Abstract Antimicrobial peptides (AMPs) secreted by the innate immune system are prevalent as the effective first-line of defense to overcome recurring microbial invasions. They have been widely ...accepted as the blueprints for the development of new antimicrobial agents for the treatment of drug resistant infections. However, there is also a growing concern that AMPs with a sequence that is too close to the host organism’s AMP may inevitably compromise its own natural defense. In this study, we design a series of synthetic (non-natural) short α-helical AMPs to expand the arsenal of the AMP families and to gain further insights on their antimicrobial activities. These cationic and amphiphilic peptides have a general sequence of (XXYY)n (X: hydrophobic residue, Y: cationic residue, and n: the number of repeat units), and are designed to mimic the folding behavior of the naturally-occurring α-helical AMPs. The synthetic α-helical AMPs with 3 repeat units, (FFRR)3 , (LLRR)3 , and (LLKK)3 , are found to be more selective towards microbial cells than rat red blood cells, with minimum inhibitory concentration (MIC) values that are more than 10 times lower than their 50% hemolytic concentrations (HC50 ). They are effective against Gram-positive B. subtilis and yeast C. albicans ; and the studies using scanning electron microscopy (SEM) have elucidated that these peptides possess membrane-lytic activities against microbial cells. Furthermore, non-specific immune stimulation assays of a typical peptide shows negligible IFN-α, IFN-γ, and TNF-α inductions in human peripheral blood mononuclear cells, which implies additional safety aspects of the peptide for both systemic and topical use. Therefore, the peptides designed in this study can be promising antimicrobial agents against the frequently-encountered Gram-positive bacteria- or yeast-induced infections.
We report about rationally designed ultrashort peptide bioinks, overcoming severe limitations in current bioprinting procedures. Bioprinting is increasingly relevant in tissue engineering, ...regenerative and personalized medicine due to its ability to fabricate complex tissue scaffolds through an automated deposition process. Printing stable large-scale constructs with high shape fidelity and enabling long-term cell survival are major challenges that most existing bioinks are unable to solve. Additionally, they require chemical or UV-cross-linking for the structure-solidifying process which compromises the encapsulated cells, resulting in restricted structure complexity and low cell viability. Using ultrashort peptide bioinks as ideal bodylike but synthetic material, we demonstrate an instant solidifying cell-embedding printing process via a sophisticated extrusion procedure under true physiological conditions and at cost-effective low bioink concentrations. Our printed large-scale cell constructs and the chondrogenic differentiation of printed mesenchymal stem cells point to the strong potential of the peptide bioinks for automated complex tissue fabrication.
Self‐assembling proteins and peptides are increasingly gaining interest for potential use as scaffolds in tissue engineering applications. They self‐organize from basic building blocks under mild ...conditions into supramolecular structures, mimicking the native extracellular matrix. Their properties can be easily tuned through changes at the sequence level. Moreover, they can be produced in sufficient quantities with chemical synthesis or recombinant technologies to allow them to address homogeneity and standardization issues required for applications. Here. recent advances in self‐assembling proteins, peptides, and peptide amphiphiles that form scaffolds suitable for tissue engineering are reviewed. The focus is on a variety of motifs, ranging from minimalistic dipeptides, simplistic ultrashort aliphatic peptides, and peptide amphiphiles to large “recombinamer” proteins. Special emphasis is placed on the rational design of self‐assembling motifs and biofunctionalization strategies to influence cell behavior and modulate scaffold stability. Perspectives for combination of these “bottom‐up” designer strategies with traditional “top‐down” biofabrication techniques for new generations of tissue engineering scaffolds are highlighted.
Recent advances in self‐assembling proteins, peptides, and peptide amphiphiles that form scaffolds suitable for tissue engineering are discussed. Rational design and biofunctionalization strategies for a variety of motifs ranging from minimalistic dipeptides, ultrashort aliphatic peptides, and peptide amphiphiles to large “recombinamer” proteins are reviewed and challenges and perspectives for their widespread adoption in applications are highlighted.
Blood vessel generation is an essential process for tissue formation, regeneration, and repair. Notwithstanding, vascularized tissue fabrication in vitro remains a challenge, as current fabrication ...techniques and biomaterials lack translational potential in medicine. Naturally derived biomaterials harbor the risk of immunogenicity and pathogen transmission, while synthetic materials need functionalization or blending to improve their biocompatibility. In addition, the traditional top-down fabrication techniques do not recreate the native tissue microarchitecture. Self-assembling ultrashort peptides (SUPs) are promising chemically synthesized natural materials that self-assemble into three-dimensional nanofibrous hydrogels resembling the extracellular matrix (ECM). Here, we use a modular tissue-engineering approach, embedding SUP microgels loaded with human umbilical vein endothelial cells (HUVECs) into a 3D SUP hydrogel containing human dermal fibroblast neonatal (HDFn) cells to trigger angiogenesis. The SUPs IVFK and IVZK were used to fabricate microgels that gel in a water-in-oil emulsion using a microfluidic droplet generator chip. The fabricated SUP microgels are round structures that are 300–350 μm diameter in size and have ECM-like topography. In addition, they are stable enough to keep their original size and shape under cell culture conditions and long-term storage. When the SUP microgels were used as microcarriers for growing HUVECs and HDFn cells on the microgel surface, cell attachment, stretching, and proliferation could be demonstrated. Finally, we performed an angiogenesis assay in both SUP hydrogels using all SUP combinations between micro- and bulky hydrogels. Endothelial cells were able to migrate from the microgel to the surrounding area, showing angiogenesis features such as sprouting, branching, coalescence, and lumen formation. Although both SUP hydrogels support vascular network formation, IVFK outperformed IVZK in terms of vessel network extension and branching. Overall, these results demonstrated that cell-laden SUP microgels have great potential to be used as a microcarrier cell delivery system, encouraging us to study the angiogenesis process and to develop vascularized tissue-engineering therapies.
Abstract There is an unmet clinical need for wound dressings to treat partial thickness burns that damage the epidermis and dermis. An ideal dressing needs to prevent infection, maintain skin ...hydration to facilitate debridement of the necrotic tissue, and provide cues to enhance tissue regeneration. We developed a class of ‘smart’ peptide hydrogels, which fulfill these criteria. Our ultrashort aliphatic peptides have an innate tendency to self-assemble into helical fibers, forming biomimetic hydrogel scaffolds which are non-immunogenic and non-cytotoxic. These nanofibrous hydrogels accelerated wound closure in a rat model for partial thickness burns. Two peptide hydrogel candidates demonstrate earlier onset and completion of autolytic debridement, compared to Mepitel® , a silicone-coated polyamide net used as standard-of-care. They also promote epithelial and dermal regeneration in the absence of exogenous growth factors, achieving 86.2% and 92.9% wound closure respectively, after 14 days. In comparison, only 62.8% of the burnt area is healed for wounds dressed with Mepitel® . Since the rate of wound closure is inversely correlated with hypertrophic scar formation and infection risks, our peptide hydrogel technology fills a niche neglected by current treatment options. The regenerative properties can be further enhanced by incorporation of bioactive moieties such as growth factors and cytokines.
Polyethyleneimine (PEI) is widely regarded as one of the most efficient non-viral transfection agents commercially available. However, a key concern is its pronounced cytotoxicity, ascribed mainly to ...its high amine content and cationic charge density. Significant past efforts to mitigate its toxicity usually involved lengthy synthetic procedures. We now propose a simple strategy using hydrogen peroxide (H2O2) to oxidize the amine groups. PEI/DNA complexes were first formed before some amine groups were removed with H2O2. This reduced surface charge while the remaining cationic charges still allowed for efficient transfection. The DNA was not damaged and remained bound after oxidation. Furthermore, H2O2 was quantitatively removed with sodium pyruvate prior to cell culture. Oxidized complexes caused no cytotoxicity even at high polymer concentrations. Compared to non-oxidized complexes used at subtoxic doses, oxidized complexes mediated significantly more GFP expression. A key strength of this approach is its simplicity as it involves only simple mixing of solutions. This strategy promises to further realize the potential of using PEI for the delivery of nucleic acids or other cargos.
Short synthetic peptide amphiphiles have recently been explored as effective nanobiomaterials in applications ranging from controlled gene and drug release, skin care, nanofabrication, ...biomineralization, membrane protein stabilization to 3D cell culture and tissue engineering. This range of applications is heavily linked to their unique nanostructures, remarkable simplicity and biocompatibility. Some peptide amphiphiles also possess antimicrobial activities whilst remaining benign to mammalian cells. These attractive features are inherently related to their selective affinity to different membrane interfaces, high capacity for interfacial adsorption, nanostructuring and spontaneous formation of nano-assemblies. Apart from sizes, the primary sequences of short peptides are very diverse as they can be either biomimetic or de novo designed. Thus, their self-assembling mechanistic processes and the nanostructures also vary enormously. This critical review highlights recent advances in studying peptide amphiphiles, focusing on the formation of different nanostructures and their applications in diverse fields. Many interesting features learned from peptide self-organisation and hierarchical templating will serve as useful guidance for functional materials design and nanobiotechnology (123 references).
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A short tetramer peptide, Ac-IVKC, spontaneously formed a hydrogel in water. Disulfide bonds were introduced via hydrogen peroxide (H2O2)-assisted oxidation, resulting in (Ac-IVKC)2 ...dimers. The extent of disulfide bond formation and gel stiffness increased with the amount of H2O2 used and 100% dimerization was achieved with 0.2% H2O2. The resultant gel achieved an elastic modulus of ∼0.9 MPa, which to our knowledge, has not been reported for peptide-based hydrogels. The enhanced mechanical property enabled the fabrication of thin and transparent membranes. The hydrogel could also be handled with forceps at mm thickness, greatly increasing its ease of physical manipulation. Excess H2O2 was removed and the membrane was then infused with cell culture media. Various cells, including primary human corneal stromal and epithelial cells, were seeded onto the hydrogel membrane and demonstrated to remain viable. Depending on the intended application, specific cell combination or membrane stacking order could be used to engineer layered biostructures.
A short tetramer peptide – Ac-IVKC – spontaneously formed a hydrogel in water and disulfide bonds were introduced via hydrogen peroxide (H2O2)-assisted oxidation. The extent of disulfide-bond formation and gel stiffness were modulated by the amount of H2O2. At maximum disulfide-bond formation, the hydrogel achieved an elastic modulus of ∼0.9 MPa, which to our knowledge, has not been reported for peptide-based hydrogels. The enhanced mechanical property enabled the fabrication of thin transparent membranes that can be physically manipulated at mm thickness. The gels also supported 3D cell growth, including primary human corneal stromal and epithelial cells. Depending on the intended application, specific combination of cells or individual membrane stacking order could be used to engineer layered biostructures.
Three-dimensional (3D) bioprinting is a disruptive technology for creating organotypic constructs for high-throughput screening and regenerative medicine. One major challenge is the lack of suitable ...bioinks. Short synthetic self-assembling peptides are ideal candidates. Several classes of peptides self-assemble into nanofibrous hydrogels resembling the native extracellular matrix. This is a conducive microenvironment for maintaining cell survival and physiological function. Many peptides also demonstrate stimuli-responsive gelation and tuneable mechanical properties, which facilitates extrusion before dispensing and maintains the shape fidelity of the printed construct in aqueous media. The inherent biocompatibility and biodegradability bodes well for in vivo applications as implantable tissues and drug delivery matrices, while their short length and ease of functionalization facilitates synthesis and customization. By applying self-assembling peptide inks to bioprinting, the dynamic complexity of biological tissue can be recreated, thereby advancing current biomedical applications of peptide hydrogel scaffolds.
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