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DLVO interaction potential calculated from eqn (8) for pH = 3.5 and 4 (thick lines). The thin lines are for pH = 3, 5, 6 and 7, from the top to the bottom line. The left axis is for a characteristic surface of interaction s = 10 nm 2 , whereas the right axis is for s = 100 nm 2 . The
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Hydrogels formed from the self-assembly of oligopeptides are being extensively studied for biomedical applications. The kinetics of their gelation, as well as a quantitative description of the forces controlling the rate of assembly has not yet been addressed. We report here the use of multiple particle tracking to measure the self-assembly kinetic...
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... carboxylate-modified probes in a solution of KFE8 powder at a concentration of 0.1 wt% with pH set at 3.5 (same as in the previous section). At this pH, carboxylate-modified probes are negatively charged whereas the amine-modified particles are positively charged. The peptide in solution at this pH being itself positively charged (see inset of Fig. 5 and later in the text), we evaluate here the influence of a possible interaction between the bead and the peptide. Moreover, this comparison is performed with similar particle sizes, so we truly isolate the effect of surface chemistry. We present in Fig. 2 the kinetics results at the lag time t 2 = 1 s using these two different surface ...
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... (6) in conjunction with eqn (7), can be used to calculate s and y s at every pH. The results are given in the inset of Fig. 5 with a TFA concentration [T] 0 = 10 23 M (see the ESI{). We see that the peptide is positively charged for pH = 4 and negatively charged for pH > 11. In these domains of pH, we expect that the electrostatic repulsion between the peptides slows down the self- assembly process. The bulk rheology measurements would conclude to a ...
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... The bulk rheology measurements would conclude to a non-elastic state of the material unless the sample is assessed after a prohibitively long equilibration time. This observation is in agreement with the rheology results obtained by Caplan et al. with the peptide KFE12 10 that would share a similar theoretical curve as the one presented in Fig. ...
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... Debye length k 21 (10 nm) is bigger than the typical radius (3.5 nm) of the cross-section of the KFE8 double-layer ribbon (see Fig. S1 in the ESI{). In Fig. 5 we plot the interaction potential given by eqn (8) in the planar geometry at various pH, and for s = 10 nm 2 and s = 100 nm 2 corresponding to dimensions of y3 6 3 nm and y10 6 10 nm ,respectively, for the surface of blocks' interaction. This last characteristic size of 10 nm is intended to include the electrostatic layer on the side ...
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... observe in Fig. 5 that the potential barrier value U max DLVO is decreasing as the pH increases from 3 to 7, as expected from the corresponding decrease of the surface-charge density s on the interacting blocks (see inset). At pH . 6, we observe that U max DLVO , k B T, meaning that the potential barrier is not significant and that the block monomers ...
Citations
... Alternatively, the Rouse prediction of ( ) − / G t t 1 2 has been successfully used to identify the critical gel point of gelling systems [25,[28][29][30]. At the gel point, t gel , the elastic (solid-like) storage modulus, ′ G , and the viscous (liquid-like) loss modulus, ″ G , crossover such that ′ G will dominate. ...
The maintenance of hemostasis to ensure vascular integrity is dependent upon the rapid conversion of zymogen species of the coagulation cascade to their enzymatically active forms. This process culminates in the generation of the serine protease thrombin and polymerization of fibrin to prevent vascular leak at sites of endothelial cell injury or loss of cellular junctions. Thrombin generation can be initiated by the extrinsic pathway of coagulation through exposure of blood to tissue factor at sites of vascular damage, or alternatively by the coagulation factor (F) XII activated by foreign surfaces with negative charges, such as glass, through the contact activation pathway. Here, we used transient particle tracking microrheology to investigate the mechanical properties of fibrin in response to thrombin generation downstream of both coagulation pathways. We found that the structural heterogeneity of fibrin formation was dependent on the reaction kinetics of thrombin generation. Pharmacological inhibition of FXII activity prolonged the time to form fibrin and increased the degree of heterogeneity of fibrin, resulting in fibrin clots with reduced mechanical properties. Taken together, this study demonstrates a dependency of the physical biology of fibrin formation on activation of the contact pathway of coagulation.
... 50 The exact mechanism by which the cyclotides coalesce to form a network is not fully understood, but it is known that self-assembly is sensitive to the medium condition. 51 The results commendably revealed that pH, temperature, and time play a decisive role in the process of cyclotide self-assembly, and the assembly rate of these peptides could be efficiently controlled by the optimization of these variables to achieve a desired size and shape of the nanostructures. The main questions are: how one can ascertain that cyclotides can be self-associated and what drives the self-assembly of the model system cyclotides? ...
... 33,57,58 It could be hypothesized that the self-assembly was promoted by the aid of hydrophobic effects but hindered by electrostatic repulsion. 51 Due to the presence of the hydrophobic patch on the surface of the cyclotides, the electrostatic repulsion caused by the charged residues is typically weaker and does not suffice to overcome the hydrophobic interactions. So there is no need for salt/surfactant to initiate the aggregation of the cyclotides. ...
Self-assembling peptides have attracted researchers' attention recently. They are classified as biomedical materials with unique properties formed in response to environmental conditions. Cyclotides are macrocyclic plant-derived peptides containing 28-37 amino acids that have the ability to self-assemble. Herein, we investigated the effect of pH, time, and temperature on the self-assembling properties of the cyclotides extracted from Viola odorata. For this purpose, the cyclotides were dispersed in aqueous trifluoroacetic acid at pH 2, 4, or 6 and incubated at 25 or 37 °C for 1, 2, 3, 5, 7 or 10 days, and the morphology of the self-assembled structures was identified by optical microscopy, polarized optical microscopy, scanning electron microscopy, transmission electron microscopy, and fluorescence microscopy. At pH 2 and 4, the self-assembly process of cyclotides comprises a number of steps, starting with the formation of spherical peptide nanostructures followed by hierarchically assembled nanotubes, and then shifting to nanofibers after 10 days. At pH 6, amorphous structures were produced even after 10 days. The temperature also could affect the self-assembly mechanism of the cyclotides. At 25 °C, the spherical peptide micelles formed firstly and then merged to form nanotubes, while at 37 °C the cyclotides showed crystallization followed by an increase in length with time. The fluorescence microscopy images showed that the nanotubes could efficiently entrap the hydrophobic molecules of coumarin. This comparative study on the self-assembly of the cyclotides extracted from Viola odorata is the first example exploring the capacity of these cyclotides to adopt precise nanostructures. The nanotubes and nanofibers obtained with these cyclotides might find interesting applications in drug delivery and tissue engineering.
... The crossover point of G′ and G″ has also been traditionally used to identify the gel point of materials. For a material exhibit a power law behavior such that G′(ω) ∝ ω α and G″(ω) ∝ ω α , the Rouse dynamic model of α 0.5 has been used to identify the gel point that G′(ω) G″(ω) (Savin and Doyle, 2007;Wehrman et al., 2017). For a probe particle that follows Eq. 4, this scaling can be converted to 〈Δr 2 (τ)〉~τ 0.5 . ...
... For a probe particle that follows Eq. 4, this scaling can be converted to 〈Δr 2 (τ)〉~τ 0.5 . Transient microrheology of gelling materials has been performed to identify the critical point of α 0.5 (Savin and Doyle, 2007;Xing et al., 2018). ...
... The SLB here is the diffusive exponent (α) of the MSD. The relation 〈Δr 2 (t)〉~t 0.5 has been used in microrheology to determine the gel point based on the Rouse model (Savin and Doyle, 2007), as discussed in Section 2.1.4. ...
Microrheology encompasses a range of methods to measure the mechanical properties of soft materials. By characterizing the motion of embedded microscopic particles, microrheology extends the probing length scale and frequency range of conventional bulk rheology. Microrheology can be characterized into either passive or active methods based on the driving force exerted on probe particles. Tracer particles are driven by thermal energy in passive methods, applying minimal deformation to the assessed medium. In active techniques, particles are manipulated by an external force, most commonly produced through optical and magnetic fields. Small-scale rheology holds significant advantages over conventional bulk rheology, such as eliminating the need for large sample sizes, the ability to probe fragile materials non-destructively, and a wider probing frequency range. More importantly, some microrheological techniques can obtain spatiotemporal information of local microenvironments and accurately describe the heterogeneity of structurally complex fluids. Recently, there has been significant growth in using these minimally invasive techniques to investigate a wide range of biomedical systems both in vitro and in vivo. Here, we review the latest applications and advancements of microrheology in mammalian cells, tissues, and biofluids and discuss the current challenges and potential future advances on the horizon.
... It took minutes to form hydrogel instead of hours. However, the mechanism by which the hydrogel formed remained the same 177 . Short chain peptides such as FKFEFKFE are more sensitive to the ionic strength of the solution and pH due to the presence of ionizable side chains. ...
The applications of nanoparticulate drug delivery have received abundant interest in the field of cancer diagnosis and treatment. By virtue of their unique features and design, nanomedicines have made remarkable progress in eliminating dreadful tumors. Research in cancer nanomedicine has spanned multitudes of drug delivery systems that possess high tumor targeting ability, sensitivity towards tumor microenvironments and improved efficacy. Various nanocarriers have been developed and approved for anti-tumor drug targeting. These nanocarriers, i.e., liposomes, micelles, nanotubes, dendrimers and peptide, offer a wide range of advantages, such as high selectivity, multi-functionality, specificity, biocompatibility and precise control of drug release. Nanomedicines based on self-assembled peptide carrier systems have been developed in recent years for cancer targeting. Self-assembled peptides have tremendous properties of forming targeted drug delivery vehicles, such as nanohydrogels with unique features and functionality. In this review article, we discuss some developments in cancer nanomedicine. We present the diverse range of nanotargeted drug-delivery systems.
... 30 The sol-gel transition (gel point) was determined by calculating the logarithmic slope of the mean-squared displacement, a=log10(áDr 2 (t)ñ)/log10(t). We used a critical value of n=0.5 where the gel point is defined as the time when measured α < n. 31 ...
We report the design of a mucin hydrogel created using a thiol-based cross-linking strategy. By using a cross-linking reagent capable of forming disulfide linkages between mucins, the mucin-based hydrogels possess viscoelastic properties comparable to native mucus as measured by bulk rheology. We confirmed disulfide cross-links mediate gel formation in our system using chemical treatments to block and reduce cysteines where we found mucin hydrogel network formation was inhibited and disrupted, respectively. Particle tracking microrheology was used to investigate the kinetics and evolution of microstructure and viscoelasticity within the hydrogel as it formed. We found that the rate of gel formation could be tuned by varying the mucin to crosslinker ratio, producing network pore sizes in the range measured previously in human mucus. The results of this work provide a new, simple method for creating mucin hydrogels with physiologically relevant properties using readily available reagents.
... An analogy to CSAC has been drawn to the critical micelle concentration (CMC) of amphiphilic molecules such as surfactants. An amphiphilic peptide may display similar concentration-dependent self-assembly behavior, leading to the formation of peptide nano-structures [12]. We performed conductivity measurement to determine the CSAC values of FFFFKK as a function of peptide concentration. ...
Peptide self-assembly forms different nanostructures under simple alteration in the solution environment. Understanding the mechanism of the assembly will help us to control and tailor functional nanomaterials. This study aims to investigate the influence of anions on the self-assembly morphology and shape using a synthetic peptide of FFFFKK. Circular Dichoism (CD) and Environmental Scanning Electron Microscope (ESEM) were used to determine the secondary structure and self-assembly morphology, while Image J imaging software was used to measure diameter size. In the absence of anion, FFFFKK formed anti-parallel β-sheet that adopted sizeable fibrillar structure with a minimal increment over the first 7 hours of assembly. Irregular structure was observed in the presence of Iodide ion (I⁻) with a less stable secondary structure such as β-turn and β-loop. In the presence of perchlorate ion (ClO4⁻), needle-like structure was observed with predominantly β-sheet structure. Our study showed that peptide morphology can be controlled by using different anions with careful selection of amino acid residues in peptide sequence.
... More details on microrheological approaches can be found in the available literature. 2,3,7 Various paste-like systems such as Carbopol suspensions, 24 gellan gum, 25 peptide dispersions, 26 hectorite clay dispersions, 27 etc. have been studied using microrheology techniques. ...
... The kinetics of peptide self-assembly was studied using microrheology by Savin and Doyle. 26 An increase in the self-assembly of the peptide led to a subdiffusive behavior exhibited by the probe mean square displacements, suggestive of a gel formation. The gel point was determined by the congruency of the frequency dependence of the viscous and elastic moduli 29 as calculated from the mean square displacements. ...
We studied the aging dynamics of an aqueous suspension of LAPONITE®, a model time dependent soft glassy material, using a passive microrheology technique. This system is known to undergo physical aging during which its microstructure evolves progressively to explore lower free energy states. Optical microscopy is used to monitor the motion of micron-sized tracer probes embedded in a sample kept between two glass plates. The mean square displacements (MSD) obtained from the motion of the tracer particles show a systematic change from a purely diffusive behavior at short aging times to a subdiffusive behavior as the material ages. Interestingly, the MSDs at all the aging times as well as different LAPONITE® concentrations superpose remarkably to show a time-aging time master curve when the system is transformed from the real time domain to the effective time domain, which is obtained by rescaling the material clock to account for the age dependent relaxation time. The transformation of the master curve from the effective time domain to the real time domain leads to the prediction of the MSD in real time over a span of 5 decades when the measured data at individual aging times are only over 2 decades. Since the MSD obtained from microrheology is proportional to the creep compliance of a material, by using the Boltzmann superposition principle along with the convolution relation in the effective time domain, we predict the stress relaxation behavior of the system in real time. This work shows that the effective time approach applied to microrheology facilitates the prediction of long time creep and relaxation dynamics of a time dependent soft material by carrying out short time experiments at different aging times.
... In particular, the self-assembly process has been exploited to create fibrillar peptide networks, or hydrogels, that are able to swell in the presence of water [4] and, as a result, have been targeted for various biological and biomedical applications, for example as drug delivery systems [5], tissue engineering scaffolds [6], and for uses in the acceleration of hemostasis [7]. Many previous studies have shown that peptide sequences that contain alternating hydrophobic and hydrophilic residues with complementary charge distributions prompt hydrogel formation [8,9]; examples include, but are not limited to, KLD-12 (AcN-(KLDL) 3 -CNH 2 ) [10], RADA-16 (AcN-(RADA) 4 -CNH 2 ) [5], EAK-16 ((AEAEAKAK) 2 ) [6], EMK16-II ((MEMEMKMK) 2 ) [11], and (FKFE) n=2,3 [12][13][14][15]. Moreover, macroscopic hydrogels formed from such peptides typically consist of extensive β-sheet-rich fibrillar networks that are stabilized by both favorable hydrophobic and electrostatic interactions [8,16]. ...
Peptide hydrogels are promising candidates for a wide range of medical and biotechnological applications. To further expand the potential utility of peptide hydrogels, herein we demonstrate a simple yet effective strategy to render peptide hydrogels photodegradable, making controlled disassembly of the gel structure of interest feasible. In addition, we find that the high-frequency amide I' component (i.e., the peak at ~1685 cm(-1)) of the photodegradable peptide hydrogel studied shows an unusually large enhancement, in comparison to that of other peptide fibrils consisting of antiparallel β-sheets, making it a good model system for further study of the coupling-structure relationship.
... Our experiments measure changes in frequencydependent viscoelasticity over many orders of magnitude of frequency (timescales of interactions) while fibrils assemble. Previous dynamical measurements of rheological changes occurring during protein or peptide self-assembly have been limited to frequencies ,100 Hz [24,27,[30][31][32][33]; our optical-tweezers-based probe system extends the range of frequencies spanning from 1 Hz to .1 kHz, thus enabling study of interactions in more dilute systems and for more transient interactions. This dynamic range encapsulates timescales that are relevant for protein-protein interactions, molecular motor activity, protein-cell adhesion and cytoskeletal remodelling, which allow cells to sense, respond to and move through their external environments [5,34,35]. ...
Collagen is the most abundant protein in the extracellular matrix (ECM), where its structural organization conveys mechanical information to cells. Using optical-tweezers-based microrheology, we investigated mechanical properties both of collagen molecules at a range of concentrations in acidic solution where fibrils cannot form and of gels of collagen fibrils formed at neutral pH, as well as the development of microscale mechanical heterogeneity during the self-assembly process. The frequency scaling of the complex shear modulus even at frequencies of ∼10 kHz was not able to resolve the flexibility of collagen molecules in acidic solution. In these solutions, molecular interactions cause significant transient elasticity, as we observed for 5 mg/ml solutions at frequencies above ∼200 Hz. We found the viscoelasticity of solutions of collagen molecules to be spatially homogeneous, in sharp contrast to the heterogeneity of self-assembled fibrillar collagen systems, whose elasticity varied by more than an order of magnitude and in power-law behavior at different locations within the sample. By probing changes in the complex shear modulus over 100-minute timescales as collagen self-assembled into fibrils, we conclude that microscale heterogeneity appears during early phases of fibrillar growth and continues to develop further during this growth phase. Experiments in which growing fibrils dislodge microspheres from an optical trap suggest that fibril growth is a force-generating process. These data contribute to understanding how heterogeneities develop during self-assembly, which in turn can help synthesis of new materials for cellular engineering.
... To determine whether other self-assembling peptides also strongly adjuvant particular epitopes, we investigated the self-assembling peptide domain KFE8 (Ac-FKFEFKFE-Am). 38,40 We synthesized three peptides, KFE8, RGD-KFE8, and OVA-KFE8 (sequences in Figure 1a), determined their capacity to self-assemble, and measured their immunogenicity in mice. All three KFE8-containing peptides self-assembled into fibrillar structures, similar to Q11 and its derivatives (Figure 6a-c). ...
Self-assembling peptides and peptide derivatives have received significant interest for several biomedical applications, including tissue engineering, wound healing, cell delivery, drug delivery, and vaccines. This class of materials has exhibited significant variability in immunogenicity, with many peptides eliciting no detectable antibody responses but others eliciting very strong responses without any supplemental adjuvants. Presently, strategies for either avoiding strong antibody responses or specifically inducing them are not well-developed, even though they are critical for the use of these materials both within tissue engineering and within immunotherapies. Here, we investigated the molecular determinants and immunological mechanisms leading to the significant immunogenicity of the self-assembling peptide OVA-Q11, which has been shown previously to elicit strong antibody responses in mice. We show that these responses can last for at least a year. Using adoptive transfer experiments and T cell knockout models, we found that these strong antibody responses were T cell-dependent, suggesting a route for avoiding or ensuring immunogenicity. Indeed, by deleting amino acid regions in the peptide recognized by T cells, immunogenicity could be significantly diminished. Immunogenicity could also be attenuated by mutating key residues in the self-assembling domain, thus preventing fibrillization. A second self-assembling peptide, KFE8, was also nonimmunogenic, but nanofibers of OVA-KFE8 elicited strong antibody responses similar to OVA-Q11, indicating that the adjuvant action was not dependent on the specific self-assembling peptide sequence. These findings will facilitate the design of self-assembled peptide biomaterials, both for applications where immunogenicity is undesirable and where it is advantageous.
