RADA 16-I Research Articles

Bio inspired growth factor loaded self assembling peptide nano hydrogel for chronic wound healing

Self assembling peptidebased hydrogel has been explored for delivering growth factors, anticancer drugs, antibiotics etc. Here, RADA 16-I (RADARADARADARADA), an ionic self complementary peptide that forms a well defined nanohydrogel has been studied for its ability to deliver PDGF-BB in a sustained manner and to destruct biofilm formed by wound specific pathogens. Results of the structural analysis of the nanohydrogel studied through AFM, FeSEM, CD, FT-IR and Rheometry, revealed the hydrogel forming ability of RADA 16-I with stable β-sheet structure at room temperature. The nanohydrogel was also found to destruct the biofilm formed under in vitro condition using S. aureus, E. coli and P. aeruginosa. The growth factor incorporated in the nanohydrogel followed first order release kinetics and showed sustained release up to 48 h. Angiogenic potential and wound healing ability of PDGF-BB incorporated nanohydrogel was confirmed in both in vitro and in vivo conditions. The animals treated with PDGF-BB incorporated nanohydrogel exhibited 99.5% wound closure at day 21. The content of hydroxyproline and ascorbic acid was significantly high in the treated animals when compared to control and untreated animals. Overall, the study provides the essential information and data for using RADA 16-I for treating chronic wounds.

Bone Tissue Engineering in a Perfusion Bioreactor Using Dexamethasone-Loaded Peptide Hydrogel.

The main goal of this study was the formation of bone tissue using dexamethasone (DEX)-loaded [COCH3]-RADARADARADARADA-[CONH2] (RADA 16-I) scaffold that has the ability to release optimal DEX concentration under perfusion force. Bone-marrow samples were collected from three patients during a hip arthroplasty. Human mesenchymal stem cells (hMSCs) were isolated and propagated in vitro in order to be seeded on scaffolds made of DEX-loaded RADA 16-I hydrogel in a perfusion bioreactor. DEX concentrations were as follows: 4 × 10−3, 4 × 10−4 and 4 × 10−5 M. After 21 days in a perfusion bioreactor, tissue was analyzed by scanning electron microscopy (SEM) and histology. Markers of osteogenic differentiation were quantified by real-time polymerase chain reaction (RT-PCR) and immunocytochemistry. Minerals were quantified and detected by the von Kossa method. In addition, DEX release from the scaffold in a perfusion bioreactor was assessed. The osteoblast differentiation was confirmed by the expression analysis of osteoblast-related genes (alkaline phosphatase (ALP), collagen I (COL1A1) and osteocalcin (OC). The hematoxylin/eosin staining confirmed the presence of cells and connective tissue, while SEM revealed morphological characteristics of cells, extracellular matrix and minerals—three main components of mature bone tissue. Immunocytochemical detection of collagen I is in concordance with given results, supporting the conclusion that scaffold with DEX concentration of 4 × 10−4 M has the optimal engineered tissue morphology. The best-engineered bone tissue is produced on scaffold loaded with 4 × 10−4 M DEX with a perfusion rate of 0.1 mL/min for 21 days. Differentiation of hMSCs on DEX-loaded RADA 16-I scaffold under perfusion force has a high potential for application in regenerative orthopedics.

Assembly Pathway Selection of Designer Self-Assembling Peptide and Fabrication of Hierarchical Scaffolds for Neural Regeneration.

The self-assembling peptide (SAP) RADA 16-I has been modified with various functional motifs to improve its performances in biomedical applications. Nevertheless, the assembly mechanisms of designer functional RADA 16-I SAPs (F-SAPs) have not been clearly illustrated. The main problem is the difficulty in preparing a completely molecular aqueous solution of F-SAP. In the current study, we demonstrated that different procedures for preparing the F-SAP solution could result in the formation of different conformations and consequently micro/macroscopic morphologies. F-SAP was molecularly dissolved in an appropriate solvent, such as hexafluoroisopropanol (HFIP), as evidenced by random coil conformation characterized by circular dichroism spectroscopy and morphologies under transmission electron microscopy. The monomers were induced into monolayers when the F-SAP solution in HFIP was adsorbed on mica as observed by atomic force microscopy. However, nanoscaled filaments containing β-sheets dominated in the F-SAP aqueous solution, in which case water acted as a poor solvent of F-SAP. Furthermore, the results of molecular dynamics simulation implicated that water facilitated F-SAP aggregation, whereas HFIP inhibited it. The β-sheet assemblies formed in water exhibited a high kinetic stability and did not disassemble rapidly after the addition of HFIP. Our study indicated that selecting the right assembly pathway of F-SAP required for targeted functions, for example, delivery of hydrophobic drugs in aqueous conditions, could be achieved by optimizing the preparation protocol in addition to molecular design. Moreover, hierarchical scaffolds mimicking the natural extracellular matrix could be fabricated by the direct electrospinning of F-SAP molecular solution in HFIP and biodegradable polymer for applications in neural regeneration by promoting neural differentiation, neurite outgrowth, and synapse formation.

Investigating the Structural Stability of RADA16-I Peptide Conjugated to Gold Nanoparticles

RADA 16-I is an amphiphilic peptide which can form macroscopic scaffolds through self-assembly and has found many applications in tissue regeneration and hemostasis. It is still unclear in which conditions this peptide can perform self-assembly more effectively while maintaining its stability. Interaction between peptides and gold surfaces has been increasingly regarded for biotechnological applications. In order to better understand the effect of this conjugation on the application of RADA16-I, properties of peptide-modified gold nanoparticles were studied using circular dichroism, DLS, DSC and UV–Visible imaging to monitor changes in conformation of the conjugated peptide. The bioinformatics studies showed that the peptides exhibit a predominantly helical structure as monomer. UV–Visible spectrum of the gold nanoparticles showed an absorption peak at around 520 nm. Spectral and DLS analyses indicated precision of conjugation and the stronger tendency of conjugated peptide, as compared to its unconjugated form, toward aggregation or self-assembly. CD analysis showed a slight increase in β-strands of the conjugated peptides. Furthermore, DSC data suggested enthalpy-driven nature of the conjugation process minor changes in peptide stability. These findings can help researchers in the design of future nano-biomaterials, such as biosensors, based on β-sheets, scaffolds, and gold nanoparticles. The present study may serve as a scientific basis for improving efficacy and application of peptide as hemostat.

Investigating the Stability of RADA16 Peptide Nanofibers Using CD Spectra

RADA 16-I is a synthetic amphiphilic peptide which self-assembles into nanofibers and scaffolds in favor of cell growth, hemostasis and tissue engineering. However, it is still unclear in which conditions the peptide maintains its stability and structural consistency in aqueous solutions during storage time. This study investigates dynamic behavior of RADA 16-I using circular dichroism, so as to monitor changes in conformation of the peptides dispersed in water (pH 5) as well as 0.003 M (pH 4) and 0.02 M (pH 3) acetic acid solutions for various incubation times (0.5, 60 and 120 days), concentrations (0.1, 0.3 and 0.5%) and temperatures (4 and 25 °C). The results showed that the peptides exhibit a predominantly helical structure immediately after making their solutions. However, it was seen that, when exposed to solutions with pH 3 and 4, the peptides started to lose the helical structure, with increased amounts of aggregation at these two acidic pH values; this was attributed to increased hydrophobicity. The stable RADA 16-I peptides were identified at pH 5, 0.1% solution and 4 °C, with their secondary structure remained mostly unchanged during the 120-day test period. Furthermore, statistical analysis showed that, the concentration had little effect while pH and temperature had significant effects on the peptide stability, while acidic pH enhanced aggregation. These results may serve as a scientific basis for the processing and application of peptide.

Evaluation of the hemocompatibility of RADA 16-I peptide.

RADA 16-I is an ionic self-assembling peptide that can form macroscopic scaffolds through β-sheet structures which are used in favor of cell growth and tissue engineering. This peptide has also the ability to stop bleeding effectively and quickly (∼20 seconds) when applied directly to the injuries. This study is focused on coagulation process, platelet aggregation, C3 and C4 concentrations, CBC counting, hemolysis, and white blood cell morphology tests to analyze hemocompatibility of RADA 16-I at different concentrations - 0.1, 0.2, 0.3 and 0.5%. According to the results, RADA 16-I hydrogel decreased the number of blood cells, slightly increased clot formation time and platelet aggregation, and yielded negligible hemolysis and only small changes in C3 and C4 concentrations and white blood cell morphology. All by all, the in vitro tests of hemocompatibility showed no perturbation in the blood composition when the peptides were in contact with the blood. The observed rapid hemostasis might be a result of increasing local concentrations of molecules involved in the formation of clot near the peptide hydrogel, thereby making a barrier which ended up with complete hemostasis. In conclusion, our experiments strongly supported further development of biomaterials based on RADA 16-I peptide.

Functional self-assembling peptide nanofiber hydrogel for peripheral nerve regeneration.

Peripheral nerves are fragile and easily damaged, usually resulting in nervous tissue loss, motor and sensory function loss. Advances in neuroscience and engineering have been significantly contributing to bridge the damage nerve and create permissive environment for axonal regrowth across lesions. We have successfully designed two self-assembling peptides by modifying RADA 16-I with two functional motifs IKVAV and RGD. Nanofiber hydrogel formed when combing the two neutral solutions together, defined as RADA 16-Mix that overcomes the main drawback of RADA16-I associated with low pH. In the present study, we transplanted the RADA 16-Mix hydrogel into the transected rat sciatic nerve gap and effect on axonal regeneration was examined and compared with the traditional RADA16-I hydrogel. The regenerated nerves were found to grow along the walls of the large cavities formed in the graft of RADA16-I hydrogel, while the nerves grew into the RADA 16-Mix hydrogel toward distal position. RADA 16-Mix hydrogel induced more axons regeneration and Schwann cells immigration than RADA16-I hydrogel, resulting in better functional recovery as determined by the gait-stance duration percentage and the formation of new neuromuscular junction structures. Therefore, our results indicated that the functional SAP RADA16-Mix nanofibrous hydrogel provided a better environment for peripheral nerve regeneration than RADA16-I hydrogel and could be potentially used in peripheral nerve injury repair.

Self-assembly behaviors of molecular designer functional RADA16-I peptides: influence of motifs, pH, and assembly time

In the current study, we present three designer self-assembling peptides (SAPs) by appending RADA 16-I with epitopes IKVAV, RGD, and YIGSR, which have different net charges and amphiphilic properties at neutral pH. The self-assembly of the designer SAPs is intensively investigated as a function of pH, canion type, and assembly time. The morphologies of the designer SAPs were studied by atomic force microscope. The secondary structure was investigated by circular dichroism. The dynamic viscoelasticity of designer SAP solutions was examined during titration with different alkaline reagents. Our study indicated that both electrostatic and hydrophilic/hydrophobic interactions of the motifs exhibited influences on the self-assembly, consequentially affecting the fiber morphologies and rheological properties. Moreover, NaOH induced a quicker assembly/reassembly of the designer SAPs than Tris because of its strong ionic strength. Therefore, our study gained comprehensive insight into the self-assembling mechanism as references for developing RADA 16-I–based functional SAPs.

Investigation of human cell response to covalently attached RADA16-I peptide on silicon surfaces

We described a modification of the ionic (RADARADARADARADA)1 peptide or RADA16-I with 4-azidophenyl isothiocyanate via a specific and gentle reaction. The azidated peptide was covalently immobilized on an alkyne-terminated monolayer on Si(111) via the Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction. Detailed characterization using Impedance spectroscopy (IS), X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR) spectroscopy demonstrated high coverage of the RADA 16-I peptide on silicon surfaces. Scanning electron microscopy (SEM) and methyl tetrazole sulfate (MTS) assay were used to characterize the morphology and proliferation ability of human fibroblast cells on surfaces. Cell adhesion assay was performed to examine cell-substrate interactions. Significant differences in fibroblast cell morphology, adhesion, and viability were observed on the RADA16-I peptide modified surfaces compared to the control surfaces. These results may suggest a potential application of RADA16-I peptide modified surfaces in biomedical applications.

Functional Self-Assembling Peptide Nanofiber Hydrogels Designed for Nerve Degeneration.

Self-assembling peptide (SAP) RADA16-I (Ac-(RADA)4-CONH2) has been suffering from a main drawback associated with low pH, which damages cells and host tissues upon direct exposure. In this study, we presented a strategy to prepare nanofiber hydrogels from two designer SAPs at neutral pH. RADA16-I was appended with functional motifs containing cell adhesion peptide RGD and neurite outgrowth peptide IKVAV. The two SAPs were specially designed to have opposite net charges at neutral pH, the combination of which created a nanofiber hydrogel (-IKVAV/-RGD) characterized by significantly higher G' than G″ in a viscoelasticity examination. Circular dichroism, Fourier transform infrared spectroscopy, and Raman measurements were performed to investigate the secondary structure of the designer SAPs, indicating that both the hydrophobic/hydrophilic properties and electrostatic interactions of the functional motifs play an important role in the self-assembling behavior of the designer SAPs. The neural progenitor cells (NPCs)/stem cells (NSCs) fully embedded in the 3D-IKVAV/-RGD nanofiber hydrogel survived, whereas those embedded within the RADA 16-I hydrogel hardly survived. Moreover, the -IKVAV/-RGD nanofiber hydrogel supported NPC/NSC neuron and astrocyte differentiation in a 3D environment without adding extra growth factors. Studies of three nerve injury models, including sciatic nerve defect, intracerebral hemorrhage, and spinal cord transection, indicated that the designer -IKVAV/-RGD nanofiber hydrogel provided a more permissive environment for nerve regeneration than the RADA 16-I hydrogel. Therefore, we reported a new mechanism that might be beneficial for the synthesis of SAPs for in vitro 3D cell culture and nerve regeneration.

Contribution of Electrostatics in the Fibril Stability of a Model Ionic-Complementary Peptide.

In this work we quantified the role of electrostatic interactions in the self-assembly of a model amphiphilic peptide (RADA 16-I) into fibrillar structures by a combination of size exclusion chromatography and molecular simulations. For the peptide under investigation, it is found that a net charge of +0.75 represents the ideal condition to promote the formation of regular amyloid fibrils. Lower net charges favor the formation of amorphous precipitates, while larger net charges destabilize the fibrillar aggregates and promote a reversible dissociation of monomers from the ends of the fibrils. By quantifying the dependence of the equilibrium constant of this reversible reaction on the pH value and the peptide net charge, we show that electrostatic interactions contribute largely to the free energy of fibril formation. The addition of both salt and a charged destabilizer (guanidinium hydrochloride) at moderate concentration (0.3-1 M) shifts the monomer-fibril equilibrium toward the fibrillar state. Whereas the first effect can be explained by charge screening of electrostatic repulsion only, the promotion of fibril formation in the presence of guanidinium hydrochloride is also attributed to modifications of the peptide conformation. The results of this work indicate that the global peptide net charge is a key property that correlates well with the fibril stability, although the peptide conformation and the surface charge distribution also contribute to the aggregation propensity.

A Colloidal Description of Intermolecular Interactions Driving Fibril-Fibril Aggregation of a Model Amphiphilic Peptide.

We apply a kinetic analysis platform to study the intermolecular interactions underlying the colloidal stability of dispersions of charged amyloid fibrils consisting of a model amphiphilic peptide (RADA 16-I). In contrast to the aggregation mechanisms observed in the large majority of proteins and peptides, where several elementary reactions involving both monomers and fibrils are present simultaneously, the system selected in this work allows the specific investigation of the fibril-fibril aggregation process. We examine the intermolecular interactions driving the aggregation reaction at pH 2.0 by changing the buffer composition in terms of salt concentration, type of ion as well as type and concentration of organic solvent. The aggregation kinetics are followed by dynamic light scattering, and the experimental data are simulated by Smoluchowski population balance equations, which allow to estimate the energy barrier between two colliding fibrils in terms of the Fuchs stability ratio (W). When normalized on a dimensionless time weighted on the Fuchs stability ratio, the aggregation profiles under a broad range of conditions collapse on a single master curve, indicating that the buffer composition modifies the aggregation kinetics without affecting the aggregation mechanism. Our results show that the aggregation process does not occur under diffusion-limited conditions. Rather, the reaction rate is limited by the presence of an activation energy barrier that is largely dominated by electrostatic repulsive interactions. Such interactions could be reduced by increasing the concentration of salt, which induces charge screening, or the concentration of organic solvent, which affects the dielectric constant. It is remarkable that the dependence of the activation energy on the ionic strength can be described quantitatively in terms of charge screening effects in the frame of the DLVO theory, although specific anion and cation effects are also observed. While anion effects are mainly related to the binding to the positive groups of the fibril surface and to the resulting decrease of the surface charge, cation effects are more complex and involve additional solvation forces.

All-Atom Molecular Dynamics Study of Four RADA 16-I Peptides: The Effects of Salts on Cluster Formation

RADA 16-I is a synthetic amphiphilic peptide, composed of 16 amino acids, designed to self-assemble in a controlled way into fibrils and higher ordered structures depending on solvent condition. This peptide has many applications in tissue engineering and wound healing. We have studied the cluster formation of four RADA 16-I peptide chains in the presence of different concentrations of NaCl and CaCl2 as solvents, using all-atom molecular dynamics simulation. Nine independent simulations over 20 ns were performed. The results show that the fastest cluster formation rate occurs in the presence of 0.2 M NaCl following by water. In these simulations cluster formation doesn’t occur in the presence of CaCl2. It is also found that the cluster formation depends on two solvent-related factors: (a) salt concentration and (b) salt type. The effect of salts may act through changing the compactness and secondary structure of this peptide. In fact NaCl helps each peptide to adopt transitional expanded α-helical conformation in order to induce better interaction sites for self-assembly. The results gave insight into the effect of ionic strength in self-assembly mechanism of RADA 16-I in the bulk solution.

Sol–gel transition of charged fibrils composed of a model amphiphilic peptide

We characterized the sol–gel transition of positively charged fibrils composed of the model amphiphilic peptide RADARADARADARADA (RADA 16-I) using a combination of microscopy, light scattering, microrheology and rheology techniques, and we investigated the dependence of the hydrogel formation on fibril concentration and ionic strength. The peptide is initially present as a dispersion of short rigid fibrils with average length of about 100nm. During incubation, the fibrils aggregate irreversibly into longer fibrils and fibrillar aggregates. At peptide concentrations in the range 3–6.5g/L, the fibrillar aggregates form a weak gel network which can be destroyed upon dilution. Percolation occurs without the formation of a nematic phase at a critical peptide concentration which decreases with increasing ionic strength. The gel structure can be well described in the frame of the fractal gel theory considering the network as a collection of fibrillar aggregates characterized by self-similar structure with a fractal dimension of 1.34.

End-to-End Self-Assembly of RADA 16-I Nanofibrils in Aqueous Solutions

RADARADARADARADA (RADA 16-I) is a synthetic amphiphilic peptide designed to self-assemble in a controlled way into fibrils and higher ordered structures depending on pH. In this work, we use various techniques to investigate the state of the peptide dispersed in water under dilute conditions at different pH and in the presence of trifluoroacetic acid or hydrochloric acid. We have identified stable RADA 16-I fibrils at pH 2.0–4.5, which have a length of ∼200–400 nm and diameter of 10 nm. The fibrils have the characteristic antiparallel β-sheet structure of amyloid fibrils, as measured by circular dichroism and Fourier transform infrared spectrometry. During incubation at pH 2.0–4.5, the fibrils elongate very slowly via an end-to-end fibril-fibril aggregation mechanism, without changing their diameter, and the kinetics of such aggregation depends on pH and anion type. At pH 2.0, we also observed a substantial amount of monomers in the system, which do not participate in the fibril elongation and degrade to fragments. The fibril-fibril elongation kinetics has been simulated using the Smoluchowski kinetic model, population balance equations, and the simulation results are in good agreement with the experimental data. It is also found that the aggregation process is not limited by diffusion but rather is an activated process with energy barrier in the order of 20 kcal/mol.

Temperature and pH effects on biophysical and morphological properties of self‐assembling peptide RADA16‐I

It has been found that the self-assembling peptide RADA 16-I forms a beta-sheet structure and self-assembles into nanofibers and scaffolds in favor of cell growth, hemostasis and tissue-injury repair. But its biophysical and morphological properties, especially for its beta-sheet and self-assembling properties in heat- and pH-denatured conditions, remain largely unclear. In order to better understand and design nanobiomaterials, we studied the self-assembly behaviors of RADA16-I using CD and atomic force microscopy (AFM) measurements in various pH and heat-denatured conditions. Here, we report that the peptide, when exposed to pH 1.0 and 4.0, was still able to assume a typical beta-sheet structure and self-assemble into long nanofiber, although its beta-sheet content was dramatically decreased by 10% in a pH 1.0 solution. However, the peptide, when exposed to pH 13.0, drastically lost its beta-sheet structure and assembled into different small-sized globular aggregates. Similarly, the peptide, when heat-denatured from 25 to 70 degrees C, was still able to assume a typical beta-sheet structure with 46% content, but self-assembled into small-sized globular aggregates at much higher temperature. Titration experiments showed that the peptide RADA16-I exists in three types of ionic species: acidic (fully protonated peptide), zwitterionic (electrically neutral peptide carrying partial positive and negative charges) and basic (fully deprotonated peptide) species, called 'super ions'. The unordered structure and beta-turn of these 'super ions' via hydrogen or ionic bonds, and heat Brownian motion under the above denatured conditions would directly affect the stability of the beta-sheet and nanofibers. These results help us in the design of future nanobiomaterials, such as biosensors, based on beta-sheets and environmental changes. These results also help understand the pathogenesis of the beta-sheet-mediated neuronal diseases such as Alzheimer's disease and the mechanism of hemostasis.