increasing Ce amount will generate oxygen vacancy defects and induce the valence change of Mo6+/Mo5+ and Ce4+/Ce3+, which leads to strong near-infrared absorption with high photothermal conversion efficiency of 71.31 and 49.86% for 808 and 1064 nm. Other than photothermal conversion, the materials demonstrate pH-/glutathione (GSH)-activated photoacoustic (PA) imaging capability in vitro. In addition, Ce-MoOv acts as a CDT reagent capable of converting endogenous H2O2 to two types of reactive oxygen species (•OH, 1O2) while depleting GSH. Ce-MoOv demonstrates an excellent therapeutic effect against HCT116 cells and effectively reduces the intracellular GSH level and significantly increases the number of reactive radicals under 1064 nm laser irradiation as compared with the no-laser group in vitro. This work provides a new paradigm using lanthanide-doped polymetallic oxides for pH-/GSH-responsive photothermal/chemodynamic therapy with PA imaging ability.

Structuring supramolecular hyaluronan hydrogels via peptide self-assembly for modulating the cell microenvironment.

The use of synthetic extracellular matrices (ECMs) in fundamental in vitro cell culture studies has been instrumental for investigating the interplay between cells and matrix components. To provide cells with a more native environment in vitro, it is desirable to design matrices that are biomimetic and emulate compositional and structural features of natural ECMs. Here, the supramolecular fabrication of peptide-hyaluronan (HA) hydrogels is presented as potential ECM surrogates, combining native HA and rationally designed cationic amphipatic peptides [(KI)nK, lysine (K), isoleucine (I), n â€‹= â€‹2-6] whose mechanical properties and microstructure are tunable by the peptide sequence. (KI)nK peptides adopt ß-sheet configuration and self-assemble into filamentous nanostructures triggered by pH or ionic strength. The self-assembly propensity of (KI)nK peptides increases with the sequence length, forming single phase hydrogels (shorter peptides) or with phase separation (longer peptides) in presence of the anionic polyelectrolyte HA through electrostatic complexations. The gel phase formed in (KI)nK-HA complexes exhibits viscoelastic behavior and triggers the formation of human mesenchymal stem cell (MSC) spheroids which disassemble over the time. It is anticipated that these (KI)nK-HA hydrogels with tunable physical and biochemical properties offer a promising platform for in vitro applications and in stem cell therapy.

DUCNP@Mn-MOF/FOE as a Highly Selective and Bioavailable Drug Delivery System for Synergistic Combination Cancer Therapy.

Heterostructures comprising lanthanide-doped upconversion nanoparticles (DUCNPs) and metal-organic frameworks (MOFs) are emerging as promising nanosystems for integrating medical diagnosis and treatment. Here, the DUCNP@Mn-MOF nanocarrier was developed, which showed good efficiency for loading and delivering a cytotoxic antitumor agent (3-F-10-OH-evodiamine, FOE). The combined advantages of the pH-responsive and peroxidase-like properties of Mn-MOF and the unique optical features of DUCNPs granted the DUCNP@Mn-MOF/FOE system synergistic chemodynamic and chemotherapeutic effects. The DUCNP@Mn-MOF nanocarrier effectively overcame the intrinsic limitations of FOE, such as its unfavorable physicochemical properties and limited in vivo potency. This complexed nanosystem was responsive to the tumor microenvironment and showed excellent tumor targeting capability. Thus, DUCNP@Mn-MOF/FOE exhibited highly selective and bioavailable drug delivery properties and is promising for cancer therapy. In a mouse breast cancer model, DUCNP@Mn-MOF/FOE inhibited tumor growth without significant toxicity. Therefore, the proposed nanosystem represents a promising theragnostic platform for multimodal combination diagnosis and therapy of tumors.

Polymyxin B-Triggered Assembly of Peptide Hydrogels for Localized and Sustained Release of Combined Antimicrobial Therapy.

Repurposing old antibiotics into more effective and safer formulations is an emergent approach to tackle the growing threat of antimicrobial resistance. Herein, a peptide hydrogel is reported for the localized and sustained release of polymyxin B (PMB), a decade-old antibiotic with increasing clinical utility for treating multidrug-resistant Gram-negative bacterial infections. The hydrogel is assembled by additing PMB solution into a rationally designed peptide amphiphile (PA) solution and its mechanical properties can be adjusted through the addition of counterions, envisioning its application in diverse infection scenarios. Sustained release of PMB from the hydrogel over a 5-day period and prolonged antimicrobial activities against Gram-negative bacteria are observed. The localized release of active PMB from the hydrogel is shown to be effective in vivo for treating Pseudomonas aeruginosa infection in the Galleria mellonella burn wound infection model, dramatically reducing the mortality from 93% to 13%. Complementary antimicrobial activity against Gram-positive Staphylococcus aureus and enhanced antimicrobial effect against the Gram-negative Acinetobacter baumannii are observed when an additional antibiotic fusidic acid is incorporated into the hydrogen network. These results demonstrate the potential of the PMB-triggered PA hydrogel as a versatile platform for the localized and sustained delivery of combined antimicrobial therapies.

Hyaluronan (HA)-inspired glycopolymers as molecular tools for studying HA functions.

Hyaluronic acid (HA), the only non-sulphated glycosaminoglycan, serves numerous structural and biological functions in the human body, from providing viscoelasticity in tissues to creating hydrated environments for cell migration and proliferation. HA is also involved in the regulation of morphogenesis, inflammation and tumorigenesis through interactions with specific HA-binding proteins. Whilst the physicochemical and biological properties of HA have been widely studied for decades, the exact mechanisms by which HA exerts its multiple functions are not completely understood. Glycopolymers offer a simple and precise synthetic platform for the preparation of glycan analogues, being an alternative to the demanding synthetic chemical glycosylation. A library of homo, statistical and alternating HA glycopolymers were synthesised by reversible addition-fragmentation chain transfer polymerisation and post-modification utilising copper alkyne-azide cycloaddition to graft orthogonal pendant HA monosaccharides (N-acetyl glucosamine: GlcNAc and glucuronic acid: GlcA) onto the polymer. Using surface plasmon resonance, the binding of the glycopolymers to known HA-binding peptides and proteins (CD44, hyaluronidase) was assessed and compared to carbohydrate-binding proteins (lectins). These studies revealed potential structure-binding relationships between HA monosaccharides and HA receptors and novel HA binders, such as Dectin-1 and DEC-205 lectins. The inhibitory effect of HA glycopolymers on hyaluronidase (HAase) activity was also investigated suggesting GlcNAc- and GlcA-based glycopolymers as potential HAase inhibitors.

Molecular Engineering of Polymyxin B for Imaging and Treatment of Bacterial Infections.

The emergence of multi-drug resistant bacteria and the lack of novel antibiotics to combat them have led to the revival of polymyxin B, a previously abandoned antibiotic due to its potential nephrotoxicity and neurotoxicity. To facilitate its widely clinical applications, increasing effort has been devoted to molecularly engineer polymyxin B for the targeted imaging and effective treatment of bacterial infections. Herein, the molecular engineering strategies will be summarized in this mini review, with selected recent advances for illustration. Perspective of the challenges and trends in this exciting and eagerly anticipated research area will also be provided in the end. We hope this mini review will inspire researchers from diverse fields to bring forward the next wave of exploiting molecular engineering approaches to propel the "old" polymyxin B to "new" clinical significance in combating bacterial infections.

Unravelling the Enzymatic Degradation Mechanism of Supramolecular Peptide Nanofibers and Its Correlation with Their Internal Viscosity.

Enzyme-responsive supramolecular peptide biomaterials have attracted growing interest for disease diagnostics and treatments. However, it remains unclear whether enzymes target the peptide assemblies or dissociated peptide monomers. To gain further insight into the degradation mechanism of supramolecular peptide amphiphile (PA) nanofibers, cathepsin B with both exopeptidase and endopeptidase activities was exploited here for degradation studies. Hydrolysis was found to occur directly on the PA nanofibers as only surface amino acid residues were cleaved. The number of cleaved residues and the degradation efficiency was observed to be negatively correlated with the internal viscosity of the PA nanofibers, quantified to be between 200-800 cP (liquid phase) using fluorescence lifetime imaging microscopy combined with an environmentally sensitive molecular rotor, BODIPY-C10. These findings enhance our understanding on the enzymatic degradation of supramolecular PA nanofibers and have important implications for the development of PA probes for the real-time monitoring of disease-related enzymes.

An Invertebrate Burn Wound Model That Recapitulates the Hallmarks of Burn Trauma and Infection Seen in Mammalian Models.

The primary reason for skin graft failure and the mortality of burn wound patients, particularly those in burn intensive care centers, is bacterial infection. Several animal models exist to study burn wound pathogens. The most commonly used model is the mouse, which can be used to study virulence determinants and pathogenicity of a wide range of clinically relevant burn wound pathogens. However, animal models of burn wound pathogenicity are governed by strict ethical guidelines and hindered by high levels of animal suffering and the high level of training that is required to achieve consistent reproducible results. In this study, we describe for the first time an invertebrate model of burn trauma and concomitant wound infection. We demonstrate that this model recapitulates many of the hallmarks of burn trauma and wound infection seen in mammalian models and in human patients. We outline how this model can be used to discriminate between high and low pathogenicity strains of two of the most common burn wound colonizers Pseudomonas aeruginosa and Staphylococcus aureus, and multi-drug resistant Acinetobacter baumannii. This model is less ethically challenging than traditional vertebrate burn wound models and has the capacity to enable experiments such as high throughput screening of both anti-infective compounds and genetic mutant libraries.

Magnetic Field-Induced Alignment of Nanofibrous Supramolecular Membranes: A Molecular Design Approach to Create Tissue-like Biomaterials.

A molecular design approach to fabricate nanofibrous membranes by self-assembly of aromatic cationic peptides with hyaluronic acid (HA) and nanofiber alignment under a magnetic field is reported. Peptides are designed to contain a block composed of four phenylalanine residues at the C-terminus, to drive their self-assembly by hydrophobic association and aromatic stacking, and have a positively charged domain of lysine residues for electrostatic interaction with HA. These two blocks are connected by a linker with a variable number of amino acids and the ability to adopt distinct conformations. Zeta potential measurements and circular dichroism confirm their positive charge and variable conformation (random coil, ß-sheet, or α-helix), which depend on the pH and sequence. Their self-assembly, examined by fluorescence spectroscopy, small-angle X-ray scattering, and transmission electron microscopy, show the formation of fiberlike nanostructures in the micromolar range. When the peptides are combined with HA, hydrogels or flat membranes are formed. The molecular structure tunes the mechanical behavior of the membranes and the nanofibers align in the direction of magnetic field due to the high diamagnetic anisotropy of phenylalanine residues. Mesenchymal stem cells cultured on magnetically aligned membranes elongate in direction of the nanofibers supporting their application for soft tissue engineering.

Disordered protein-graphene oxide co-assembly and supramolecular biofabrication of functional fluidic devices.

Supramolecular chemistry offers an exciting opportunity to assemble materials with molecular precision. However, there remains an unmet need to turn molecular self-assembly into functional materials and devices. Harnessing the inherent properties of both disordered proteins and graphene oxide (GO), we report a disordered protein-GO co-assembling system that through a diffusion-reaction process and disorder-to-order transitions generates hierarchically organized materials that exhibit high stability and access to non-equilibrium on demand. We use experimental approaches and molecular dynamics simulations to describe the underlying molecular mechanism of formation and establish key rules for its design and regulation. Through rapid prototyping techniques, we demonstrate the system's capacity to be controlled with spatio-temporal precision into well-defined capillary-like fluidic microstructures with a high level of biocompatibility and, importantly, the capacity to withstand flow. Our study presents an innovative approach to transform rational supramolecular design into functional engineering with potential widespread use in microfluidic systems and organ-on-a-chip platforms.

Tuning the matrix metalloproteinase-1 degradability of peptide amphiphile nanofibers through supramolecular engineering.

Matrix metalloproteinases (MMPs) are a family of endopeptidases capable of degrading extracellular matrix (ECM) components. They are known to play crucial roles during the ECM turnover in both physiological and pathological processes. As such, their activities are utilized as biological stimuli to engineer MMP-responsive peptide-based biomaterials such as self-assembled peptide amphiphiles (PAs). Although previous studies have unveiled the role of PAs secondary structure on the mechanical and biological properties of their self-assembled nanostructures, the effect on the degradability of their assemblies by MMP-1 has not been reported. Herein, a series of PAs are designed and synthesized, all comprising the same MMP-1 cleavable domain but with variable structural segments, to decipher the role of PA's secondary structure on the MMP-1 degradability of their assemblies. This study reveals a correlation between the MMP-1 degradation efficiency and the ß-sheet content of the self-assembled PA nanofibers, with the MMP-1 cleavability being significantly reduced in the PA nanofibers with stronger ß-sheet characteristics. These results shed light on the role of supramolecular cohesion in PA assemblies on their hydrolysis by MMP-1 and open up the possibility to control the degradation rate of PA-based nanostructures by MMP-1 through tweaking their molecular sequences.

Enhancing the Potency of Antimicrobial Peptides through Molecular Engineering and Self-Assembly.

Healthcare-associated infections resulting from bacterial attachment and biofilm formation on medical implants are posing significant challenges in particular with the emergence of bacterial resistance to antibiotics. Here, we report the design, synthesis and characterization of self-assembled nanostructures, which integrate on their surface antibacterial peptides. The antibacterial WMR peptide, which is a modification of the native sequence of the myxinidin, a marine peptide isolated from the epidermal mucus of hagfish, was used considering its enhanced activity against Gram-negative bacteria. WMR was linked to a peptide segment of aliphatic residues (AAAAAAA) containing a lipidic tail (C19H38O2) attached to the ε-amino of a terminal lysine to generate a peptide amphiphile (WMR PA). The self-assembly of the WMR PA alone, or combined with coassembling shorter PAs, was studied using spectroscopy and microscopy techniques. The designed PAs were shown to self-assemble into stable nanofiber structures and these nanoassemblies significantly inhibit biofilm formation and eradicate the already formed biofilms of Pseudomonas aeruginosa (Gram-negative bacteria) and Candida albicans (pathogenic fungus) when compared to the native WMR peptide. Our results provide insights into the design of peptide based supramolecular assemblies with antibacterial activity, and establish an innovative strategy to develop self-assembled antimicrobial materials for biomedical applications.

Targeted Delivery of Sildenafil for Inhibiting Pulmonary Vascular Remodeling.

Pulmonary arterial hypertension is a fatal lung disease caused by the progressive remodeling of small pulmonary arteries (PAs). Sildenafil can prevent the remodeling of PAs, but conventional sildenafil formulations have shown limited treatment efficacy for their poor accumulation in PAs. Here, glucuronic acid (GlcA)-modified liposomes (GlcA-Lips) were developed to improve the delivery of sildenafil to aberrant over-proliferative PA smooth muscle cells via targeting the GLUT-1 (glucose transport-1), and, therefore, inhibiting the remodeling of PAs in a monocrotaline-induced PA hypertension model. GlcA-Lips encapsulating sildenafil (GlcA-sildenafil-Lips) had a size of 90 nm and a pH-sensitive drug release pattern. Immunostaining assay indicated the overexpression of GLUT-1 in PA smooth muscle cells. Cellular uptake studies showed a 1-fold increase of GlcA-Lips uptake by PA smooth muscle cells and pharmacokinetics and biodistribution experiments indicated longer blood circulation time of GlcA-Lips and increased ability to target PAs by 1-fold after 8 hours administration. Two-week treatment indicated GlcA-sildenafil-Lips significantly inhibited the remodeling of PAs, with a 32% reduction in the PA pressure, a 41% decrease in the medial thickening, and a 44% reduction of the right ventricle cardiomyocyte hypertrophy, and improved survival rate. Immunohistochemical analysis showed enhanced expression of caspase-3, after administration of GlcA-sildenafil-Lips, and reduced expression of P-ERK1/2 (phosphorylated ERK1/2) and HK-2 (hexokinase-2), and increased level of eNOS (endothelial nitric oxide synthase) and cyclic GMP (cGMP). In conclusion, targeted delivery of sildenafil to PA smooth muscle cells with GlcA-Lips could effectively inhibit the remodeling of PAs in the monocrotaline-induced PA hypertension.

Multifunctional Self-Assembling Peptide-Based Nanostructures for Targeted Intracellular Delivery: Design, Physicochemical Characterization, and Biological Assessment.

Peptide amphiphiles (PAs), consisting of a hydrophobic alkyl chain covalently bound to a hydrophilic peptide sequence, possess a versatile molecular design due to their combined self-assembling features of amphiphile surfactants and biological functionalities of peptides. Through rational design, PAs can self-assemble into a variety of nanostructures with controlled shape, size, and biological functionality to deliver therapeutic and imaging agents to target cells. Here, we describe principles to design multifunctional PAs for self-assembly into micellar nanostructures and targeted intracellular delivery. The PA micelles are designed to display a tumour targeting sequence on their surfaces and direct their interactions with specific cells. This targeting sequence includes an enzymatic sensitive sequence that can be cleaved upon exposure to matrix metalloproteinase 2 (MMP-2), an enzyme overexpressed in tumor environment, allowing the presentation of a cell-penetrating domain. The presentation of this domain will then facilitate the delivery of therapeutics for cancer treatment inside targeted cells. Methods to characterize the key physicochemical properties of PAs and their assemblies, including secondary structure, critical micelle concentration, shape and size, are described in detail. The enzyme responsiveness of PA assemblies is described with respect to their degradation by MMP-2. Protocols to evaluate the cytotoxicity and cellular uptake of the micellar carriers are also included.

Empowering the Potential of Cell-Penetrating Peptides for Targeted Intracellular Delivery via Molecular Self-Assembly.

Cell-penetrating peptides (CPPs) have been widely explored as an effective tool to deliver a variety of molecules and nanoparticles into cells due to their intrinsic property to translocate across cell membranes. CPPs are easier to synthesize and functionalize, and their incorporation into delivery vehicles could be achieved by both non-covalent and covalent methods. Recent advances in molecular self-assembly have demonstrated the possibility to fabricate various nanostructures with precise control over the shape, size and presentation of diverse functionalities. Through rational design, CPPs could be used as a building block for the nanostructure formation via self-assembly, while providing the functionality for intracellular delivery. In this book chapter, we will describe strategies to design self-assembling CPP conjugates and illustrate how their self-assembled nanostructures are manipulated for effective intracellular delivery. Fundamental knowledge on CPPs and molecular self-assembly will also be described.