RESUMEN
In recent decades, peptide amphiphiles (PAs) have established themselves as promising self-assembling bioinspired materials in a wide range of medical fields. Herein, we report a dual-therapeutic system constituted by an antimicrobial PA and a cylindrical protease inhibitor (LJC) to achieve broad antimicrobial spectrum and to enhance therapeutic efficacy. We studied two strategies: PA-LJC nanostructures (Encapsulation) and PA nanostructures + free LJC (Combination). Computational modeling using a molecular theory for amphiphile self-assembly captures and explains the morphology of PA-LJC nanostructures and the location of encapsulated LJC in agreement with transmission electron microscopy and two-dimensional (2D) NMR observations. The morphology and release profile of PA-LJC assemblies are strongly correlated to the PA:LJC ratio: high LJC loading induces an initial burst release. We then evaluated the antimicrobial activity of our nanosystems toward gram-positive and gram-negative bacteria. We found that the Combination broadens the spectrum of LJC, reduces the therapeutic concentrations of both agents, and is not impacted by the inoculum effect. Further, the Encapsulation provides additional benefits including bypassing water solubility limitations of LJC and modulating the release of this molecule. The different properties of PA-LJC nanostructures results in different killing profiles, and reduced cytotoxicity and hemolytic activity. Meanwhile, details in membrane alterations caused by each strategy were revealed by various microscopy and fluorescent techniques. Last, in vivo studies in larvae treated by the Encapsulation strategy showed better antimicrobial efficacy than polymyxin B. Collectively, this study established a multifunctional platform using a versatile PA to act as an antibiotic, membrane-penetrating assistant, and slow-release delivery vehicle.
Asunto(s)
Antiinfecciosos , Nanoestructuras , Antibacterianos/farmacología , Antibacterianos/química , Bacterias Gramnegativas , Bacterias Grampositivas , Nanoestructuras/químicaRESUMEN
Antimicrobial peptide amphiphiles (PAs) are a promising class of molecules that can disrupt the bacterial membrane or act as drug nanocarriers. In this study, we prepared 33â PAs to establish supramolecular structure-activity relationships. We studied the morphology and activity of the nanostructures against different Gram-positive and Gram-negative bacterial strains (such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii). Next, we used principal component analysis (PCA) to determine the key contributors to activity. We found that for S. aureus, the zeta potential was the major contributor to the activity while Gram-negative bacteria were more influenced by the partition coefficient (LogP) with the following order P. aeruginosa>E. coli>A. baumannii. We also performed a study of the mechanism of action of selected PAs on the bacterial membrane assessing the membrane permeability and depolarization, changes in zeta potential and overall integrity. We studied the toxicity of the nanostructures against mammalian cells. Finally, we performed an inâ vivo study using the wax moth larvae to determine the therapeutic efficacy of the active PAs. This study shows cationic PA nanostructures can be an intriguing platform for the development of nanoantibacterials.
Asunto(s)
Antiinfecciosos , Staphylococcus aureus , Animales , Antibacterianos/farmacología , Antibacterianos/química , Escherichia coli , Antiinfecciosos/farmacología , Péptidos , Relación Estructura-Actividad , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa , MamíferosRESUMEN
Self-assembled nanostructures such as those formed by peptide amphiphiles (PAs) are of great interest in biological and pharmacological applications. Herein, a simple and widely applicable chemical modification, a urea motif, was included in the PA's molecular structure to stabilize the nanostructures by virtue of intermolecular hydrogen bonds. Since the amino acid residue nearest to the lipid tail is the most relevant for stability, we decided to include the urea modification at that position. We prepared four groups of molecules (13 PAs in all), with varying levels of intermolecular cohesion, using amino acids with distinct ß-sheet promoting potential and/or containing hydrophobic tails of distinct lengths. Each subset contained one urea-modified PA and nonmodified PAs, all with the same peptide sequence. The varied responses of these PAs to variations in pH, temperature, counterions, and biologically related proteins were examined using microscopic, X-ray, spectrometric techniques, and molecular simulations. We found that the urea group contributes to the stabilization of the morphology and internal arrangement of the assemblies against environmental stimuli for all peptide sequences. In addition, microbiological and biological studies were performed with the cationic PAs. These assays reveal that the addition of urea linkages affects the PA-cell membrane interaction, showing the potential to increase the selectivity toward bacteria. Our data indicate that the urea motif can be used to tune the stability of a wide range of PA nanostructures, allowing flexibility on the biomaterial's design and opening a myriad of options for clinical therapies.
Asunto(s)
Enlace de Hidrógeno , Urea , Urea/química , Interacciones Hidrofóbicas e Hidrofílicas , Péptidos/química , Péptidos/farmacología , Nanoestructuras/química , Tensoactivos/químicaRESUMEN
AIMS: We developed three new analogs of the antimicrobial peptide (AMP) Citropin 1.1: DAN-1-13, AJP-1-1, and HHX-2-28, and tested their potential antimicrobial and antibiofilm activities against Staphylococcus aureus and S. pseudintermedius. Potential cytotoxic or hemolytic effects were determined using cultured human keratinocytes and erythrocytes to determine their safety. METHODS AND RESULTS: To assess the antimicrobial activity of each compound, minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) were determined against methicillin-resistant and methicillin-susceptible strains of S. aureus and S. pseudintermedius. Activity against newly formed and mature biofilms was determined in two clinical isolates using spectrophotometry and scanning electron microscopy (SEM). All three compounds exhibited antimicrobial and bactericidal activity against all studied S. aureus and S. pseudintermedius strains, with MICs ranging from 4-32 µg ml-1 and MBCs ranging from 8-128 µg ml-1. Subinhibitory concentrations of all compounds also showed ant-biofilm activity in the two tested isolates. All compounds exhibited limited cytotoxic and hemolytic activity. CONCLUSIONS: Novel analogs of Citropin 1.1 exhibit antimicrobial and bactericidal activities against S. aureus and S. pseudintermedius isolates and inhibit the biofilm formation of these bacteria.
Asunto(s)
Antibacterianos , Biopelículas , Pruebas de Sensibilidad Microbiana , Staphylococcus aureus , Staphylococcus , Biopelículas/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Humanos , Antibacterianos/farmacología , Staphylococcus/efectos de los fármacos , Péptidos Antimicrobianos/farmacología , Péptidos Antimicrobianos/química , Eritrocitos/efectos de los fármacos , Queratinocitos/efectos de los fármacosRESUMEN
Gram-negative strains are intrinsically resistant to most antibiotics due to the robust and impermeable characteristic of their outer membrane. Self-assembling cationic peptide amphiphiles (PAs) have the ability to disrupt bacteria membranes, constituting an excellent antibacterial alternative to small molecule drugs that can be used alone or as antibiotic adjuvants to overcome bacteria resistance. PA1 (C16KHKHK), self-assembled into micelles, which exhibited low antibacterial activity against all strains tested, and showed strong synergistic antibacterial activity in combination with Vancomycin with a Fractional Inhibitory Concentration index (FICi) of 0.15 against E. coli. The molecules, PA2 (C16KRKR) and PA3 (C16AAAKRKR), also self-assembled into micelles, displayed a broad-spectrum antibacterial activity against all strains tested, and low susceptibility to resistance development over 21 days. Finally, PA1, PA 2 and PA3 displayed low cytotoxicity against mammalian cells, and PA2 showed a potent antibacterial activity and low toxicity in preliminary in vivo models using G. mellonella. The results show that PAs are a great platform for the future development of effective antibiotics to slow down the antibiotic resistance and can act as antibiotic adjuvants with synergistic mechanism of action, which can be repurposed for use with existing antibiotics commonly used to treat gram-positive bacteria to treat infections caused by gram-negative bacteria.
RESUMEN
Supramolecular nanostructures with tunable properties can have applications in medicine, pharmacy, and biotechnology. In this work, we show that the self-assembly behavior of peptide amphiphiles (PAs) can be effectively tuned by replacing the carboxylic acids exposed to the aqueous media with isosteres, functionalities that share key physical or chemical properties with another chemical group. Transmission electron microscopy, atomic force microscopy, and small-angle X-ray scattering studies indicated that the nanostructure's morphologies are responsive to the ionization states of the side chains, which are related to their pKa values. Circular dichroism studies revealed the effect of the isosteres on the internal arrangement of the nanostructures. The interactions between diverse surfaces and the nanostructures and the effect of salt concentration and temperature were assessed to further understand the properties of these self-assembled systems. These results indicate that isosteric replacements allow the pH control of supramolecular morphology by manipulating the pKa of the charged groups located on the nanostructure's surface. Theoretical studies were performed to understand the morphological transitions that the nanostructures underwent in response to pH changes, suggesting that the transitions result from alterations in the Coulomb forces between PA molecules. This work provides a strategy for designing biomaterials that can maintain or change behaviors based on the pH differences found within cells and tissues.
Asunto(s)
Nanoestructuras , Dicroismo Circular , Microscopía Electrónica de Transmisión , Péptidos , AguaRESUMEN
Although cardiovascular diseases (CVD) are the leading cause of global mortality, there is a lack of therapies that target and revert underlying pathological processes. Mitochondrial dysfunction is involved in the pathophysiology of CVD, and thus is a potential target for therapeutic development. To target the mitochondria and improve therapeutic efficacy, nanoparticle-based delivery systems have been proposed as promising strategies for the delivery of therapeutic agents to the mitochondria. This review will first discuss how mitochondrial dysfunction is related to the progression of several CVD and then delineate recent progress in mitochondrial targeting using nanoparticle-based delivery systems including peptide-based nanosystems, polymeric nanoparticles, liposomes, and lipid nanoparticles. In addition, we summarize the advantages of these nanocarriers and remaining challenges in targeting the mitochondria as a therapeutic strategy for CVD treatment.
Asunto(s)
Enfermedades Cardiovasculares , Enfermedades Mitocondriales , Nanopartículas , Humanos , Enfermedades Mitocondriales/tratamiento farmacológico , Enfermedades Cardiovasculares/tratamiento farmacológico , Mitocondrias , Sistemas de Liberación de MedicamentosRESUMEN
Hydrogen bonding plays a critical role in the self-assembly of peptide amphiphiles (PAs). Herein, we studied the effect of replacing the amide linkage between the peptide and lipid portions of the PA with a urea group, which possesses an additional hydrogen bond donor. We prepared three PAs with the peptide sequence Phe-Phe-Glu-Glu (FFEE): two are amide-linked with hydrophobic tails of different lengths and the other possesses an alkylated urea group. The differences in the self-assembled structures formed by these PAs were assessed using diverse microscopies, nuclear magnetic resonance (NMR), and dichroism techniques. We found that the urea group influences the morphology and internal arrangement of the assemblies. Molecular dynamics simulations suggest that there are about 50% more hydrogen bonds in nanostructures assembled from the urea-PA than those assembled from the other PAs. Furthermore, in silico studies suggest the presence of urea-π stacking interactions with the phenyl group of Phe, which results in distinct peptide conformations in comparison to the amide-linked PAs. We then studied the effect of the urea modification on the mechanical properties of PA hydrogels. We found that the hydrogel made of the urea-PA exhibits increased stability and self-healing ability. In addition, it allows cell adhesion, spreading, and growth as a matrix. This study reveals that the inclusion of urea bonds might be useful in controlling the morphology, mechanical, and biological properties of self-assembled nanostructures and hydrogels formed by the PAs.