Thermal Responsiveness & Cell Alignment of Nanofibers from Peptide Solutions, Notas de estudo de Engenharia Elétrica

Baixe Thermal Responsiveness & Cell Alignment of Nanofibers from Peptide Solutions e outras Notas de estudo em PDF para Engenharia Elétrica, somente na Docsity! ARTICLES PUBLISHED ONLINE: 13 JUNE 2010 | DOI: 10.1038/NMAT2778 A self-assembly pathway to aligned monodomain gels Shuming Zhang1†, Megan A. Greenfield2†, Alvaro Mata3‡, Liam C. Palmer4, Ronit Bitton3, Jason R. Mantei1, Conrado Aparicio3, Monica Olvera de la Cruz1,2,3,4 and Samuel I. Stupp1,3,4,5* Aggregates of charged amphiphilic molecules have been found to access a structure at elevated temperature that templates alignment of supramolecular fibrils over macroscopic scales. The thermal pathway leads to a lamellar plaque structure with fibrous texture that breaks on cooling into large arrays of aligned nanoscale fibres and forms a strongly birefringent liquid. By manually dragging this liquid crystal from a pipette onto salty media, it is possible to extend this alignment over centimetres in noodle-shaped viscoelastic strings. Using this approach, the solution of supramolecular filaments can be mixed with cells at physiological temperatures to form monodomain gels of aligned cells and filaments. The nature of the self-assembly process and its biocompatibility would allow formation of cellular wires in situ that have any length and customized peptide compositions for use in biological applications. Inspired largely by biological systems, molecular self-assemblycontinues to be a theme of great interest in science. Thetargets differ broadly, from accessing ordered materials and self-assembling devices1–3 to understanding howmisfolded proteins self-assemble into stable fibres linked to human disease4,5. Long- range alignment of extracellular fibrils and cells in the heart6, brain and spinal cord7 must involve highly complex self-assembly mechanisms that remain largely unknown. Access to similar three- dimensional (3D) artificial systems of aligned fibrils and cells is therefore of scientific and biomedical interest8–10. Spontaneous long-range alignment of molecules is known to occur in liquid crystals11 but its fixation in the solid state normally requires chemical reactions12,13 that are not likely to be compatible with living cells. Electrospinning of polymers faces similar challenges because it requires the use of high mechanical and electrical energies14 that are not highly compatible with encapsulation of living cells. We report the discovery of a thermal pathway that leads highly designable peptide-based small molecules in water to form two-dimensional (2D) plaques with filamentous texture that spontaneously template long-range alignment of bundled nanofibres on cooling. This liquid crystal of supramolecular filaments can be mixed with cells at physiological temperatures and drawn by hand from a pipette into salt solutions to form monodomain gels of aligned filaments. Cells remain viable during the process and the monodomain gels can be drawn to arbitrary lengths and geometrical contours. We hypothesize that divalent ions and slow relaxation times of the long nanofibre bundles generated by this self-assembly pathway enable formation of the macroscopic monodomains. We prepared 0.5–1.0 wt% aqueous solutions of peptide amphiphiles known to self-assemble into high-aspect-ratio nanofibres15,16. One peptide amphiphile molecule investigated contains the peptide sequence V3A3E3(CO2H) and a C16 alkyl tail 1Department of Materials Science and Engineering, Northwestern University, Evanston, Illinois 60208, USA, 2Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208, USA, 3Institute for BioNanotechnology in Medicine, Northwestern University, Chicago, Illinois 60611, USA, 4Department of Chemistry, Northwestern University, Evanston, Illinois 60208, USA, 5Department of Medicine, Northwestern University, Chicago, Illinois 60611, USA. †These authors contributed equally to this work. ‡Present address: Nanotechnology Platform, Parc Cientific, 08028 Barcelona, Spain. *e-mail: s-stupp@northwestern.edu. at the peptide’s N-terminus, and its self-assembly into nanofibres is triggered by ions that screen the charged amino-acid residues, resulting in the formation of gels. The diameter of these nanofibres, which contain β-sheets near their hydrophobic core, is roughly equivalent to the length of two peptide amphiphile molecules and lengths in excess of micrometres. We heated the aqueous solutions unscreened by added ions to 80 ◦C and kept them at this temperature for 30min before cooling to 25 ◦C. After this heat treatment, the solution viscosity increased threefold from 5 to 15 cP. When calcium chloride was added to the heated and cooled peptide amphiphile solution, we observed the formation of a gel that was at least fourfold stiffer than one formed from an unheated solution (see Supplementary Information). Using polarized optical microscopy, we also found that gels or films formed from heated solutions contained large birefringent domains (tenths ofmillimetres; Fig. 1), whereas those formed fromunheated solutions appeared completely isotropicwith no birefringence. We observed that noodle-like strings of arbitrary length could be formed by manually drawing the aqueous peptide amphiphile solution into a salty medium from a pipette (Fig. 1a,b). When the solution was dragged on a surface covered by a thin layer of this medium (Fig. 1c), uniform birefringence was observed along the length of the string (Fig. 1h,i). This observation suggested that macroscopic alignment extending over centimetres was achieved. Using the same methods, the unheated solutions did not form mechanically stable string gels or show any birefringence. Scanning electron microscopy (SEM) indicated that strings formed from heated peptide amphiphile solutions contained extraordinarily long arrays of aligned nanofibre bundles (Fig. 2a,b). In great contrast, unheated peptide amphiphile solutions formed matrices of randomly entangled nanofibres (Fig. 2d,e). To verify this orientational order, we carried out small-angle X-ray scattering (SAXS) experiments and found that only strings generated from the 594 NATURE MATERIALS | VOL 9 | JULY 2010 | www.nature.com/naturematerials NATURE MATERIALS DOI: 10.1038/NMAT2778 ARTICLES a d e f g h i b c 5 mm 5 mm 5 mm 2 mm 4 mm 200 µm 200 µm 100 µm 200 µm Figure 1 | Strings and gels with long-range internal alignment. a,b, A peptide amphiphile solution coloured with trypan blue injected into phosphate-buffered saline after heat treatment. c, The same solution dragged through a thin layer of aqueous CaCl2 to form a noodle-like string. d, A knot made with peptide amphiphile string. e, Birefringence of a bubble gel observed between cross polars suggesting the presence of macroscopically aligned domains. f, Similar domains in a gel film. g, Peptide amphiphile noodle spirals prepared on a spin coater. h, Birefringence of a single string suggesting alignment along the string axis. i, Light extinction between cross polars at the crosspoint of two noodles demonstrating uniform alignment in each. heated solutions revealed alignment (Fig. 2c,f). In contrast, simply dragging a peptide amphiphile solution that had not been heated does not lead to significant alignment. Similar strings can be made with other peptide amphiphile molecules, including those with bioactive epitopes, although their structural integrity depended on the amino-acid sequence (Supplementary Information). To gain a mechanistic understanding of the observed trans- formations, we examined the effects of heating on the peptide amphiphile solution structure by quick-freeze/deep-etch (QFDE) transmission electron microscopy17 (TEM). QFDE is a sample preparation technique that allows high-resolution imaging of hy- drated structures while minimizing disruption of the sample result- ing from fixation or processing. The freshly dissolved peptide am- phiphile solution contained a variety of nanoscale, elongated objects less than amicrometre in length (Supplementary Information).Mi- crographs of the peptide amphiphile solution equilibrated at 80 ◦C for 30min showed that the small aggregates largely disappeared; in- stead we observed thin ‘plaque-like’ structures up tomicrometres in both length and width (Fig. 3a). Some portions of these 2D plaques had a periodic surface texture with a characteristic spacing of about 7.5 nm, which corresponds to the expected diameter of a single canonical nanofibre formed by the peptide amphiphile molecules used15,18 (Fig. 3b). After cooling to room temperature, solutions were clearly composed of aligned filaments (Fig. 3d). The filaments did not have the diameter of canonical nanofibres (7–8 nm) but were instead tens of nanometres in diameter, and are therefore bundles of many cylindrical nanofibres. To further visualize the 3D structure of the plaque, we fixed the structure by adding calcium chloride at 80 ◦C and imaged the resulting structure by SEM. Although most of the sample cooled to ambient temperature was composed of large arrays of aligned nanofibres, some plaques were captured as well (Fig. 3e). These plaques measured about 40 nm in thickness and had lengths and widths comparable to those observed by QFDE-TEM. They often contained long parallel striations and, in some cases, appeared to be cracking into fibre bundles (Fig. 3f). These plaque structures were not observedwithout heating. We explored the effect of heating on structure in peptide amphiphile solutions using SAXS, circular dichroism, differential scanning calorimetry and Fourier-transform infrared spectroscopy. The X-ray scattering curve of peptide amphiphile solutions at 25 ◦C (Fig. 3c) shows a q−4 dependence within the low q range, indicative of the presence of large aggregates. On heating to 80 ◦C, the−4 slope NATURE MATERIALS | VOL 9 | JULY 2010 | www.nature.com/naturematerials 595 ARTICLES NATURE MATERIALS DOI: 10.1038/NMAT2778 than individual canonical fibres with diameters of about 8 nm. The diameter of the bundles corresponds to the plaque’s thickness observed by SEM (Fig. 3e). The possibility of a transition from the plaque to a fused nanofi- bre bundle can be understood by computing the contributions to the difference in free energy per thermal energy kBT per amphiphile for a cylindrical fibre F f and a lamella F l, F f−F l= (Fef−Fel)− (Fcf−Fcl) where Fef and Fel are the electrostatic free energies of a fibre and a lamella, respectively, and Fcf and Fcl are the cohesive free energies of molecules in these two different morphologies. The fraction of ions condensed on the surface of peptide amphiphile fibres and plaques is estimated from the modified Poisson–Boltzmann equation21, which shows that the charges are neutralized by counterions for both the plaque and the fibre (see Supplementary Information). Therefore, the transition should be dominated by the difference in cohesive energies of lamellae and fibres,1Fc=Fcf−Fcl, which is given by, 1Fc= 1HPA kBT −[1SPA+1Swater] where 1HPA is the enthalpy difference per peptide amphiphile molecule between lamellar and fibre aggregates, and 1SPA and 1Swater are the entropy differences of the peptide amphiphile and water molecules, respectively. As the β-sheet signature in the circular dichroism spectrum does not change significantly during cooling, we assumed that the internal energy of the β-sheet is similar in the fibre and the plaque. Therefore, the enthalpy difference between lamellae and fibres must originate from the coupling of interactions among peptide segments and hydrophobic tails. This coupling is supported by our previous spectroscopic experiments that showed order can exist in the hydrophobic core of peptide amphiphile nanofibres and is enhanced by β-sheet orientation along the fibre axis22. The fibre architecture could also optimize interactions among peptide segments and alkyl segments. We estimate1HPA to be dominated by van der Waals forces, which are of the order of thermal energy. However, the increase in entropy of the peptide amphiphile and water molecules in the plaque state can offset the enthalpy difference at elevated temperature. Specifically, the higher entropy in the plaque can originate in greater translation of water molecules (1Swater), because there is less water interface per peptide amphiphile molecule than in the fibre structure, and therefore fewer restricted water molecules per amphiphile. This would reasonably predict a transition temperature from fibrous to planar assemblies of the peptide amphiphiles (see Supplementary Information). The observed rupture of the plaque at lower temperatures into bundles of fibres that give rise to an aqueous lyotropic liquid crystal at an unusually low concentration suggests an unusual mechanism of membrane rupture. The plaque observed by QFDE-TEM at 80 ◦C reveals a periodic surface texture with a characteristic spacing of about 7.5 nm, which corresponds to the expected diameter of a single canonical nanofibre15. This strongly suggests the plaque results from the fusion of nanofibres as the dehydration occurs at elevated temperature. Themicroscopy also revealed the existence of ripples in the plaque of larger dimension than the fibres (Fig. 3a,b). We propose that fluctuations of the anisotropic plaque structure with 1D fibrous texture are crucial for its metamorphosis into arrays of highly aligned nanofibres. It is known that typically only membranes in curved geometries such as cylinders break by Rayleigh instabilities23; flat membranes generally rupture by creating holes24. Therefore, the possible breaking mechanism of a plaque into bundled fibres that gives rise to a lyotropic liquid crystal is unique given its underlying anisotropic 1D substructure imparted by the nanofibres composed of β-sheets. Long-range forces have been proposed to cause the rupture of surfactant membranes by means of concentration fluctuations25–27. The fibrous texture on the surface, however, generates an anisotropic surface tension, which may lead to the formation of waves of fluctuating composition on the surface similar to binary immiscible lipid membranes28. The waves resulting from the membrane tension appear as surface ripples when the β-sheets align, and this may generate the concentration of fluctuations required for rupture. However, the size of the successful composition fluctuation has to be large enough (larger than the membrane thickness D) to generate a critical size of a neck for rupture; otherwise the strain generated by the composition fluctuations in the internal structure (the interpenetrated bilayers) opposes the growth of the fluctuation29, and the lateral composition fluctuations are restored. One can assume that overall the breaking of the surface is due to a decrease in the overall surface tension γ , which is the change of free energy (F) as the interface area (A) increases, ∂F/∂A. That is, the bundled-fibre surface tension γf is lower than that of the lamellar γl because of the increase of hydration and order of peptide amphiphile molecules within the bundle. Unfortunately, the linear theory that assumes γ is constant under a deformation of the interface is not appropriate to describe the breaking of a lamella. To a first-order approximation one can assume that the interfacial energy of the fluctuation that leads to the rupture of the plaque is of the order of the γf. Consider fluctuations perpendicular to the lamellar surface plane (x,y) described by a function h(x,y), which induce a decrease in γl–γf that may result in the rupture of the plaque. To induce rupture, the resulting free-energy change1F=Ff−F , is negative, or 1F<0,whereFf is the free energy of the fluctuating plaque given by Ff= (1/2) ∫ dxdy γf(1+hx 2+hy 2)1/2 (1) where hx = ∂h/∂x and hy = ∂h/∂y , and F the flat-plaque free energy is given by, F = (γl/2) ∫ dxdy If we assume a 1D fluctuation along x of maximum amplitude h0, which is half the thickness of the lamella D (h0 = D/2) and wavelength λ, then h= h0exp(i2πx/λ) (2) Therefore, by approximating (1+ hx 2+ hy 2)1/2 in equation (1) as (1+ hx 2/2) and assuming γf is a constant in the integrant of Ff, we find in equation (2) that after the integration of both Ff and F , breaking occurs if1F/A6 0, or 1γ/2+γf(2πh0/λ)2/46 0 (3) where1γ =γf−γl<0 andA is the total area of the plaque of volume V = AD. Equation (3) gives a bound for the characteristic size λc above which fluctuations lead to rupture, given by (2πh0/λc)2 = −21γ/γf or λc = h0π(2γf/(γl− γf))1/2; that is, all λ> λc lead to rupture and all λ<λc would lead to stable plaques. As h0=D/2 at rupture and there is no long-range diffusion in the rupture process, λc is the most probable size for rupture, and, given that γf/(γl−γf) is of the order of one (see Supplementary Information), λc is of the order of themembrane thicknessD, in agreement with the expected size of the breaking of a cylinder through aRayleigh instability. It has been shown previously that liquid crystals can be aligned with elongational flow30. Theoretical models suggest that during uniaxial stretching of polymeric nematics, if the product of strain rate ε̇ and the conformational relaxation time λ is greater than unity, one can expect high degrees of uniaxial 598 NATURE MATERIALS | VOL 9 | JULY 2010 | www.nature.com/naturematerials NATURE MATERIALS DOI: 10.1038/NMAT2778 ARTICLES ∗ ∗ ∗ a b c d e f 0 ms 80 ms 160 ms 240 ms 320 ms 400 ms 500 μm 500 nm2 μm 150 μm 250 μm 1 cm 2,000 ms 1 2 3 Figure 4 | Cell alignment in strings of aligned filaments. a, Preferential alignment of encapsulated hMSCs along the string axis. b, Calcein-labelled aligned cells cultured in string. c, SEM images at different magnifications of a single cell in a string (inset is the zoom-out view; the arrow indicates the alignment direction). d, A conductive black string formed by dispersing carbon nanotubes in peptide amphiphile solutions before heating. The SEM micrograph on the right shows aligned nanofibre bundles in the black string. e, Top: calcium fluorescence image of HL-1 cardiomyocytes encapsulated in a noodle-like string. Below: successive spatial maps of calcium fluorescence intensity travelling at 80-ms intervals, showing the propagation of an electrical signal throughout the entire string and demonstrating a functional cardiac syncytium. f, Calcium fluorescence intensity signal in time at three points in the string marked by colour in e, showing repeated propagating action potentials. orientation31. In the context of this model, the product (known as the Weissenberg number) would be greater than unity for thermally treated peptide amphiphile gel strings and less than unity for gel strings formed with unheated samples. As we used the same longitudinal strain rate for both samples (Fig. 2c,d), the aggregates in thermally treated solutions must have longer relaxation times. Thus, we hypothesize the formation of larger diameter supramolecular bundles derived from the rupture of the plaque enables the fixing of macroscopic orientation over arbitrary length in hand-drawn string gels. It is also interesting to note that the low strain rate used here (1–4 s−1) is ideal for supramolecular aggregates, because their non-covalent interactions may not be stable in some cases at the large strain rates commonly used in electrospinning of polymers (104–105 s−1; ref. 32). The mechanism proposed here is fundamentally different from that observed in gel spinning of polymers33 and the polymerization of monomers in heated liquid-crystalline solution34. Although these strings were produced by hand drawing, this methodology could be extended to automated printing technology. We used the strings of aligned peptide amphiphile nanofibres to direct the orientation of cells in 3D environments. After dispersing human mesenchymal stem cells (hMSCs) in heated and cooled peptide amphiphile solutions, we manually dragged these solutions onto salty media (160mM NaCl and 5–10mM CaCl2) to form noodle-shaped strings with encapsulated stem cells. These cells remain viable during the process of string formation and start NATURE MATERIALS | VOL 9 | JULY 2010 | www.nature.com/naturematerials 599 ARTICLES NATURE MATERIALS DOI: 10.1038/NMAT2778 to elongate along the string director axis within 12 h. Optical, fluorescence and electron microscopy (Fig. 4a–c) demonstrated that both cell bodies and filopodia were aligned with peptide amphiphile nanofibre bundles in the extracellular space. These effects on hMSCorientation are hypothesized to result from contact guidance along the preferentially orientedmatrix35. A ‘cellular wire’ based on the noodle-like string could also serve as a bridge to direct cells spatially for function or migration from one site to another; current methods to generate long-range 3D alignment of a syn- thetic, fibrous matrix over arbitrary lengths in the presence of living cells are limited. To demonstrate further biological applications of the noodle construct, we formed a string in the presence of HL-1 cardiomyocytes, a cell line with spontaneous electrical activity that requires extensive cell–cell contacts to propagate signals, and then observed the cellular behaviour over time36. The cells survived and proliferated extensively to fill the entire structure. By fluorescently visualizing intracellular calcium concentration, we observed pock- ets of spontaneous electrical activity by day 6 and action potentials that were able to propagate throughout the entire macroscopic structure by day 10 (Fig. 4e). Plots of the calcium fluorescence over time show these propagating signals, demonstrating the presence of a continuous functional cardiac syncytium (Fig. 4f). This finding suggests potential for these ‘cellular wires’ in regenerative medicine applications in which restoring electrical communication in tissues is of critical importance, for example, in the treatment of cardiac arrhythmia or spinal cord injury. In addition to this ability to propagate electric signals, it is clear that the strong cell alignment of the constructs, as found in muscle and nervous tissue, will also be helpful in developing regenerative strategies. In another example we created amonodomain peptide amphiphile string gel containing dispersed carbon nanotubes that retained the same degree of align- ment as in pure peptide amphiphile strings (Fig. 4d). These black strings had electrical conductivities of the order of 1–10 S cm−1 in a dry state, depending on the concentration of carbon nanotubes. The incorporation of carbon nanotubes into the structure without disrupting the final construct suggests the use of the methodology to align 1Dnanostructures within biocompatiblematrices. We found in this work that supramolecular architectures of self-assembling molecules can markedly change in a pathway- dependent manner. We specifically discovered a thermal pathway that converts isotropic solutions of peptide-containing molecules to liquid crystals in which molecules group into long filaments of bundled nanofibres. This transformation allows the formation of monodomain fibrous gels, which may contain viable cells as well. These systems offer the potential to study phenomena in macroscopically aligned arrays of cells and could also lead to the development of therapies that require directed cell migration, directed cell growth or the spatial cell interconnections in tissues such as heart, brain and spinal cord. 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Acknowledgements This work was supported by the US Department of Energy-Basic Energy Sciences (DE-FG02-00ER45810, DE-FG02-08ER46539), National Institutes of Health (5-R01-EB003806, 5-R01-DE015920, 5-P50-NS054287), National Science Foundation (DMR-0605427), Department of Homeland Security Fellowship (M.A.G.), Non-Equilibrium Energy Research Center (NERC), an Energy Frontier Research Center funded by DOE-BES (award number DE-SC0000989 for L.C.P), Northwestern University’s NIH Biotechnology Training Program (pre-doctoral fellowship to J.R.M.), Ben Gurion University of Negev, Israel (post-doctoral fellowship for R.B.) and 600 NATURE MATERIALS | VOL 9 | JULY 2010 | www.nature.com/naturematerials