Characterizing the influence of fragment crystallization (Fc) and antigen-binding fragment (Fab) on monoclonal antibody (mAb) adsorption at the air/water interface is an important step to understanding liquid mAb drug product stability during manufacture, shipping, and storage. Here, neutron reflection is used to study the air/water adsorption of a mAb and its Fc and Fab fragments. By varying the isotopic contrast, the adsorbed amount, thickness, orientation, and immersion of the adsorbed layers could be determined unambiguously. While Fc adsorption reached saturation within the hour, its surface adsorbed amount showed little variation with bulk concentration. In contrast, Fab adsorption, was slower and the adsorbed amount was concentration dependent. The much higher Fc adsorption, as compared to Fab, was linked to its lower surface charge. Time and concentration dependence of mAb adsorption was dominated by Fab behavior, although both Fab and Fc behaviors contributed to the amount of mAb adsorbed. Changing the pH from 5.5 to 8.8 did not much perturb the adsorbed amount of Fc, Fab, or mAb. However, a small decrease in adsorption was obseived for the Fc over pH 8-8.8 and vice versa for the Fab and mAb, consistent with a dominant Fab behavior. As bulk concentration increased from S to 50 ppm, the thicknesses of the Fc layers were almost constant at 40 angstrom, while Fab and mAb layers increased from 45 to 50 angstrom. These results imply that the adsorbed mAb, Fc, and Fab all retained their globular structures and were oriented with their short axial lengths perpendicular to the interface.

The aim of this work is to examine how adhered individual cells could detach from the patterned, discontinuous thermoresponsive coating substrate and how different patterns in the form of thermoresponsive squares and gaps would affect cell detachment. Microgels prepared from copolymerization of N-isopropylacrylamide and styrene (pNIPAAmSt) were spin-coated on polyethylenimine (PEI) precoated glass coverslips to form a uniform microgel monolayer; then a surface-moisturized PMDS stamp was used to contact the microgel monolayer at room temperature. The thin layer of water on the PDMS stamp surface worked as an ink to penetrate the microgels so that any microgels in direct contact with the wet stamp surface became swollen and could be peeled away, while uncontacted microgels formed patterns. Using this method, various patterns with different thermoisland diameters and gaps could be fabricated. NIH3T3 fibroblast cells were then cultured on these patterns to study their detachment behavior. It was found that cells could detach not only from these discontinuous thermoresponsive coatings, but also from the patterned surfaces with the thermoresponsive area being as low as 20% of the cell spread area.

The artificially designed amphiphilic peptide G(IIKK)(3)I-NH2 has been shown to be highly effective at killing bacteria and inhibiting the growth of tumor cells whilst remaining benign to normal mammalian cells. Herein we report how the side chain length and branching of constituent amino acids affect these bioactivities. Two peptide groups were designed by utilizing G(IIKK)(3)I-NH2 as the base template. In Group 1, hydrophobic residues were replaced from Ile to Leu, Nle (norleucine), or Val. It was found that an increase in the side chain carbon number from 3 (Val) to 4 (Leu, Ile or Nle) substantially enhanced their antibacterial and antitumor activities, but different branching in the butyl side chain showed very different cytotoxicities to host mammalian cells, with the gamma-branching in Leu eliciting the highest potency. Group 2 covered those cationic Lys residues which were replaced by synthetic homologues with shorter side chains, namely, Orn, Dab and Dap containing 3, 2 and 1 methylene units, respectively. The replacement did not affect their antibacterial activities much, but their anticancer activities were maximized in Orn and Dab. On the other hand, their cytotoxicities also became higher, indicating a multi-faceted role played by the cationic residues. Thus, changes in both the side chain length and branching strongly affected the amphiphilicity of the short peptides and their interactions with different membranes. This work has revealed a strong relationship among side chain structures, amphiphilicity and selective bioactivities of the short peptide amphiphiles.

Short designed peptide amphiphiles are attractive at killing bacteria and inhibiting cancer cell growth, and the flexibility in their structural design offers a great potential for improving their potency and biocompatibility to mammalian host cells. Amino acid sequences such as G(IIKK)nI-NH2 (n=E2=89=A53) have been shown to be membrane lytic, but terminal amino acid modifications could impose a huge influence on their performance. We report in this work how terminal amino acid modifications to G(IIKK)3I-NH2 influence its alpha-helical structure, membrane penetrating ability, and selective actions against different cell types. Deletion of an N-terminal Gly or a C-terminal Ile did not affect their antibacterial activity much, an observation consistent with their binding behavior to negatively charged membrane lipid monolayers. However, the cytotoxicity against mammalian cells was much worsened by the N-terminal Gly deletion, consistent with an increase in its helical content. Despite little impact on the antibacterial activity of G(IIKK)3I-NH2, deletion of both terminal amino acids greatly reduced its antitumor activity. Cholesterol present in tumor cell membrane-mimic was thought to constrain (IIKK)3-NH2 from penetrating into the cancerous membranes, evident from its lowest surface physical activity at penetrating model lipid membranes. On the other hand, its low toxicity to normal mammalian cells and high antibacterial activity in vitro and in vivo made it an attractive antibacterial agent. Thus, terminal modifications can help rebalance the different interactions involved and are highly effective at manipulating their selective membrane responses.=20

During the formulation of therapeutic monoclonal antibodies (mAbs), nonionic surfactants are commonly added to attenuate structural rearrangement caused by adsorption/desorption at interfaces during processing, shipping, and storage. We examined the adsorption of a mAb (COE-3) at the SiO2/water interface in the presence of pentaethylene glycol monododecyl ether (C12E5), polysorbate 80 (PS80-20EO), and a polysorbate 80 analogue with seven ethoxylates (PS80-7EO). Spectroscopic ellipsometry was used to follow COE-3 dynamic adsorption, and neutron reflection was used to determine interfacial structure and composition. Neither PS80-20EO nor C12E5 had a notable affinity for COE-3 or the interface under the conditions studied and thus did not prevent COE-3 adsorption. In contrast, PS80-7EO did coadsorb but did not influence the dynamic process or the equilibrated amount of absorbed COE-3. Near equilibration, COE-3 underwent structural rearrangement and PS80-7EO started to bind the COE-3 interfacial layer and subsequently formed a well-defined surfactant bilayer via self-assembly. The resultant interfacial layer comprised an inner mAb layer of about 70 A thickness and an outer surfactant layer of a further 70 A, with distinct transitional regions across the mAb-surfactant and surfactant-bulk water boundaries. Once formed, such interfacial layers were very robust and worked to prevent further mAb adsorption, desorption, and structural rearrangement. Such robust interfacial layers could be anticipated to exist for formulated mAbs stored in type II glass vials; further research is required to understand the behavior of these layers for siliconized glass syringes.=20

Monodisperse poly(N-isopropylacrylamide-styrene) (PNIPAAmSt) microgels with different St/NIPAAm ratios have been synthesized via a one-step surfactant-free emulsion polymerization process. The resulting microgel dispersions were used to fabricate 2D arrays on the surface of silicon wafers/glass coverslips through dip coating. The thermal responsiveness of the PNIPAAmSt microgel arrays was examined by spectroscopic ellipsometry and the results unraveled that the thermoresponsive behavior of the arrays was highly consistent with the microgels dispersed in the bulk, showing high dependence on the content of styrene. The structure of the films varied from nonclose-packed 2D arrays to close-packed 2D arrays, depending on both properties of the microgels and array fabrication conditions. When the weight ratio of styrene was below 40%, the microgel arrays demonstrated effective control for cell growth and detachment across their volume phase transition temperatures (around 28 =C2=B0C). The extent of swelling of the microgels was the key factor to determine whether the cells could detach from the film easily. For the rather close-packed 2D arrays prepared by the same kind of PNIPAAmSt microgels, the gaps between microgel particles showed no obvious effect on the rate of cell detachment. =20

Peptide self-assembly is a hierarchical process, often starting with the formation of alpha-helices, beta-sheets or beta-hairpins. However, how the secondary structures undergo further assembly to form higher-order architectures remains largely unexplored. The polar zipper originally proposed by Perutz is formed between neighboring beta-strands of poly-glutamine via their side-chain hydrogen bonding and helps to stabilize the sheet. By rational design of short amphiphilic peptides and their self-assembly, here we demonstrate the formation of polar zippers between neighboring beta-sheets rather than between beta-strands within a sheet, which in turn intermesh the beta-sheets into wide and flat ribbons. Such a super-secondary structural template based on well-defined hydrogen bonds could offer an agile route for the construction of distinctive nanostructures and nanomaterials beyond beta-sheets.=20

In this study, Saccharomyces cerevisiae was genetically engineered to harbor the capability of utilizing celluloses for bioethanol production by displaying active cellulolytic enzymes on the cell surface. An endo-1,4--glucanase gene egX was cloned from Bacillus pumilus C-9 and its expression products, the EGX cellulases, were displayed on the cell surface of S. cerevisiae by fusing egX with aga2 that encodes the binding subunit of the S. cerevisiae cell wall protein -agglutinin. To achieve high gene copies and stability, multicopy integration was obtained by integrating the fusion aga2-egX gene into the rDNA region of the S. cerevisiae chromosome. To achieve high expression and surface display efficiency, the aga2-egX gene was expressed under the control of a strong promoter. The presence of the enzymatically active cellulase fusion proteins on the S. cerevisiae cell surface was verified by carboxymethyl cellulase activity assay and immunofluorescence microscopy. This work presented a promising strategy to genetically engineer yeasts to perform efficient fermentation of cellulosic materials for bioethanol production.

Fabrication of antibacterial materials with sustained release of active components is of great importance for long-term antibacterial applications. Graphene oxide (GO) has been found to be an excellent carrier for accumulating the antibacterial peptide of G(IIKK)4I-NH2 and mediating its loading into the layer-by-layer (LBL) films for sustained release applications. G(IIKK)4I-NH2 takes random coiled conformation in monomeric state below 0.17 mM but self-assembles into supramolecular aggregates with alpha-helical secondary structure at higher concentrations. It can bind onto GO surface in both monomeric and aggregate states to form stable GO@G(IIKK)4I-NH2 composites. Upon binding, the local amphiphilic environment of GO surface induces a conformational transition of G(IIKK)4I-NH2 monomers from random coils to alpha-helix. The aggregate binding enhances the loading amount greatly. GO (1 mg) can load as high as 1.7 mg of peptide at saturation. This enables the GO@G(IIKK)4I-NH2 composites to serve as reservoirs for sustained release of active G(IIKK)4I-NH2 monomers. Moreover, G(IIKK)4I-NH2 itself shows low efficiency in LBL assembly, whereas the GO@G(IIKK)4I-NH2 composites are ideal LBL assembling units with highly enhanced loading efficiency of G(IIKK)4I-NH2. The LBL films involving degradable poly(beta-amino esters) can realize sustained release of G(IIKK)4I-NH2 for bacteria killing in a well-controlled manner. This study demonstrates an efficient strategy for fabrication of long-durable antibacterial materials and surface coatings by using GO as the carrier for drug accumulation and loading.=20