Multidomain Peptide Research Articles

Abstract No. 430 Self-assembling hydrogel platform improves intratumoral drug delivery and retention

Direct intratumoral (IT) delivery of immunotherapeutics is an increasingly important intersection between interventional radiology and oncology. However, the challenge of IT drug deposition and retention has become apparent as drugs delivered in low viscosity fluids localize poorly in solid tumors. We hypothesized that drugs embedded in a multidomain peptide (MDP) hydrogel would exhibit significantly improved delivery and retention within tumors following percutaneous delivery. The cationic lysine-based MDP K2 (SL)6K2 (or K2-MDP) was used to load and test the biodistribution of the contrast agent iohexol. The radio-opacity of various hydrogel formulations containing 24, 48, and 120 mgI/mL iohexol and 1% wtK2-MDP stock peptide prepared in a buffered solution were tested via CT. Next, we generated a tumor model by subcutaneously implanting 5x106 McA-7777 cells in the 2 flanks of female Buffalo rats (n = 10). Once the tumors reached 1 cm in diameter, the MDP-iohexol hydrogel was injected using a 29G needle at a rate of 50 uL/sec under fluoroscopy, followed by immediate post injection CT scan. Injections of free iohexol in the contralateral tumors were used as controls. All formulations tested here were fully homogeneous and successfully formed hydrogels. The radio-opacity intensity curve showed that the optimal concentration for in vivo experiments was 60 mgI/mL within 1% wt K2-MDP. Fluoroscopic imaging during in vivo injection of free iohexol demonstrated leakage along the tumor capsule as well as venous intravasation. In marked contrast, there was substantially improved localization of the MDP-iohexol hydrogel within the tumor without intravasation. This was confirmed by subsequent CT imaging which demonstrated an approximately 5-fold improvement in the percentage of tumor volume containing the agent (38% vs. 8%, P < 0.001). Our K2-MDP hydrogel significantly improve intratumoral delivery of iohexol, suggesting that embedding immunotherapeutics in this hydrogel has the potential to improve the delivery and outcome in patients receiving intratumoral immunotherapy.

Modular design and self-assembly of multidomain peptides towards cytocompatible supramolecular cell penetrating nanofibers.

The discovery of cell penetrating peptides (CPPs) with unique membrane activity has inspired the design and synthesis of a variety of cell penetrating macromolecules, which offer tremendous opportunity and promise for intracellular delivery of a variety of imaging probes and therapeutics. While cell penetrating macromolecules can be designed and synthesized to have equivalent or even superior cell penetrating activity compared with natural CPPs, most of them suffer from moderate to severe cytotoxicity. Inspired by recent advances in peptide self-assembly and cell penetrating macromolecules, in this work, we demonstrated a new class of peptide assemblies with intrinsic cell penetrating activity and excellent cytocompatibility. Supramolecular assemblies were formed through the self-assembly of de novo designed multidomain peptides (MDPs) with a general sequence of Kx(QW)6Ey in which the numbers of lysine and glutamic acid can be varied to control supramolecular assembly, morphology and cell penetrating activity. Both supramolecular spherical particles and nanofibers exhibit much higher cell penetrating activity than monomeric MDPs while supramolecular nanofibers were found to further enhance the cell penetrating activity of MDPs. In vitro cell uptake results suggested that the supramolecular cell penetrating nanofibers undergo macropinocytosis-mediated internalization and they are capable of escaping from the lysosome to reach the cytoplasm, which highlights their great potential as highly effective intracellular therapeutic delivery vehicles and imaging probes.

Chemical functionality of multidomain peptide hydrogels governs early host immune response

Multidomain Peptide (MDP) hydrogels are nanofibrous materials with many potential biomedical applications. The peptide sequence design of these materials offers high versatility and allows for the incorporation of various chemical functionalities into the nanofibrous scaffold. It is known that host response to biomaterials is strongly affected by factors such as size, shape, stiffness, and chemistry. However, there is a lack of fundamental understanding of the host response to different MDP hydrogels. In particular, it is unknown what effect the chemical functionality displayed on the nanofiber has on biological activity. Here we evaluated the early inflammatory host response to four MDP hydrogels displaying amines, guanidinium ions, and carboxylates in a subcutaneous injection model. While all the studied peptide materials possess similar nanostructure and physical properties, they trigger markedly different inflammatory responses. These were characterized by immunophenotyping of the cellular infiltrate using multi-color flow cytometry. The negatively-charged peptides elicit minimal inflammation characterized by tissue-resident macrophage infiltration, fast remodeling, and no collagen deposition or blood vessel formation within the implants. In contrast, the positively-charged peptides are highly infiltrated by immune cells, are remodeled at a slower rate, promote angiogenesis, and result in a high degree of collagen deposition. The presence of dynamic cell phenotypes characterizes the inflammation caused by the lysine-based peptide, including inflammatory monocytes, macrophages, and lymphoid cells, which is seen to be resolving over time. The arginine-based hydrogel shows higher inflammatory response with a persistent and significant infiltration of polymorphonuclear myeloid-derived cells, even ten days after implantation. This understanding of the immune response to peptide biomaterials improves our ability to design effective materials and to tailor their use for specific biomedical applications.

Drug-Mimicking Nanofibrous Peptide Hydrogel for Inhibition of Inducible Nitric Oxide Synthase.

In this work, we develop a drug-mimicking nanofibrous peptide hydrogel that shows long-term bioactivity comparable to a small-molecule inhibitor of inducible nitric oxide synthase (iNOS). The iNOS inhibitor, N 6-(1-iminoethyl)-l-lysine (l-NIL), is a positively charged amino acid whose structure could be readily integrated into the framework of a positively charged multidomain peptide (MDP) through the modification of lysine side chains. This new l-NIL-MDP maintains the self-assembling properties of the base peptide, forming β-sheet nanofibers, which entangle into a thixotropic hydrogel. The l-NIL-MDP hydrogel supports cell growth in vitro and allows syringe-directed delivery that persists in a targeted location in vivo for several weeks. Multiple characterization assays demonstrate the bioactivity of the l-NIL-MDP hydrogel to be comparable to the l-NIL small molecule. This includes iNOS inhibition of macrophages in vitro, reduced nitrotyrosine immunostaining in murine subcutaneous histology, and reduced serum levels of vascular endothelial growth factor in vivo. This study expands the toolbox of available peptide hydrogel scaffold designs that can modify biological activity without the need for any additional small-molecule drugs, proteins, or cells.

Aggregation Behavior of Nanoparticle-Peptide Systems Affects Autophagy.

The aggregation of nanoparticle colloidal dispersions in complex biological environments changes the nanoparticle properties, such as size and surface area, thus affecting the interaction of nanoparticles at the interface with cellular components and systems. We investigated the effect of nanoparticle aggregation on autophagy, the main catabolic pathway that mediates degradation of nanosized materials and that is activated in response to internalization of foreign nanosized materials. We used carboxylated polystyrene nanoparticles (100 nm) and altered the nanoparticle aggregation behavior through addition of a multidomain peptide, thus generating a set of nanoparticle-peptide mixtures with variable aggregation properties. Specifically, modulating the peptide concentration resulted in nanoparticle-peptide mixtures that are well dispersed extracellularly but aggregate upon cellular internalization. We monitored the effect of internalization of nanoparticle-peptide mixtures on a comprehensive set of markers of the autophagy pathway, ranging from transcriptional regulation to clearance of autophagic substrates. The nanoparticle-peptide mixtures were found to activate the transcription factor EB, a master regulator of autophagy and lysosomal biogenesis. We also found that intracellular aggregation of nanoparticle colloidal dispersions causes blockage of autophagic flux. This study provides important insights on the effect of the aggregation properties of nanoparticles on cells and, particularly, on the main homeostatic pathway activated in response to nanoparticle internalization. These results also point to the need to control the colloidal stability of nanoparticle systems for a variety of biomedical applications.

Self-Assembling Multidomain Peptides: Design and Characterization of Neutral Peptide-Based Materials with pH and Ionic Strength Independent Self-Assembly.

Self-assembly of peptides is a powerful method of preparing nanostructured materials. These peptides frequently utilize charged groups as a convenient switch for controlling self-assembly in which pH or ionic strength determines the assembly state. Multidomain peptides have been previously designed with charged domains of amino acids, which create molecular frustration between electrostatic repulsion and a combination of supramolecular forces including hydrogen bonding and hydrophobic packing. This frustration is eliminated by the addition of multivalent ions or pH adjustment, resulting in a self-assembled hydrogel. However, these charged functionalities can have profound, unintended effects on the properties of the resulting material. Access to neutral self-assembled nanostructured hydrogels may allow for distinct biological properties that are not available to highly charged analogues. Here, we designed a series of peptides to determine if self-assembly could be mediated by the steric interactions created by neutral hydroxyproline (O) domains, eliminating the need for charged residues and creating a neutral peptide hydrogel. The series of peptides, O n (SL)6O n , was studied to determine the effect of oligo-hydroxyproline on peptide self-assembly and nanostructure. We show that peptide solubility and nanofiber length increase with a higher number of hydroxyproline residues. Within this series, O5(SL)6O5 displayed the optimal properties for self-assembly and hydrogelation. In vitro, this hydrogel supports cell viability of fibroblasts, while in vivo it is infiltrated with cells and easily degraded over time without promoting a strong inflammatory response. This neutral self-assembling peptide hydrogel shows promising properties for biomedical, cell preservation, and tissue regeneration applications.

Regulation of Osteoblast Differentiation by Affinity Peptides of TGF-β1 Identified via Phage Display Technology.

Transforming growth factor β1 (TGF-β1) plays a dual role in bone formation. In addition to promoting early differentiation of osteogenesis, it may also lead to uncontrolled extracellular matrix synthesis, inhibition of bone mineralization in the late stage, and aberrant bone remodeling. In this work, affinity peptides of TGF-β1 (Tβms) were identified from a phage display library to modify the TGF-β1 signal transduction. Tβms with more order and compact structures tended to have a higher affinity to TGF-β1 but maintained a greater immunoreactivity of TGF-β1. Tβms promoted the early osteoblast proliferation and had a negligible effect on the osteoblast differentiation. In synergy with exogenous TGF-β1, Tβms reduced the alkaline phosphatase (ALP) mRNA expression but significantly improved the expression of osteocalcin (OCN), along with impaired phosphorylation of Smad2/3. Moreover, osteoblasts showed an overall increase in ALP activity and Ca deposition than the blank control. These results demonstrated that Tβms could weaken the inhibition of TGF-β1 on osteogenic differentiation in the late stage. Depending on the impact features of Tβms on TGF-β1 response, these peptides may help to modify the implant surfaces to optimize the bone remodeling of interface, and be of interest in design of multidomain peptides.

A Single-Step Self-Assembly Approach for the Fabrication of Aligned and Multilayered Three-Dimensional Tissue Constructs Using Multidomain Peptide Hydrogel.

Hydrogels are homogenous materials that are limited in their ability to form oriented multilayered architecture in three-dimensional (3D) tissue constructs. Current techniques have led to advancements in this area. Such techniques often require extra devices and/or involve complex processes that are inaccessible to many laboratories. Here is described a one-step methodology that permits reliable alignment of cells into multiple layers using a self-assembling multidomain peptide (MDP) hydrogels. We characterized the structural features, viability, and molecular properties of dental pulp cells fabricated with MDP and demonstrated that manipulation of the layering of cells in the scaffolds was achieved by decreasing the weight by volume percentage (w/v%) of MDP contained within the scaffold. This approach allows cells to remodel their environment and enhanced various gene expression profiles, such as cell proliferation, angiogenesis, and extracellular matrix (ECM) remodeling-related genes. We further validated our approach for constructing various architectural configurations of tissues by fabricating cells into stratified multilayered and tubular structures. Our methodology provides a simple, rapid way to generate 3D tissue constructs with multilayered architectures. This method shows great potential to mimic in vivo microenvironments for cells and may be of benefit in modeling more complex tissues in the field of regenerative medicine.

Multidomain Peptide Hydrogel Accelerates Healing of Full-Thickness Wounds in Diabetic Mice.

In vivo, multidomain peptide (MDP) hydrogels undergo rapid cell infiltration and elicit a mild inflammatory response which promotes angiogenesis. Over time, the nanofibers are degraded and a natural collagen-based extracellular matrix is produced remodeling the artificial material into natural tissue. These properties make MDPs particularly well suited for applications in regeneration. In this work, we test the regenerative potential of MDP hydrogels in a diabetic wound healing model. When applied to full-thickness dermal wounds in genetically diabetic mice, the MDP hydrogel resulted in significantly accelerated wound healing compared to a clinically used hydrogel, as well as a control buffer. Treatment with the MDP hydrogel resulted in wound closure in 14 days, formation of thick granulation tissue including dense vascularization, innervation, and hair follicle regeneration. This suggests the MDP hydrogel could be an attractive choice for treatment of wounds in diabetic patients.

STINGel: Controlled release of a cyclic dinucleotide for enhanced cancer immunotherapy

Recent advancements in the field of immunotherapy have yielded encouraging results for the treatment of advanced cancers. Cyclic dinucleotides (CDNs) are a powerful new class of immunotherapy drugs known as STING (Stimulator of Interferon Genes) agonists, currently in clinical trials. However, previous studies of CDNs in murine cancer models have required multiple injections, and improve survival only in relatively nonaggressive tumor models. Therefore, we sought to improve the efficacy of CDN immunotherapy by developing a novel biomaterial we call “STINGel.” STINGel is an injectable peptide hydrogel that localizes and provides controlled release of CDN delivery, showing an 8-fold slower release rate compared to a standard collagen hydrogel. The carrier hydrogel is a positively charged, MultiDomain Peptide (MDP) which self-assembles to form a nanofibrous matrix and is easily delivered by syringe. The highly localized delivery of CDN from this nanostructured biomaterial affects the local histological response in a subcutaneous model, and dramatically improves overall survival in a challenging murine model of head and neck cancer compared to CDN alone or CDN delivered from a collagen hydrogel. This study demonstrates the feasibility of biomaterial-based immunotherapy platforms like STINGel as strategies for increasing the efficacy of CDN immunotherapies.

Self-assembly of Filamentous Cell Penetrating Peptides for Gene Delivery.

Cell penetrating peptides (CPPs) have been proven to be an effective vector to deliver a variety of membrane-impermeable macromolecules, such as DNAs, siRNAs, and proteins. Conventional single-chain CPPs typically suffer from severe protease degradation and fast clearance rate for in vivo therapeutic delivery application. In this chapter, we show that supramolecular assembly of de novo designed cationic multidomain peptides (MDPs) leads to nanostructured filaments with increased proteolytic stability and potent membrane activity necessary for improved transfection efficiency.

Design and fabrication of reduction-sensitive cell penetrating nanofibers for enhanced drug efficacy.

This work demonstrates a modular design strategy based on the supramolecular assembly of multidomain peptides to fabricate reduction-responsive cell penetrating nanofibers (CPNs), which hold great promise for selective targeting of cancer therapeutics to tumor cells.

Fabrication of self-assembling nanofibers with optimal cell uptake and therapeutic delivery efficacy

Effective strategies to fabricate finite organic nanoparticles and understanding their structure-dependent cell interaction is highly important for the development of long circulating nanocarriers in cancer therapy. In this contribution, we will capitalize on our recent development of finite supramolecular nanofibers based on the self-assembly of modularly designed cationic multidomain peptides (MDPs) and use them as a model system to investigate structure-dependent cell penetrating activity. MDPs self-assembled into nanofibers with high density of cationic charges at the fiber-solvent interface to interact with the cell membrane. However, despite the multivalent charge presentation, not all fibers led to high levels of membrane activity and cellular uptake. The flexibility of the cationic charge domains on self-assembled nanofibers plays a key role in effective membrane perturbation. Nanofibers were found to sacrifice their dimension, thermodynamic and kinetic stability for a more flexible charge domain in order to achieve effective membrane interaction. The increased membrane activity led to improved cell uptake of membrane-impermeable chemotherapeutics through membrane pore formation. In vitro cytotoxicity study showed co-administering of water-soluble doxorubicin with membrane-active peptide nanofibers dramatically reduced the IC50 by eight folds compared to drug alone. Through these detailed structure and activity studies, the acquired knowledge will provide important guidelines for the design of a variety of supramolecular cell penetrating nanomaterials not limited to peptide assembly which can be used to probe various complex biological processes.

Covalent Capture of Aligned Self-Assembling Nanofibers.

A great deal of effort has been invested in the design and characterization of systems which spontaneously assemble into nanofibers. These systems are interesting for their fundamental supramolecular chemistry and have also been shown to be promising materials, particularly for biomedical applications. Multidomain peptides are one such assembler, and in previous work we have demonstrated the reversibility of their assembly under mild and easily controlled conditions, along with their utility for time-controlled drug delivery, protein delivery, cell encapsulation, and cell delivery applications. Additionally, their highly compliant criteria for sequence selection allows them to be modified to incorporate protease susceptibility and biological-recognition motifs for cell adhesion and angiogenesis. However, control of their assembly has been limited to the formation of disorganized nanofibers. In this work, we expand our ability to manipulate multidomain-peptide assembly into parallel-aligned fiber bundles. Albeit this alignment is achieved by the shearing forces of syringe delivery, it is also dependent on the amino acid sequence of the multidomain peptide. The incorporation of the amino acid DOPA (3,4-dihydroxyphenylalanine) allows the self-assembled nanofibers to form an anisotropic hydrogel string under modest shear stress. The hydrogel string shows remarkable birefringence, and highly aligned nanofibers are visible in scanning electronic microscopy. Furthermore, the covalent linkage induced by DOPA oxidation allows covalent capture of the aligned nanofiber bundles, enhancing their birefringence and structural integrity.

Self-Assembling Multidomain Peptide Nanofibers for Delivery of Bioactive Molecules and Tissue Regeneration.

ConspectusMultidomain peptides (MDPs) are a class of self-assembling peptides that are organized in a β-sheet motif, resulting in a nanofibrous architecture. This structure is stabilized by hydrophobic packing in the fiber core and a hydrogen-bonding network down the fiber long axis. Under easily controllable conditions, regulated by electrostatic interactions between the peptides and the pH and salt composition of the solvent, the nanofiber length can be dramatically extended, resulting in fiber entanglement and hydrogel formation. One of the chief strengths of this supramolecular material is that the design criteria governing its structure and assembly are robust and permit a wide range of modifications without disruption. This allows the MDPs to be tailored to suit a wide range of applications, particularly in biomedical engineering. For example, delivery of small molecules, proteins, and cells is easily achievable. These materials can be trapped within the matrices of the hydrogel or trapped within the hydrophobic core of the nanofiber, depending on the cargo and the design of the MDP. Interactions between the nanofibers and their cargo can be tailored to alter the release profile, and in the most sophisticated cases, different cargos can be released in a cascading time-dependent fashion. The MDP hydrogel and its cargo can be targeted to specific locations, as the thixotropic nature of the hydrogel allows it to be easily aspirated into a syringe and then delivered from a narrow-bore needle. The sequence of amino acids making up the MDP can also be modified to permit cross-linking or enzymatic degradation. Selection of sequences with or without these modifications allows one to control the rate of degradation in vivo from as rapidly as 1 week to well over 6 weeks as the MDP nanofibers are degraded to their amino acid components. MDP sequences can also be modified to add biomimetic sequences derived from growth factors and other signaling proteins. These chemical signals are displayed at a very high density on the fibers’ surface, where they contribute to the modification of cellular behavior. We have used this approach to drive blood vessel formation, which is critical for tissue regeneration generally and more specifically for the treatment of diseases related to poor blood flow. MDPs represent an ideal case of bottom-up design where control of chemical structure leads to control of self-assembly and nanostructure and thereby control of material properties that collectively can control biological function.

"Missing Tooth" Multidomain Peptide Nanofibers for Delivery of Small Molecule Drugs.

The clinical administration of many small molecule hydrophobic drugs is challenged by the insolubility of these drugs under physiological conditions. Because of this, the development of biocompatible scaffolds capable of effectively delivering hydrophobic drug molecules is of particular interest. Multidomain peptides (MDPs) provide biocompatible hydrogel scaffolds that are injectable and space-conforming, allowing for in situ delivery of a variety of drugs. Here we demonstrate that through manipulation of peptide primary sequence, a molecular cavity can be incorporated into the hydrophobic core of these peptide nanofibers allowing for encapsulation and delivery of small molecule drugs with poor water solubility. Using SN-38, daunorubicin, diflunisal, etodolac, levofloxacin, and norfloxacin, we demonstrate drug encapsulation and release from multidomain peptide fibers. Steady-state fluorescence and drug release studies show that hydrogels loaded with SN-38, diflunisal, and etodolac exhibit prolonged drug release profiles due to intrafibrillar drug encapsulation. This study establishes multidomain peptides as promising carriers for localized in situ delivery of small molecule drugs with poor water solubility.

Rational design of fiber forming supramolecular structures.

Recent strides in the development of multifunctional synthetic biomimetic materials through the self-assembly of multi-domain peptides and proteins over the past decade have been realized. Such engineered systems have wide-ranging application in bioengineering and medicine. This review focuses on fundamental fiber forming α-helical coiled-coil peptides, peptide amphiphiles, and amyloid-based self-assembling peptides; followed by higher order collagen- and elastin-mimetic peptides with an emphasis on chemical / biological characterization and biomimicry.

Inter-molecular β-sheet structure facilitates lung-targeting siRNA delivery.

Size-dependent passive targeting based on the characteristics of tissues is a basic mechanism of drug delivery. While the nanometer-sized particles are efficiently captured by the liver and spleen, the micron-sized particles are most likely entrapped within the lung owing to its unique capillary structure and physiological features. To exploit this property in lung-targeting siRNA delivery, we designed and studied a multi-domain peptide named K-β, which was able to form inter-molecular β-sheet structures. Results showed that K-β peptides and siRNAs formed stable complex particles of 60 nm when mixed together. A critical property of such particles was that, after being intravenously injected into mice, they further associated into loose and micron-sized aggregates, and thus effectively entrapped within the capillaries of the lung, leading to a passive accumulation and gene-silencing. The large size aggregates can dissociate or break down by the shear stress generated by blood flow, alleviating the pulmonary embolism. Besides the lung, siRNA enrichment and targeted gene silencing were also observed in the liver. This drug delivery strategy, together with the low toxicity, biodegradability, and programmability of peptide carriers, show great potentials in vivo applications.

Self-assembled bioactive membranes for bone regeneration

Event Abstract Back to Event Self-assembled bioactive membranes for bone regeneration Sofia C. Ribeiro1, 2, 3, Daniela S. Ferreira3, 4, Rogério P. Pirraco1, 2, Isabel B. Leonor1, 2, Rui L. Reis1, 2, Álvaro Mata3, 4 and Helena S. Azevedo1, 2, 3, 4 1 University of Minho, 3B’s Research Group – Biomaterials, Biodegradables and Biomimetics, Portugal 2 University of Minho, ICVS/3B’s – PT Government Associate Laboratory, Portugal 3 Queen Mary University of London, School of Engineering and Materials Science, United Kingdom 4 Queen Mary University of London, Institute of Bioengineering, United Kingdom Introduction: Peptide self-assembly offers the possibility of fabricating highly organized biomaterials with the ability of recreating the nano-architecture and functionality of the extracellular matrix of tissues. The self-assembly of peptides with the negatively charged biopolymer hyaluronan (HA) has been explored to fabricate membranes with different biochemical and physical features[1]-[3]. Periosteum consists of microvascularized connective tissue covering the outer surface of cortical bone formed by two distinct layers: an outer fibrous layer that contains fibroblasts and Sharpey’s fibers; an inner layer called cambium, which contains multipotent mesenchymal stem cells and osteoprogenitor cells that contribute to normal bone growth, healing, and regeneration. To mimic the structure and function of periosteum, we designed new multi-domain peptides (MPDs) known to form filamentous nano-structures by self-assembly[4], to fabricate bilayer, periosteum-like, membranes by self-assembly with HA. Materials and Methods: MPDs used in this study are composed of a positively charged block (to interact with HA), a β-sheet forming segment and bioactive domain aiming to promote cell adhesion (RGDS), mineralization (EE) and osteogenic differentiation (YGFGG) (Fig.1-A). The peptides were synthesised by solid phase and purified as described elsewhere[3]. Their secondary structure and charge were analysed by circular dichroism and zeta-potential, respectively. Membranes were obtained by interfacial self-assembly between aqueous solutions of HA (700 kDa, 2%) and the various MPDs (3%) (Fig.1-B). The microstructure of the membranes was analysed by scanning electron microscopy (SEM) and a preliminary cell adhesion assay was performed using periosteum-derived cells (PDCs) to assess their ability to support cell adhesion in the absence of serum. The cells were seeded on top of the membranes and cultured for 24 h. To examine the morphology of adherent cells, cells were first stained with TRITC-conjugated phalloidin and DAPI and then observed under a confocal microscope. For cell proliferation studies, PDCs were cultured with serum for 14 days. The samples were fixed and dehydrated for observation by SEM. Results and Discussion: All designed peptides were successfully synthesised, maintaining the ability to form beta-sheet secondary structures. The HA-peptide membranes show a well-defined stratified organization with a thickness of ∼55 μm (Fig.1-C, cross-section) and a nanofibrillar structure. The presence of RGDS clearly enhances the adhesion of PDCs, when compared with control membranes (Fig.2). After 14 days of culture, a large number of well-spread cells with an elongated morphology are observed adhered to the membrane. Conclusions: We anticipate that these highly organized biomimetic membranes can provide biochemical signals for stem cell growth and differentiation and be used to promote bone regeneration. This work was supported by the Portuguese Foundation for Science and Technology under the scope of the project PTDC/CTM-BIO/0814/2012 and by the European Regional Development Fund (ERDF) through the Operational Competitiveness Programme “COMPETE” (FCOMP-01-0124-FEDER-028491).

Controlled Angiogenesis in Peptide Nanofiber Composite Hydrogels.

Multidomain peptide (MDP) nanofibers create scaffolds that can present bioactive cues to promote biological responses. Orthogonal self-assembly of MDPs and growth-factor-loaded liposomes generate supramolecular composite hydrogels. These composites can act as delivery vehicles with time-controlled release. Here we examine the controlled release of placental growth factor-1 (PlGF-1) for its ability to induce angiogenic responses. PlGF-1 was loaded either in MDP matrices or within liposomes bound inside MDP matrices. Scaffolds showed expected rapid infiltration of macrophages. When released through liposomes incorporated in MDP gels (MDP(Lipo)), PlGF-1 modulates HUVEC VEGF receptor activation in vitro and robust vessel formation in vivo. These loaded MDP(Lipo) hydrogels induce a high level of growth-factor-mediated neovascular maturity. MDP(Lipo) hydrogels offer a biocompatible and injectable platform to tailor drug delivery and treat ischemic tissue diseases.