Interfaces between materials and cells play a critical role in cell biomedical applications. Here, a simple, robust, and cost-effective method is developed to fabricate patterned thermoresponsive poly(N-isopropylacrylamide-co-styrene) microgel strips on a polyethyleneimine-precoated, non-thermoresponsive cell-adherent glass coverslip. The aim is to investigate whether cell sheets could be harvested from these cell-adherent surfaces patterned with thermoresponsive strips comprised of the microgels. We hypothesize that if the cell-to-cell interaction is strong enough to retain the whole cell sheet from disintegration, the cell segments growing on the thermoresponsive strips may drag the cell segments growing on the cell-adherent gaps to detach, ending with a whole freestanding and transferable cell sheet. Critical value concerning the width of the thermoresponsive strip and its ratio to the non-thermoresponsive gap may exist for cell sheet recovery from this type of surface pattern. To obtain this critical value, a series of strip patterns with various widths of thermoresponsive strip and non-thermoresponsive gap were prepared using negative microcontact printing technology, with COS7 fibroblast cells being used to test the growth and detachment. The results unraveled that COS7 cells preferentially attached and proliferated on the cell-adherent, non-thermoresponsive gaps to form patterned cell layers and that they subsequently proliferated to cover the microgel strips to form a confluent cell layer. Intact COS7 cell sheets could be recovered when the width of the thermoresponsive strip is no smaller than that of the non-thermoresponsive gap. Other cells such as HeLa, NIH3T3, 293E, and L929 could grow similarly; that is, they showed initial preference to the non-thermoresponsive gaps and then migrated to cover the entire patterned surface. However, it was difficult to detach them as cell sheets due to the weak interactions within the cell layers formed. In contrast, when COS7 and HeLa cells were cultured successively, they formed the cocultured cell layer that could be detached together. These freestanding patterned cell sheets could lead to the development of more elaborate tumor models for drug targeting and interrogation.
The distribution of counterions at a charged surface has been measured directly using neutron specular reflection with isotopic labeling being used to highlight the scattering from the counterions. The system investigated was the soluble surfactant tetramethylammonium dodecyl sulfate with and without added tetramethylammonium chloride. Depending on the conditions a fraction of ions were found to penetrate the surfactant head-group region. The majority of the counterions observed formed a layer adjacent to the head-group layer which must be part of the diffuse layer. These two layers did not, however, account for all the counterions, and the remainder of the diffuse layer could not be observed with certainty. The reason for this is not understood, although it is probably associated with the roughness of the diffuse layer. Using the measured charge density and surface coverage as parameters, the counterion distribution calculated from the Stern−Gouy−Chapman model of the electrical double layer appears to account quite well for the shape of the distribution, although the effect of the "missing" ions made a detailed quantitative comparison impossible.
Short synthetic peptide amphiphiles have recently been explored as effective nanobiomaterials in applications ranging from controlled gene and drug release, skin care, nanofabrication, biomineralization, membrane protein stabilization to 3D cell culture and tissue engineering. This range of applications is heavily linked to their unique nanostructures, remarkable simplicity and biocompatibility. Some peptide amphiphiles also possess antimicrobial activities whilst remaining benign to mammalian cells. These attractive features are inherently related to their selective affinity to different membrane interfaces, high capacity for interfacial adsorption, nanostructuring and spontaneous formation of nano-assemblies. Apart from sizes, the primary sequences of short peptides are very diverse as they can be either biomimetic or de novo designed. Thus, their self-assembling mechanistic processes and the nanostructures also vary enormously. This critical review highlights recent advances in studying peptide amphiphiles, focusing on the formation of different nanostructures and their applications in diverse fields. Many interesting features learned from peptide self-organisation and hierarchical templating will serve as useful guidance for functional materials design and nanobiotechnology (123 references).
We report a new class of cationic amphiphilic peptides with short sequences, G(IIKK)nI-NH2 (n = 1–4), that can kill Gram-positive and Gram-negative bacteria as effectively as several well-known antimicrobial peptides and antibiotics. In addition, some of these peptides possess potent antitumor activities against cancer cell lines. Moreover, their hemolytic activities against human red blood cells (hRBCs) remain remarkably low even at some 10-fold bactericidal minimum inhibitory concentrations (MICs). When bacteria or tumor cells are cocultured with NIH 3T3 fibroblast cells, G(IIKK)3I-NH2 showed fast and strong selectivity against microbial or tumor cells, without any adverse effect on NIH 3T3 cells. The high selectivity and associated features are attributed to two design tactics: the use of Ile residues rather than Leu and the perturbation of the hydrophobic face of the helical structure with the insertion of a positively charged Lys residue. This class of simple peptides hence offers new opportunities in the development of cost-effective and highly selective antimicrobial and antitumor peptide-based treatments.
In a previous study of BSA adsorption onto the hydrophilic silica/water interface using neutron reflection, we examined the concentration dependence of the surface excess of BSA at a pH close to its isoelectric point (IP). The surface excess was found to reach a plateau at a very low bulk protein concentration, suggesting a high affinity of BSA for the oxide surface. This work has now been extended to an investigation of the structure and composition of the BSA layer above and below its IP. It is found that adsorption of BSA is strongly dependent on pH, although the protein concentration has little influence on the surface excess at pH 3 and 7. Changing the pH from the IP substantially reduces the surface excess. The structure of the adsorbed layers below a bulk BSA concentration of 0.5 g dm-3 can be fitted to a single uniform layer distribution over all pH conditions studied, which suggests that there is no significant denaturation. Denaturation generally leads to a more fragmented peptide distribution and a nonuniform density distribution normal to the surface. The thicknesses of the layers below 0.5 g dm-3 were all smaller than the dimension of the short axis of the globular solution structure for BSA, indicating that the molecules are adsorbed sideways-on with their long axes parallel to the solid surface and that adsorption onto the hydrophilic surface results in some structural deformation. The reversibility of BSA adsorption at the hydrophilic silica/water interface was also examined directly. Adsorption was found to be irreversible with respect to changes in BSA concentration but reversible with respect to solution pH at low BSA concentrations only.
Neutron reflection has been used to study the effects of solution pH and ionic strength on the surface excess and layer thickness of lysozyme layers adsorbed at the air/water interface. All the measurements were made in null reflecting water (NRW) so that all the specular signal arose from the protein layers. At the low ionic strength of 0.02 M, the adsorption was found to reach a maximum at the protein isoelectric point (IP) of pH 11, with the effect of pH on the adsorbed amount and layer thickness being more pronounced at the higher lysozyme concentration. At the low lysozyme concentration of 0.03 g dm-3, the thicknesses of the adsorbed layers are 30 ± 3 Å over almost the entire pH range, close to the short axial length of the globular dimension of lysozyme, and the area per molecule is 1700 ± 200 Å2, suggesting the formation of a sideways-on monolayer. At the high lysozyme concentration of 1 g dm-3, a number of conformational transitions occur within the adsorbed layers with respect to pH and these variations correlate well with the change in the number of net charges within lysozyme with pH, suggesting that the preferred conformation of protein molecules is dominated by the combined effect of steric and electrostatic repulsion within the adsorbed layer. Subsequent measurements at the high ionic strength of 1 M showed no obvious variation in either layer thickness or surface excess with pH or with bulk protein concentration. The thickness was found to be constant at 30 ± 3 Å and the area per molecule to be 1500 ± 100 Å2, corresponding to the formation of a close-packed sideways-on monolayer. These results clearly show that salt addition has screened the charges within lysozyme molecules.
De novo peptide surfactant (I3K) gels provide an ideal system to study the complex dynamics of lightly cross-linked semiflexible fibers because of their large contour lengths, simple chemistry, and slow dynamics. We used single-molecule fluorescence microscopy to record individual fibers and Fourier decomposition of the fiber dynamics to separate thermal contributions to the persistence length from compressive states of prestress (SPS). Our results show that SPS in the network depend strongly on peptide concentration, buffer, and pH and that the fibril energies in SPS follow a Lévy distribution. The presence of SPS in the network imply that collective states of self-stress are also present. Therefore, semiflexible polymer gels need to be considered as complex load-bearing structures and the mean field models for polymer gel elasticity and dynamics often applied to them will not be fully representative of the behavior at the nanoscale. We quantify the impact of cross-links on reptation tube dynamics, which provides a second population of tube fluctuations in addition to those expected for uncross-linked entangled solutions.
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